J. Mol. Hiol. (1991) 219, 533-554

Crystal Structure of Cholesterol OxidFse from Brevibacterium sterolicum Refined at 143A Resolution Alice Vrielinkf-,

Lesley F. Lloyd and David M. Blow1 Blackett Laboratory Imperial College London S W7 ZBZ, England

(Received 30 August 1990; accepted 5 February

1991)

Cholestrrol oxidase (3B-hydroxysteroid oxidase, EC 1.1.3.6) is an FAD-dependent enzyme that carries out the oxidation and isomerization of steroids with a trans A : B ring junction. The crystal structure of the enzyme from Brevibacterium sterolicum has been determined using the method of isomorphous replacement and refined to 1.8 A resolution. The refined model includes 492 amino acid residues, the FAD prosthetic group and 453 solvent molecules. The crystallographic R-factor is 153% for all reflections between l@O A and I.8 A resolution. The structure is made up of two domains: an FAD-binding domain and a steroid-binding domain. The FAD-binding domain consists of three non-continuous segments of sequence, including both the N terminus and the C terminus, and is made up of a six-stranded B-sheet sandwiched between a four-stranded B-sheet and three a-helices. The overall topology of this domain is very similar to other FAD-binding proteins. The steroidbinding domain consists of two non-continuous segments of sequence and contains a sixstranded antiparallel P-sheet forming the “roof’ of the active-site cavity. This large /?-sheet structure and the connections between the strands are topologically similar to the substratebinding domain of the FAD-binding protein para-hydroxybenzoate hydroxylase. The active site lies at the interface of the two domains, in a large cavity filled with a well-ordered latt,ice of 13 solvent molecules. The flavin ring system of FAD lies on the “floor” of the cavity with X-5 of the ring system exposed. The ring system is t’wisted from a planar conformation by an angle of approximately 17”, allowing hydrogen-bond interactions between t,he protein and t,he pyrimidine ring of FAD. The amino acid residues that line the active site are predominantly hydrophobic along the side of the cavity nearest the benzene ring of the flavin ring system, and are more hydrophilic on the opposite side near the pyrimidine ring. The cavity is buried inside the protein molecule, but three hydrophobic loops at the surface of the molecule show relatively high temperature factors, suggesting a flexible region that may form a possible path by which the substrate could enter the cavity. The active-sitr cavlt’y contains one charged residue, Glu361, for which the side-chain electron density suggests a high degree of mobility for the side-chain. This residue is appropriately positioned t’o act as the proton acceptor in the proposed mechanism for the isomerization step.

Kqwords: cholesterol

oxidase;

steroid

enzyme;

1. Introduction (:holesterol oxidase (3fi-hydroxysteroid lC(’ 1.1.3.6) catalyses the oxidation and

flavoenzyme:

crystal

struct)ure

tion of A5-ene-3/l-hydroxysteroids with a trans A : K ring junction. to give A4-%ketosteroids. The enzyme is a flavin-dependent oxidase containing one mole of tightly bound flavin adenine dinucleotide (FAD4) per iole of protein. The enzyme carries out the stoichiometric oxidation and ” isomerizat,ion steps shown in Figure 1, yielding one mole of hydrogen peroxide per mole of steroid oxidized. The enzyme has been isolated from a number of different soil bact,eria that are capable of using cholesterol as t.hrir sole source of carbon and energy (Stadtman et al.. 1!)54; Fukuda et al., 1973; Uwajima et al., 1973; Fukuyama & Miyake, 1979; Tnouye et al., 1982).

oxidase, isomeriza-

t Present address: Molecular Riology Institute and Department of Chemistry and Biochemistry, University of California, Lox Angeles, CA 90024, U.S.A. 1 Author to whom all correspondence should be addressed. Q Abbreviations used: FAD, flavin adenine dinuclcotide; NapCMB, sodium p-chloromercuribenzoak; m.i.r.. multiple isomorphous replacement: r.m.s., root-mean-square. 533 0022

-2X36/91

/l 105:K--%2

%03.00~0

(C; 1991

Aradcmic~

Prrss

I,imitrd

Cholesterol t5-cholestan-3-d)

5-Cholesten-3-one 02

4-Cholesten-3-one

HA’,

Figure 1. The enzymic reaction catalysed by cholesterol oxidase with cholesterol as the steroid substrate. The reaction consists of an oxidation step that utilizes tightly bound FAD, followed bp an isomerization step. The reduced FADH, is rcvxidized by molecular oxygen. The steroid numbering scheme referred to in the text is shown.

Cholesterol oxidase from bacterial sources is used principally in the clinical determination of serum c*holesterol concentration for the assessment of arteriosclerosis and other lipid disorders and of the risk of thrombosis. The enzyme is also used in the microanalysis of steroids in food specimens, for the determination of the steric configuration of Xb-hydroxysteroids, and in the preparation of 3-krtosteroids from their corresponding 3/l-hydroxysteroids. A broad range of steroid substrate specificities has been observed (Smith & Brooks, 1975; Kamei et al., 1978; Inouye et al., 1982). In all cases, the presence of a 3/l-hydroxyl group on the steroid substrate is essential for activity. Although the enzyme is most active towards cholesterol, a high oxidase activity is also observed for steroids without a double bond at (‘-5 of the steroid ring. Cholesterol oxidase from Rrevibacterium sterolicum has been isolated and purified by Uwajima et al. (1973). The M, determined by SDS/ polyacrylamide gel electrophoresis is 55,000 and the amino acid sequence determination of the enzyme is in progress (T. Uwajima, personal communication). However, the complete gene sequence of the enzyme from Streptomyces sp. strain SA-COO (Ishizaki et al.. 1989) has been determined. Comparison of this sequence with that of the partially determined Rrevibacterium sequence shows a high degree of homology. Although a large amount of struct,ural data is available for steroid molecules, there is very limited structural information on the proteins to which they bind. Crystallographic studies have been carried out’ on the progesterone-binding protein, uterogtobin (Mornon et al., 1980; Morize et al., 1987). This struct)ure was found to contain a large hydrophobic* pocket, on the binary axis of the molecular dimer, filled with 14 water molecules. Crystals have been reported for the NADH-dependent steroid-metabolizing enzyme 3m,20fl-hydroxysteroid dehydrogenase (Ghosh et al., 1986) and the structure determination is in progress. Westbrook et al. (1984) have determined the structure of As-3-ketosteroid isomerase from Pseudomonas testosteroni to 6 a resolution (1 A = 0.1 nm) and, more recently, to 2.3 A resolution (E. Westbrook, persona,] communicaa-

tion). This enzyme carries out t’he same isomcrization step as cholesterol oxidase using the oxidized steroid as the substrate. Kinetic and magnetit resonance studies on As-3-ketosteroid isomerase using a substrate analogue have enabled Kuliopulos et al. (1987) to identify the residues important for t,he catalysis and to suggest a mechanism for the isomerization reaction that is consistent with both covalent modification (Batzold et ccl., 1976; Ogez ct al.? 1977; Benisek et al., 1980) and affinitv labelling studies of the enzyme (Pollack et al.. 198k). A number of FAD-dependent enzymes have been studied structurally. These include glut)at,hionr reductase (Schulz et ul., 1978, 1982; Karplus & Schulz, 1987), p-hydroxybenzoate hydroxylasr (Wierenga et al., 1979; Schreuder et al., 1988. 1989) and lipoamide dehydrogenase (Takenaka et ~1.. 1988; Schierbeek, 1988). The topology of the FAD-binding domain is identical in these proteins. Although they show no global sequence homology. the FAD-binding domain in each protein contains pattern of three glycine residues. X denot’es at)) kly-X-Gly-X-X-Gly (where residue) common to NAD and FAD-binding proteins and structurally enclosed in a PCY~unit referred t,o as t,he ADP-binding /?cr.fi-fold (Wierenga et (~1.. 1985, 1986). This pattern of residues is also found near t,he Y terminus of the sequence of cholest,erol oxidase from R. sterolicum. The sequence of a protein from bovine ovary wit,h 3/3-hydroxy-5-ene-steroid dehydrogenase Mid steroid As-A4-ene-isomerase activities has been determined (Zhao et al.. 1989). Although carrying out the same enxymic reaction as cholesterol oxidase from soil bacteria, this enzyme differs in that it contains NAD+ as a co-factor rather t,han FAD. A sequence comparison between this mammalian enzyme and the bacterial cholesterol oxidasti shows no significant degree of homology. The mammalian enzyme does however contain the pattern of referred to above as thta glycine residues ADP-binding /?&fold. The present paper describes the structure solution of cholesterol oxidase at 2.7 A resolution using area detector data from five heavy-atorn derivatives. Refinement of the structure at I.8 i% resolution has facilitated a complete structural description and a

Btructure

of Cholesterol

Oxidase

Figure 2. Ort~horhombic c*rystals of cholesterol oxidase grown by vapour diffusion using the hanging-drop technique from sodium/potassium phosphate, pH 7.0. The crystals grow as clusttars of needles that can be separat,rd easily. yielding a single q&al.

discussion of t.he possible important features steroid binding and the mechanism of catalysis.

2. Experimental (a) (‘rystallization

and

of

molecule of 55.000 X, in the asymmetric unit. these dell dimensions correspond to a specific. volume. L’,,,, of 23 A3/dalton. implying a solvent conttmt of -Go& (v/v: Matthews. 1968).

Procedures space

group

determination

Cholesterol oxidasr from U. sterokum, isolated and purified using th(a methods described by IJwajima et al. (1973. 1974). was supplied by Kyowa Hakko Kogyo Ltd. in 95”, (w/w) bovine serum albumin as a freeze-dried powder. The powder was redissolved in 50 mM-potassium phosphate (pH i.0) and the bovine serum albumin removed using a Q-Sepharose ion-exchange column. The enzyme was stored at -20°C in 50% (v/v) glycerol and 50 mM-sodium/potassium phosphate (pH 7.0). Before crystallization, t#he glycerol was removed by washing with 50 mw-sodium/potassrum phosphate (pH 7.0) using a (‘entri~on 30 (Amicon) filter and the enzyme concentrated to 20 mgiml. The concentral ion of the protein was determined by measuring the absorbance at 280 nm using an extinction cdoefficient (E:&) of 17.1 (Vwajima et al.. 1974). (‘rystals suitable for X-ra), diffraction were obtained by vapour dif&sion using the hanging-drop method. Drops containing an initial protein concentration of 20 mg/ml and 0.5 to (M.5 M-s(~(~itlm/f)otassium phosphat,e were eyuilibrattltl at 1X6(’ against a well concentration of 1.3 .M-sodiunl:‘E)otassiu,n phosphate. Within 3 to 4 days. bright yellow nt>rtlle c,r,vstals of the enzyme appeared as double wedges. These most often grew as clusters with numerous small satellite needles which could be easil! removed without damage to the major needle (Fig. 2). The crystals exhibited a long lifetime in the X-ra,v beam and diffrac.trtl to better than 1.8 x resolution. The space group of the crystals was determined bJ precession photography t,o be P2,2,2, with cell dimensions (l = 67.71 X. t) = 84.58 4. c = 88.13 A. Assuming 1

(b) Henry-atom

dwiwtiw

.wmh

Hravy-atom derivatives were found by soaking native rryst,alc of the enzyme in known concentrations of heavymetal solutions dissolved in 1.3 M-sodium/potassium phosphat.e (pH 7.0) and testing for differences amongst the individual reflections. When significant differences were observed. a full data set was collecated and processed.

1)ata for t,he heavy-atom derivatives and native crystals were collected on a diffractomet.er equipped with a FAST area detector using CuKcl radiation from a rotating-anode ?i-ray generator. Data were collected with a crystal-to-detector distance of HO mm and the 20 arm of the detector positioned at, an angle of 17”. Images were processed during the data collection using the program system NADXES (Messerschmidt & I’flugrath, 1987). Images of 0.1 deg. were collected for a total crystal rotation of 1M” for 2 different crystal orientations. Scale and temperature fact)ors were calculated for bat)ches of 5” rotation and refinrd using the algorithm of Fox & Holmes (1966). The heavy-atom derivative data sets were scaled t,o the native data by applying, to the derivative data sets. a scale factor and a relative temperature fac+or. obtained from a Wilson plot (Wilson. 1950). A 1.8 .t resolution native data set was obtained at the synchrotron radiation source, Daresbury. r.K.. with the crystals cooled to a temperature of - 1O’Y’. In order to obtain ?I complrtt~ data set. 2 crystals were used. The

536

.4.

l’rielink

Table Crystal

Soak concentration (mM) Soak time (days) Source of data \Vavrlenpth (A) liesolution (A) ‘I’t~tllperaturr (“C’) Numbor of crystals Nurnhrr of reflections I ndrpendrnt reflections (‘(, (‘omplete data Merging [i-factor: (%)§ J)rrivative &factor: (%)I1 ‘l’ SKS. Synchrotron 1 FAST. Enraf-Sonius # Merging &factor:

11ljrrivative

data summary

mst 0.9 I

FAST1

1.8 -10

2.7 + 20 I 33,761 1432 O&3 54

2 149.1x7 43,094 Yfx :5x

radiation FAST

et, a1

1

of’ native and heavy-atom

FAN I.54 2.7 + “0 I 18.440 10.X80 UT!) 5.1 I-f.3

1.54

source. 1)aresbury. area det,rctor.

drrivatives

r.K

It-factor.

crgst,al morphology is such that data could he collectt~d at a number of different positions along t.hr crystal lengt’h. Oscillation photographs were collected on (‘EA Reflex film at a wavelength of 0.91 A using an ArndtWonacott oscillation camera (Enraf-Xonius. Delft). The reflections w-em recorded on Iilm packs of 3 films separated by IN pm aluminiutn foils. Scan angles of 1.0 and 1.5” were used so as to maxitnizr the number of reflections and minimize the number of overlapping reflections of racah filtn. The films were digitized by scanning on a ,Joyce -Lorbl Scandig 3 rotating drum microdensitometer controlled by a \TAX I I/750 computer. The data were processed using a MOHFLM program suite developed at, Imperial College and modified t,o inelude an algorithm for profile fit’tinp (Leslie. 1987a). The data were monit’ored for c:ryst,al slip page and the orientation matrix updated by using the (‘ommon partially recorded reflections on adjacent, films. Scale and temperature factors were calculated and rr6ned by the method of Fox & Holmes (1966) and applied to each film pack. Part,ially recorded reAections on adjacent tilms were added together and agreement between symmetry-related reflections monitored. Structure factors were calculated from the averaged intensities and tnodified according to the procedure out,lined by French & Wilson (1978), to obtain positive structure factors that follow Wilson’s distribution. Table 1 gives a summary of the data collection and processing for both the film and area detector data. (d) Heavy-atom

re$nement

and phasiny

The heavy-atom positional parameters for the sodium p-chloromercuribenzoate (NapCMB) derivative were obtained from a S-dimensional difference Patterson map using the FAST native data. The position and occupancy of the heavy-atom sites, as well as the scale and temperature factor relating the derivative data to the native data, were refined using the phase refinement program PHARE (CCP4 program suite). The anomalous scattering data

were used to determine the absolute c~onfiguration of the structure. I’sing this set of single isomorphous replacetnent phases. the positional parameters for subsequent derivatives were determined by difference Fourier methods. Only centric reflections were used for t,hcx heavy-atom refinement, whereas all reflections were used for the phase c*alculation. The final multiple isomorphous replacement (m.i.r.) phases used to calculate a “best” native Fourier map (Blow & Crick, 1959) were obtained using the refined parameters from 14 sites for 5 heavy-atom derivatives. Table 2 gives the final parameters for rarh of the heavy at,om derivatives.

(e) Solvent jlattenkg Although the solvent content. was low, density modificxation by the solvent-flattening procedure of Wang (19%) was used to improve the quality of the electron density map. The molecular envelope was determined using a reciprocal spare method (Leslie, 1987b). The calculated phases, after 8 cycles of solvent flattening were recombined with the starting m.i.r. phases (Bricogne. 1976). An electron density map was calculated using t)he csombined phases and was visually compared to an elrctron density map calculated using the m.i.r. phases. Little difference between the 2 maps was observed in sections of the map corresponding to the protein, indicating that solvent flattening did little to improve the phases. (f)

Electron density map interpretation

A 2.7 A resolution electron density map was calculated using combined phases weighted by t)he figure of merit. An initial interpretation of the map was facilitated by the interactive graphics program FRODO (-Jones, 1978). which incorporates a skeletonized electron density map (Greer, 1985). coupled with a fragment-fitting algorithm (,Iones & Thirup, 1986) using a subset of the best, refined structures from the Brookhaven Protein Data Bank.

?f Cholesterol Oxidase

Structure

537

Table 2 Heavy-atom refinement pwameters

&“lliS

Derivative

(O/o)?

Phasing power1

Nap(‘MIs K:l’t13r4

58 87

2.06 I .20

Sep(‘MB/K20s0,

65

I.76

K,OsO,

83

1.30

K,Hd,

73

I .70

t (‘ullis

Metal Hg 1 Pt 1 Pt 2 Pt 3 Hg ’ OS 1 OS 2 OS 1 OS 3 OS 4 OS 5 % 1 Hg 2 1 1 12 I 3 Hg 3

x

y

Relative occupancy

z

0.037 0.156 O-101 0663 0.365 0416 @125 0.389 0.2 18 0.012 0260 @200 0.036 0.154 0101 0474 0.120 0.010 -0.283 0.029 0016 0.474 0.119 0.012 0053 0.164 -0.139 -0.411 0.053 0.035 -@455 --0144 0.043 @042 0.156 @IO0 0151 0450 0.350 0.160 0.465 @370 ml68 0.449 0.329 0126 0.433 @352 WI96 0357 0325

190 0.94 0.47 032 0.97 0.88 0.71 0.88 0.44 0.12 0.15 0.91 0.35 059 047 0.53 0.06

the chain tracing of the emerging

progressed,

fragments

from

1

chain were found to be similar to the FAD-binding domain of p-hydroxybenzoate hydroxylase and glutathione reductase. The structure of the FAD-binding domain of p-hydroxybenzoate hydroxylase was superimposed onto the fragments of the model and the superposition used to facilitate further interpretation. The FAD molecule included in the initial model was taken from

the

structure

Cys57 Met412 Met81 Met59 Cys57 His306 Lys278 His306 (‘ys57 II Cys57 Met332 Met332 Met332 IVlet332 II

R-factor:

1 Phasmg power = r.m.s. heavy-atom structure factor:r.m.s. lack of closurr. 5 Residue in the final model to which the heavy atom bound. I/ These sit)es were not able to be verified by difference Fourier maps using the final

As region

Binding sit4

of p-hydroxybenzoate

hydroxylase.

Since t,he complete amino acid sequence of the enzyme from B. sterolicuwl was not available, the sequence of the enzyme from Strvptomyces sp. strain SP-COO (Ishizaki rt aZ., 1989) was used together with the sequence of peptide fragments from the Brevibacterium enzyme (T. Uwajima. personal communication). In this way, an initial model

refined

phases

was built that included the FAD molecule and 474 amino acid residues, of which 102 residues were modelled as alanine. since the electron density was not of sufficient quality to assign the side-chains unambiguously. (g) Refinement of the model The initial model was refined against the high-resolution film native data using the molecular dynamics program XPLOR (Briinger et al., 1987; Briinger, 1988). Initially, the refinement was carried out using only data to 2.5 A and in later rounds the resolution was gradually extended to I.8 A. The starting crystallographic R-factor for the structure was 47.1 o/o using all reflections from 100 to 2.5 A. Before

Table 3 Progress of the crystallographic rejkemrnt Refinement round la Ill 2 3 4 5 6 7 8a 8bf 9

Refinement method

Resolution (4

XPLOR XPLOR-SAt XPLOR XPLOR XPLOR XPLOR XPLOR XPLOR XPLOR PROLSQ PROLSQ

100-2.5 100-2.3 10.0-2.3 100-2.3 IWO-l.8 100~1.8 10.0-1.8 10.0-l+ 10.0~1.8 lOG1.8 10.0-1.8

Number of water molecules

t Molecular dynamics round using simulated annealing. $. The co-ordinates from refinement round 8a were further 9: (‘alculated over all reflections in the specified resolution

Number of residues

(1 0 0 26 88 206 332 391 415 415 453

refined range.

474 474 477 485 489 496 496 498 492 492 492

using

PROLSQ.

R-facto4 (%) 299 28.6 26.X 24.1 24.5 199 17.5 167 16.4 157 1,%3

538 -

A.

et al.

I’rielink

______

------.-.___

CO(Strep)

(

1)

DNCGYVPAVVICTGYGAAVSALRLGEAGVQTLMLEMCqLWNQPGPDGNIFCGMLNPDKRSSWFLNRTEAPLGSFL

CO(Brev)

(

5)

ALADGDAVPAVVIGSGYGGAVAALRLT~AGVPT~IVEMGRSWDTPGADGKIFCGMLAPDKRSMWLATKTD~PVAGFM ttttbb

bbbb

hhhhhhhhhhhh

bbbb

( 76)

WLDVVNRNIDPYAGVLDRVNYDQMSVYVGRGVGGGSLVNCDRYFPRANSMLRV

CO(Brev)

( 62)

GFGINAS-IDRYVGVLDSEAFSGISVYQGRGVGGGSLVNGGMAVTPKRSYFEEILPSVDSSEMYNKYFPRANTCLGV tt bbbbb bbbbb tttttt hhhhhhhtttt

tt (152) (157)

hhhhhhhhhhhhhhh

CO(Strep)

(153)

NHIDTKWFEDTEWYKFARVSREQAGKAGLGTVFVPNVYDFGYMQRHAAGEVPKSALATEVIYGNNHGKQSLDKTYL

CO(Brev)

(158)

NDIDQAWFESTEWYSFARTGRKTaqRSGFTTAFVPNVYDFE~KSEAAGQVTKSGLGGEVIYGNNAGKQSLDKTYL hhhhhh hhhhhhhhhhhhhh bbb tttt hhhhhhhh

CO(Strep)

(229)

AAALGTGKVTIQTLHQVKTIRQTKDGGYALTVEQKDTEGKLLATKEISCRYLFLGAGSLGSTELLVRARDTGTLPN

CO(Brev)

(234)

ASAAATGKLTITTLHKVTETAPAA hhhh bbbb bbbbbbbb

(228) (233) tttt

tttt

hh (304)

GYSI’IMEQIDEEGNVVASKTVTANRVFFAAGSVCTSKLLVSHKAQGHLPN bbbbbbbbtttt bbbbbbbbb bbbb hhhhhhhhhhhhh

CO(Strep)

(305)

LNSEVGAGWGPNGNIMTARANHMWNPTGAHQSSIPALGIDA~NSDSSVFAEIAP~AGLET~SLYLAITKNPQR

CO(Brev)

(310)

LSSQVGECWGNNGNIMTGRllNHAWDATGSSQATIP~GIDNWANPAAPIFAEIAPLPAGLETYVSLYLAITKNPER t tttt bbttttbbbbbb tttt bbbb tttt bbbbb

(309) ttt (360)

CO(Strep)

(381)

GTFVYDAATD~KLNWTRDQNAPAVNAAKALFDRINKANGTIYRYDLFGTQLKAFADDFCY~LGGCVLGKA~DY

CO(Brev)

(386)

ARFQFNAGSGKVDLTWAQSQNSKGINMALSVFDKINKKEGTIYRTDLF bbb bbb hhhhhhhhhhhhhhhhhhhhhh

(457) (462)

( 81) tttt

tttt

CO(Strep)

CO(Strep) CO(Brev)

( 75)

(365) bbbbbbbb (456)

YATWGDDFTYHPLCCVLLNSATDNF bbb bb bbbb

(461) ttt

GRVAGYKNLYVTDGSLIPGSVGVNPFVTITALAERNVERIILQDVTAS GRLPEYPGLYWDGSLVPGNVGVNPFVTITALAERNMDKIISSDV tbbb

ttttbb

tttt

(504) (506)

hhhhhhhhhhhhhhhhhhhh

Figure 3. The amino acid sequence of cholesterol rt al., 1989) and that from Rrevibacterium sterolicuwl has

I deletion

A’treptomyces

relative

to

sequence.

the

The

b denotes b-strands.

h

applying the overall

annealing,

simulated temperature

conjugate-gradient reducing the

extended ment

to

was

Simulated system

carried

denotes

minimization to 299%. further

out

and

the

assignment t denotes

the initial structure were refined using option The resolution conjugate-gradient

giving was

of structure

a-helices

factor

R-factor 2.3 A and annealing

sequence

secondary

oxidase from Streptomyces sp. strain SA-COO (CO(Strep): lshizaki (CO(Brev)) determined from the X-ray data. The X-my sequence Streptomyces enzyme. This is denoted by a dash at residue 83 in the

an performed

R-factor by

of

as defined reverse

and the

XI’LOR, was then refineof

heating

32.70;,. the

to 4000 K and then slowly cooling to 300 K. reducing the temperature by 25 K every 50 dynamic> steps (where each step corresponded to 0.5 fs). Conjugate-gradient minimization that re-optimized t’he stereochemistry further reduced the R-factor to 28%?,,. At) this stage, electron density maps were calculated using t’he Fourier coefficients (3F,,,, - 2F,,,,) and (bibs - t;(,,,J

Table 4 R-factor ltesolution rangr

versus

resolution

Number reflections 459 1419 6355 x823 10,513 3833 3934 4112 3455 42,903

of

range &factor shell

by

Kabsch

& Sander

(1983)

has

been

included;

turns.

and the model rebuilt by examining the highest differencar density peaks. Additional side-chains and some segments of the main-chain were added to the model. XPLLOR was used for subsequent rounds of standard least-squares refinement (omitting the simulated annealing refinement

step). (Konnert

Restrained

isotropic

& Hendrickson,

temperature-factor 1980)

was

alternated

with positional refinement. Target values for the trmperature factor deviations were 1.5 A* and 2.0 A2 for bonded and angle-related atoms. respectively. Aft,c:r the 2nd round of refinement,, water molecules were accounted for in the difference electron density. At the 4th round, all data from 10.0 A to 1.8 .& were included. Aft.rr caac&h round of refinement. electron density maps were cal(+ulatrd as described above and the highest differrnc*cs peaks analysed. After the 5th round, the structure was inspebd along the entire length of the chain and modified to account for the remaining difference density. A total of i rounds of XPLOR refinement and model rebuilding were carried out, reducing the K-factor to 165”,,. The final rounds of refinement were carried out using the program PR0LSQ (Hendrickson clr Konnert. 1981) and f’urther reduced the R-factor to 15.37/,. Table 3 gives an avcwunt of the progress during the refinement procedure. As the refinement progressed, wherr tliEerrnws between the Streptomyces and Hrevibacteri’unc seclut’nces became evident in the electron density. thr model was modified to account for the differences observed. The final 1.8 I% resolution map is of high yuality and enables an unambiguous assignment to be made for tht> majority ot the amino acid residues. Fig. 3 gives the amino acid sequence of the final model. The density t,he 9 residues 7Fi to 83 of the Streptomycrs

corresponding sequrnw

to c~witi

Structure

of Cholesterol

Table 5 Ikviatkns

from

ideal geometry for the final r.m.s.

Target o

deviation Distances (A) Bonds (l-2 neighbour) Angles (IL3 neighbour) Intraplanar (I-4 neighbour) Planar groups (A) (‘hiral rentres (A3) Torsion angles (“) Staggered (e.g. aliphatic xl) Transwrse (e.g. aromatic ,Q) Non-bonded contacts (A) IL4 neighhours 0th~~ Thermal factors (A’) Main-chain bond (l-2 neighbow) Main-chain angle (t-3 neighhour) Side-chain bond Sidrvhain angle

model

0.015 0.032 0.042 0.018 Cl48

0.020 0.040 0.050 0.020 0.150

14100 22900

15ooo 20900

0.162 0257

0.300 @300

1.095 I.617 3.046 4.110

1.500 2.500 3+00 4ooo

The target (r values are the input estimates of standard deviations that determine the relative weights of the corresponding geometric~ restraints (Hendrickson & Konnert, 1981).

best be fitted by 8 residues (81 to 88) in our model, but the quality of the density is not adequate to assert definitely that there is an amino acid deletion (compared with Strepfom~ycrs) in this region. Thr final R-facator is 15.3oi; for all data betwern IO+) and I.8 A (12.903 reflections). The variation of K-factor with resolution is given in Table-C. The r.m.s. phasr tliffrrrnw brtwwn the final refined phases and the rerombined m.i.r. phases is 61”. Table 5 drmonstrat’es the good

fit, of the model to ideal geometry. been deposited with the awrssion number I(:OX.

Brookhaven

Thr co-ordinates Protein

Data

have Bank,

3. Results and Discussion (;t) Quality

of the model

The final elwtron density map for the present model was calculated using the Fourier coefficients W&S - bia,c) and clearly shows density for all mainchain atoms of residues 5 to 257, 260 to 433, 437 to 501, the FAD molecule and 453 water molecules. Two loops and a few residues at the amino and carboxyl termini had ill-defined density and thus could not be modelled. Figure 4 shows two examples of the electron density map where a clear interpretation of the structure was possible. Throughout tnost of the internal region of the struct)ure an unambiguous assignment of the sidechains was possible due to the high quality of the electron density map. Since complete sequence information was available not for the Brevihacterium enzyme, the quality of the electron density map was crucial in completing the sequence interpretation. In many regions on the surface of the molecule where the density was unclear due to side-chain disorder, the sequence of the enzyme from Streptomyces sp. was usually built and the sidechain occupancies set to zero. Alternatively, where the density did not, fit the sequence of the

539

Oxidase

Streptomyces enzyme, smaller side-chains were included in the model. Q’here multiple conformations were observed such as for Va140, Ser277, Va1280 and Glu361, only one conformation was included in the model. For Ser277, both side-chain conformations give possible hydrogen-bonding interactions excluding the possibilit,y of a threonine or valine at t’his position. Glu361 is an active-site residue with poorly defined density. The electron density for the side-chain (Fig. 5) clearly shows the position of the CB atom but a break in the density between Cy and C6 as well as a disordered region of density for the carboxylate group suggests two possible conformations. Figure 6 shows a Ramachandran plot (Ramakrishman & Ramachandran, 1965) for the main-chain dihedral angles. Most of the non-glycyl residues have dihedral angles that fall in or near to the energetically preferred regions. Only Thr231 falls outside this region; however, the electron density unambiguously defines this conformation. At Thr231, the chain makes a wide turn before entering an x-helix. The $ value adopted allows a hydrogen bond to be formed between the mainchain oxygen atom of Thr231 and the main-chain nitrogen atom of Ser235 t’hus initiating the cc-helix (Fig. 7). In most cases, the x1 angles of the side-chains follow the expected trimodal distribution (Janin et aZ.. 1978). Tn certain cases, x1 values indicate less commonly observed conformations (McGregor et al., 1987); for example, Va195 (x1 = 84”), Thr168 (x1 = -168’) and Thr326 (x1 = -165”). Each of these conformations avoids an unfavourable steric interaction wit.h the rings of Trp46, Trp440 and Trp164, respectively. Va1217 is an active-site residue and also adopts a less commonly observed side-chain conformation (x1 = 67”). This conformation allows the methyl groups to point into the active-site cavity and is believed to be necessary for forming the architecture and hydrophobic nature of the cavity needed to bind the steroid substrate. The active-site residue Asn485 has been modelled with a high-energy side-chain conformation (x1 = -98”) because the electron density unambiguously defines this conformation. The observed side-chain conformation of Asn485 appears to allow it to participate in the extensive hydrogen-bonding network observed in the active site. (b) Lksrription

of the overall structure

The molecule has the approximat’e dimensions 73 ,r( x 53 A x 51 L% and is comprised of two structural domains, as shown in Figure 8, with the strands and helices labelled as referred to in the text. The FAWbinding domain is composed of residues 5 to 44, 226 to 316 and 462 to 506 and consists of a six-stranded B-pleated sheet sandwiched between a four-stranded p-sheet and three a-helices as shown in Figure S(c). A similar motif has been observed for other FAD-binding proteins in&ding p-hydroxybenzoate hydroxylase

Figure hetween between

Figure residue of the

electron density map calculated using all reflections 4. Stereo views of 2 regions of the final (2F,,,, -PC,,,) 10 and 1.8 A and phases from the final model. (a) The model of the flavin ring. (b) A salt bridge interaction Argi78 and Glu166. Electron density for water molecules is clearly visihle.

5. An electron density is located in the active-site side-chain

map (3fi6,,S -2F& cavity and may

showing be involved

the density in catalysis.

for the disordered side-chain The density suggests multiple

of Glu361. This conformations

Sructure

qf Cholesterol

541

Oxidase

to lie close to the flavin ring and facilitates hydrogen-bonding interactions between the ring and the protein main-chain. The dominant structural feature within the steroid-binding domain is a large six-stranded antiparallel b-sheet consisting of

L. 00 0

strands Bl, H2, B3, B4, B5 and B6. This sheet forms the “roof’ of the active site and includes a

number of residues that may be involved in catalysis. A similar b-sheet structure, consisting of seven strands, has been observed in p-hydroxybenzoate hydroxylase. A comparison between these struc-

-60

tures is discussed

-180 -180

-120

-60

network

0 (p (deg

(Wierenga et al., 1979) and glutathione reductase (Schulz et al., 1978). This motif includes the paa fold, involved in binding the adenine ring and inter-

acting with the pyrophosphate oxygen atoms of FAD. The second domain, the steroid-binding domain, is made up of residues 45 to 225 and 317 to 461 as shown in Figure 8(b). The chain leaves the FAD-binding domain at residue 45 to make up a two-stranded antiparallel p-sheet, Dl and D2, forming one wall of the active site. A short turn.

made

up of residues

113 to 116, is

positioned near to the ribityl chain of FAD forming hydrogen-bonding interactions and thus firmly anchoring the prosthetic group in the enzyme. A chain meander before helix H2 crossesbehind the &-face of the flavin ring. The presence of glycine residues at positions 120 and 121 enables the chain

Figure

7. Strreo

view

of hydrogen

bonds.

1

Figure 6. A f)lot of the main-chain dihedral angles for the 492 amino acid residues of the final model. The squares denote glycine residues. The continuous lines enclose areas that are fully allowed conformational regions for z(V) of’ 1 lo”, while the broken lines show the area of acceptable van der Waals contacts for t(Cb) of I 15” (Ramakrishman & Ramachandran, 1965).

3,,-helical

below.

The active site of the enzyme is located between the two domains adjacent to the flavin ring of FAD. The active-site pocket is filled with a well-ordered lat.tice of water molecules that form an intricate

X Thr231

showing

(4, II/ = - 118”, -98”). This conformation Ser23SK thus initiating an a-helix.

the disallowed allows

(c) Secondary structure The secondary structure assignments were made using

the algorithm

of Kabsch

& Sander

(1983)

and

are presented in Figure 3 with the nomenclature as shown in Figure 8(a) and described below. The structure contains a total of ten helices ranging in length from 6 to 22 residues, representing 25% of the total model. The helices are located predominantly on the external surface of the molecule surroundmg the buried p-sheets. Pro149 forms a kink in helix H3. Helices H5 and H9 are slightly bent, as shown in Figure S(a), such that they follow the curve of t.he P-sheet in the steroid-binding domain.

Residues

169 to 171 of helix

H5 and 403 to

406 of helix H9 form 3,,-type turns. Helices Hl and HlO are both oriented with their i\j termini directed towards the FAD molecule. The N terminus of helix Hl is directed towards the pyrophosphate group of FAD, possibly stabilizing the negative charge on the phosphate through the helix dipole effect (Ho1 et al., 1978). The N terminus of helix HlO is directed towards the pyrimidine ring of the flavin group of FAD. Figure 9 shows the orientation of t,hese two helices with respect to the FAD molecule. The structure contains 23 ,!I-strands, making up six p-pleated sheets and comprising 2206 of the total model. The structure is dominat)etl hy two

conformation

a hydrogen-bond

of the interaction

main-chain Tao be

dihedral angles of made b&wren Thrz3-0

Th&J1 and

Figure 8. A represent,ation of (a) the overall fold of cholesterol oxidase showing the strands and hrliws. lahelled as wf’errrtf to ill th(a text. The- F.41) n~~~lrc~ulr~ ha,. Iwen included as a stick drawing. (b) i\ strrro view showing the st~,roict-bindirlg domain. (c) X stereo vie\\ showing the FAD-binding domain The drawings were generated using the program KIKKON writt,en by Prirstlr (1988).

(b)

Structure

of Cholesterol Oxidase

543

Figure 9. A stereo view of the model showing the FAD molecule and the helices that interact with it,, HI and HlO. Both helices are positioned in such a way as to compensate the negative charges on FAD, in the case of Hl on the phosphate oxygen atoms and in the case of HlO on the pyrimidine ring.

large six-stranded P-pleated sheets. The sheet, Bl to B6, in the steroid-binding domain (Fig. 8(b)) has all six strands arranged in an antiparallel fashion and forms the roof of the active-site cavity. This sheet is surrounded on the external side by helices H4, H5 and H9. A second six-stranded p-pleated sheet, Al to A6, is located in the FAD-binding domain and has five of the six strands arranged parallel with strand A5 antiparallel. The four-stranded P-pleated sheet, Cl to C4, in the FAD-binding domain lies against the six-stranded sheet with strands C2, C3 and C4 arranged antiparallel and Cl parallel to C4. St,rands Dl. D2 and D3 make up a sheet along the

Table 6 Reverse

llrsitlur

turns

rmgr

A7-1)X-GH-III0 \‘3 I -c&3%A33-(X14 G~5O-A.51-1).5%-(~.53 1)63-K64-R65-S66 tmo-M81-GWF83 ~:111-\‘112-~1113~~:114 (:11:3-(:114-(:1l:,~s116 l,l:~T,~l’lR(i-s137-Vl:~~ s11 l-(:21%-1 A213-(:214 v%17~121x~Y21!~-~:220 S227-L22X-D~O~K230. . 1)269-F270-E271-(‘272 1 J 1,30;-1’3”8-~309-1,310 15313-\‘314-~:31r,-E316 . U32O~L‘321~(:322~L”~23 * L. H33 I -A332-W33911334 N:~.53-P3.54-~355-,~:156 1~~;3-1,454.5455.S4.56 1)4Rg-N460-F461~(:462 Y467~1’468~( :46!)-1,470 1)474-(:47,5-5476.12477 t The turn wnformat.ionitl

in, cholesterol

oxidase

42 (“1

*2 (7

43 (7

$3 (7

Typet

-- 53 - 69 - 44 -64 43 - 70 49 - 64 -~ 63 -- 68 --so -. 72 -- 67 -~ (j“ 59 -. 58 -- 62 - 56 - 76 -~49 - 64

133 -26 -50 -17 47 112 -136 -22 -26 -21 - 30 -6 -21 127 30 - 26 -25 130 -7 128 -18

91 -82 83 -70 x7 116 -57 -78 -105 -110 -63 -104 -85 101 .51 -80

-12 -4

II

-i8

3 -14 -8 -10 -22 -11 15 22 -23 7 6 -14 28 -8 -16

69 -124 104 -64

9 16 - 10 -2” -

I I I I’ 11 II’ I I I I I I II I’ I I II I II I

type is assigned un the basis of the c$*, ILz. c$~, (j13 an~lrs (Hirhardson. 1981).

“floor” of the active-site cavity. Strands Fl and F2 form a large loop and strands El and E2 form one of the links between the two domains. The structure contains 20 hydrogen-bonded reverse turns (Crawford et al., 1973; Chou & Fasman, 1977), which are listed in Table 6 using the classification adopted by Richardson ( 1981). Most of these turns are located at the surface of the structure. Four turns are internal and may be involved in interactions that are important to the tertiary structure and function of the protein. Residues 111 to 116 make up two reverse turns, located in a buried region of the structure. The t’urn at residues 474 to 477 is also buried within the structure and can be described as a 3,0 helix. Both of these turns are positioned near the pyrophosphate group on FAD with their glycine residues involved in hydrogen bonding to the pyrophosphate oxygen atoms. The loop at residues 217 to 220 is buried with the side-chains of Va1217 and Tle218 directed inwards toward the active-site cavity, where they probably contribute to the hydrophobic environment’ needed to bind the st,eroid molecule. The fourth buried turn is located between residues 320 and 323, and lies between the strands El and 132 of two different j-sheet structures. The mainchain atoms of clly319 and He324 are involved in hydrogen bonding to the neighbouring st,rands u;ithin each sheet. The side-chains of residues Asn321 and Asn323 in this turn are directed into the active sit,e and may be important in orienting the ring of His447.

(d) Temperature factors The average temperature factor for all protein atoms used in the refinement is 19.2 A’. while that for the main-chain atoms only is 8.9 AZ. Figure 10 shows the distribution of temperature factors for main-chain atoms along the chain. An unambiguous

544

A

C’rielink et al.

----___

factors with the exception of (:lu361. molecule has an average t,empttrature 4.0 A2 clearly indicating the well-ordered of the prosthetic group. (e)

o’,,,,,,,,,,,,,,,,,,,,,’ 1

Figure

loo

10. The average

200 300 Residue number atomic

400

temperature

500 factors

for

Breaks between residues 257 and 260 and between residues 433 and 437 correspond to the uninterpreted loops. Regions of high temperature factors have been labelled. the

main-chain

atoms.

interpretation of the structure was difficult or impossible in regions of the main-chain with high atomic temperature factors. In most cases this was observed for loop regions on the external surface of the molecule. Both the N-terminal and the C-terminal ends of the model also show high temperature factors. Other regions that show particularly high temperature factors are found at’ the loop defined by residues 391 through 395 and close to the uninterpreted loops between residues 257 and 260 and residues 433 and 437. The latter loop is believed to make up the entrance to the active-site cavity and the disorder may be due to the lack of interactions wit’h a substrate. Binding of the substrate is likely to order this region. Other regions of high temperature factors correspond to the chain meander from residues 47 to 90. This region also includes an external loop believed to make up the entrance to the active site from residues 80 to 90. The loops between residues 205 to 210, 302 to 316 and 350 to 355 all exhibit high temperature factors as they are located on the external surface of the structure. All the residues lining the active-site cavity are well ordered with very low t,emperature

Figure O-2’A

and

11. A stereo view of the hydrogen-bond O-3’A

of the

ribose

ring

of FAD.

interactions

Chargod

‘l’hc FXl) factor of st ructure

groups

An inspection of the position of charged sidechains shows that they occur predominantly on the external surface of the molecule with important exceptions. Glu41 is positioned near t,he ribose ring of FAD. The side-chain of Glu41 forms strong hydrogen bonds to O-2’A and 0-3’A of FAD (2.6 w and 2.8 A, respectively) as shown in Figure 11. This interaction between an acid side-chain and the ribose ring oxygen atoms is observed in other FAl) and NAD-binding proteins (Eklund et al.: 1984: Skariyriski et al., 1987: Schreuder et al., 1989). A second example of a buried side-chain is Asp274. The side-cha,in carboxylate oxygen atoms each form two hydrogen bonds. one to Qly290-N and Ser476-N and the second to Thr294-OGI and Ser476-OG, respectively. Lys225 is also found in a buried position of the structure and has very well defined side-chain density. Hydrogen bonds are formed between Lys225-NZ with Leul17-0 and Asnllg-0 as shown in Figure 12. The peptide chain between residues 117 and 122 lies near the &-side of the flavin ring and forms hydrogen-bonded int’eractSions with the pyrimidine ring of FAD. The interactions bet,ween Lys225 and Leul17 and As11119 appear to be important in st,abilizing the conformat’ion of the main-chain near the flavin ring of FAD. There is one charged group within the active site, (11~361. The poor density for this residue as shown in Figure 5 suggests multiple positions for t,he sidechain. In the final refined model Glu361 interacts with His447PNE2 via a water molecule (Wat541). There are a total of 18 intramolecular ion pairs observed in the structure, three involving bridging water molecules. The details of these ion pairs are given in Table 7. There are three occurrences of networks of ion pairs involving two acidic groups and one basic group, between Asp52, Arg65 and

made

between

the carboxylate

oxygen

atoms

ofGlu41

and

Structure

of Cholesterol Oxidase

545

Figure 12. A stereo view of the interactions made between the buried side-chain of Lys225 and the main-chain of Leull7 and Asnl19. The hydrogen-bond interactions of this charged residue define the position of this region of the main-chain with respect to the flavin ring. The hydrogen bonds made by the pyrimidine ring of FAD are shown.

Asp63 (shown in Fig. 13). between Asp459, Arg463 and Asp505 and between Asp97, Argl 10 and Glu99. A number of ion pairs appear to be important in linking elements of secondary structure together and thus maintaining the tertiary structure of the protein. Helices H4, H5 and H9 are linked via ion pairs between Glu166 and Arg178 and between Arg183 and Glu424. Helices Hl and HlO are linked via an ion pair between Arg29 and Glu495. The C-terminal helix of the protein is held alongside the strand A5 in the FAD-binding domain via an ion pair between Asp505 and Arg463. A large loop of 16

residuesis located between strands B2 and B3 of the steroid-binding domain. This loop is stabilized by an ion pair between Lys179 of helix H5 and Asp334. After the PC@fold in the FAD-binding domain the chain continues through a long meander before entering a p-sheet structure at Dl. This chain meander is made up of 54 residues and spans the surface of the molecule. The ion pair involving Asp52, Arg65 and Asp63 appears to contribute to the structural integrity of the chain meander. Three ion pairs are found between different neighbouring molecules and are described in Table 7. Two

Table 7 Zon pair interactiow Interaction distance (A)

1011 pair residues A. I rrtramo1rcu1ar

Asp52-ODl Asp52~OD2.. Asp63-ODI Asp63-OD2. Asp97-OD1 Asp97-ODl Glu99-OEl Glu132-OEl Aspl61-ODl .. GlulSG-OEl Glu166-032. (:lu266-oEl

ion

pair.7

,irg65-NH2 Arg65-NH1 Arg65-NE Arg65-NH1 Argl lo-NE ArgllO-NH1 I,ys29&NZ Arg128-NE LysBOl-NZ Arg178-NH1 Arg178-NH2 Lys249-NZ

2.83 2.99 304 2.82 2.91 2.78 2.62 285 2.89 2.78 2.78 274

13. 1 ntramolecuiar ion pair8 involving water (:lu99-OE2. Wat555.. Argl lo-NH1 Clu142-OEl Wat575.. Arg496-NE Asp499-ODP. Wat753.. Argl.50.NH1 (‘. Intrrmoleculrcr (:lu 13%OE2 Asp269-OD2 Arg284-NH1 t Interaction : Generated # (knrrated

Interaction distance (A)

Ion pair residues

ion pairs Lysl79-NZt Wat617 Wat759

Asp334-OD2.. Asp349-ODI Glu424-OEl Glu424-OE2. Asp431-ODl .. Asp43 l-OD2 Asp459-OD2.. Glu495-OEl Glu495-OE2. Asp505-ODl Asp505-ODl

Lysl79-NZ Lysl79-NZ Arg183-NH1 Arg183-NE Arg429-NE Arg429-NH Arg463-NH2 ArgSB-NE ArgPB-NH1 Arg463-NE ,4rg463-NH1

1

2.81 3.27 2.85 2.97 2.92 2.86 2.70 2.7 1 2.79 P+35

moleculrs 2.99 3.22 2.;15 2.94 2.96 3.28 2.70 2.69 2.89 3-33 2.76

Argl5O-NHlf Glu384-OE2§

generated by symmetry by symmetry operation hy symmetry operation

2.89

operation - l/2 + X, l/2 - Y, 1 - 2 - l/Z-X, - Y, - l/2 t 2. - 1 -X, -l/2Y, l/2--2.

546

Figure

A. I’rielink

13. An example of a salt-bridge

network

involving

2

of these intermolecular ion pairs involve bridging water molecules, that between OD2-Asp269 and NHl-Arg150 and between NHl-Arg284 and OE2-Glu384. The density for all three intermolecular ion pairs is well defined and the temperature factors are lower than average, suggesting a well-ordered interaction. These ion pairs may contribute to the great stability of the crystals, both mechanically and through the observed long lifetimes of the crystals in the X-ray beam, by exerting strong interactions between adjacent molecules in the crystal lattice. (f) Solvent

structure

The refined model of cholesterol oxidase includes 453 water molecules with an average temperature factor of 28.1 A2. The active-site cavity contains 13 water molecules with relatively low temperature factors (average of 20.7 b’) that form an intricate network of hydrogen bonds. Nine water molecules

Figure

14. A stereo view showing

the interaction

et

al.

acidic groups, Asp52 and Asp63, and I basic group, Arg65.

that are involved in hydrogen bonding to the FAD molecule also have a low average B-value (7.9 A2). The structure contains 47 buried water molecules that occur either as isolated molecules or as small clusters containing from two to four molecules. The average temperature factor for these water molecules is low, indicating the well-ordered nature of this buried solvent, many of which contribute to the structural integrity of the protein. A number of water molecules are involved in linking adjacent strands of /?-pleated sheets. This is particularly observed in the large P-pleated sheet structure (Bl to B6) of the steroid-binding domain and appears to be important in stabilizing t,htb pronounced twist in this sheet structure. An example showing different elements of secondary structure held together through interactions with water molecules is given in Figure 14. These inter-actions may hold the long loop made up of strands Fl and F2 at the edge of the structure to the loop between strands Dl and D2.

between water molecules Wat552

atoms of Ser102, Gly103 and Leu399. Ser102 and Gly103 is located on strand F2. The hydrogen-bond interactions strands Fl and F2 to the structure at the /?-sheet made

are located involving up of Dl,

at the loop between Wat552 and Wat558 D2 and D3.

and Wat558 and the main-chain strands Dl and D2 and Leu399 anchor the long loop made of

Structure

(g) The FAD

of Cholesterol

547

Table 8

molecule

The conformation adopted by FAD in cholesterol oxidase deviates slightly from the fully extended conformation observed in the structure of p-hydroxybenzoate hydroxylase, and is nearly identical to the FAD conformation in glutathione reductase (Table 8). The largest difference in the torsion angles is 26” between 4A of cholesterol oxidase and p-hydroxybenzoate hydroxylase, reflecting the slightly bent conformation of the prosthetic group in cholesterol oxidase as compared with the fully extended conformation in p-hydroxybenzoate hydroxylase. The FAD molecule is involved in 22 hydrogeninteractions bonding with the protein and surrounding solvent. Figure 15 shows the FAD molecule with the protein residues and solvent molecules that interact with it. The adenine and ribose rings are buried deep within the FAD-binding domain. The strong interaction of the ribose ring (O-2’A and O-3’A) with Glu41 has been discussed (Fig. 11). Interestingly, the model also indicates a hydrogen bond between O-3’A and Glylll-0 (3.18 A). This suggests that the hydroxyl proton on O-3’A is involved in a bifurcated hydrogen bond. Bifurcated hydrogen bonds

HO 3’

Oxidase

Torsion angles of FAD in cholesterol oxidase (CO) p-hydroxybenzoate hydroxylase (pHBH)t and glutathione reduetase (GR) $

co

pHBH (7

GR (7

Angle§

(7

XA t 0; VIA 6JA $5 )ir

142 -78 161 69 113 38 - 157 57

134 -57 162 59 87 55 -154 72

139 -73 163 59 112 41 -150 75

Y5 Ll Y3 x2 111

-168 180 178 -173 -74

179 161 - 171 -170 -88

179 176 -176 -175 -76

t Schreuder et al. (1989). 1 Karplus & Schulz (1987). 5 A definition of the torsion

angles is shown

in Fig. 15(a).

have been reported in a number of protein structures but they are rare (Baker & Hubbard, 1984). The pyrophosphate group is involved in eight hydrogen-bond interactions, five through water

OH

(a) (b)

Figure 15. (a) A representation of the FAD molecule showing the atomic nomenclature and the dihedral angles as referred to in the text. (b), A representation of the FAD molecule showing the protein and solvent, molecules that interact with it. Distances within 3.5 A and having good angular geometry have been included as hydrogen bonds and are shown by the broken lines.

Figure 16. A stereo view of the beginning of cl-helix, Hl, and its interactions with the FAD molecule. The hydrogenbond interactions made between the protein main-chain and the pyrophosphate group of the FAD are shown. The conserved glycine residues, Gly18, Gly20 and Gly23 are located near the PJ terminus of this helix.

molecules and three through main-chain nitrogen atoms of glycine residues. The negative charge is compensated for by the dipole of helix Hl (Ho1 et aZ., 1978). Although the common pattern of conserved glycine residues Gly-X-Gly-X-X-Gly for t’he adenine-binding region is observed at Gly18, Gly20 and Gly23, a glycine residue is also observed at position 22. The peptide nitrogen atom of this glycine residue makes a direct hydrogen bond to O-F1 of the pyrophosphate group, whereas the conserved glycine residues, Gly20 and Gly23 form hydrogen bonds to O-F1 through Wat524 (Fig. 16). It should be noted that this common pattern of conserved glycine residues is not completely homologous with t,he sequence of the Streptomyces enzyme, which consists of Gly-X-Gly-X-Gly-Ala. Glycine is also observed in the fifth position of this region of the sequence in glutathione reductase and lipoamide dehydrogenase but in p-hydroxybenzoate hydroxylase this position corresponds to serine. The peptide nitrogen atom of this serine residue hydrogen bonds directly to O-F1 of the pyrophosphate as observed group (Schreuder et al., 1989) whereas, above, the conserved glycine residues interact with O-F1 via a water molecule. The ribityl side-chain of FAD interacts with only one solvent’ molecule, Wat533. A hydrogen bond is also observed between O-2’ and the amide nitrogen at,om of Asnll9. There is only one intramolecular the FAD molecule, hydrogen bond (2.65 8) within that between O-4’ of the ribityl chain and O-Al of the pyrophosphate group. This intramolecular interaction is not observed in the structure of p-hydroxybenzoate hydroxylase but is seen in the structure of glutathione reductase (2.96 8: Karplus & Schulz. 1987). The flavin ring system shows a number of interesting structural features. The pyrimidine ring of the flavin group is positioned at the N terminus of helix HlO. A hydrogen-bond interaction is made between Phe487--N and O-2 of the pyrimidine ring. In the oxidation step, the pyrimidine ring becomes negatively charged. This charge is probably stabil-

ized by the helix dipole effect from this helix. This has also been observed in the refined structures of p-hydroxybenzoate hydroxylase and glut,at hione reductase. Tt was expected that the flavin ring system should be completely planar due to the delocalized TCelectrons through the ring. The ele(‘tron density for the flavin ring clearly shows this not to be the case. The ring system is twisted. resulting in an angle of approximately 17” bfhveen the best planes of the benzene ring and the pyrimidine ring. A deviation of 0.35 A from the best* plane defined by all 18 atoms in the flavin ring system is observed for O-4 of the pyrimidine ring. During t,htt refinement, restraints were used that imposed a planar conformation of the entire ring system but the energy terms assigned to these restraints. (*rossring dihedral terms, were set very low thus allowing the ring to twist easily. The final model of the flavin ring, using the electron density (3pobs -2f+‘,,,,: Fig. 17). clearly shows the twist) in thP flavin ring system. Nuclear magnetic resonance studies on oxidized flavin rings have reported non-planar conformation of the ring system with the angles between the benzene and pyrimidine rings ranging from 1.X’ to 7.0” (Norrestram & Stensland, 1972; Van Glehn (\i Norrestram, 1972). Deviations from planarity have been observed in ot,her flavoproteins, 3.3” in thr 1.54 A resolution structure of glutathione reducstasc (Karplus & Schulz, 1987), 10” for the 1.9 x resolution structure of the complex of p-hydroxybenzoatr hydroxylase with the substrate p-hydroxybenzoate and 19” for the 2.3 is. resolution structure of t#he complex of p-hydroxybenzoate hydroxylase with the product, X,4-dihydroxybenzoate (Schrrudchr rt al., 1989). The non-planar conformation of t,he pyrimidinr ring facilitat,es a hydrogen bond between O--t and the main-chain nitrogen atom of Met122. The sequence before Met122 is very rich in glycinr residues (113, 114, 115, 120 and 121), allowing a close approach of the main-chain to the flavin ring. Other hydrogen-bonding interactions with the flavin ring are concentrated only around the pyrimi-

AWucture

Oxidase

of Cholesterol

549

Figure 17. A stereoview showinga 3F,,, - %Fcalcelectron density mapand the modelfor the flavin ring systemof FAD in cholesteroloxidase. The twist in the pyrimidine ring is clearly observedin the density.

dine ring. Only one water molecule interacts with the ring forming a hydrogen bond between Wat518 and O-2 of FAD. The benzene ring is buried in the active site and surrounded by hydrophobic sidechains. The nitrogen atom on the flavin ring that is directly involved in the oxidation step, N-5, makes no hydrogen bond to either protein or solvent molecules. (h)

The

steroid-binding

site

The active site is located near the flavin ring system at the interface of the two domains. Figure 18(a) shows a c’” representation viewed into the active site from the top surface of the molecule. We believe this to be the entrance to the active site. The entrance is situated between a number of mobile loops that have relatively high temperature factors. The surface residues at this entrance are Phe83, l,eu369 and Leu432. The entrance to the active-site caavity is made up almost exclusively of hydrophobic residues as shown in Figure 18(b). A van der Waals’ surface of the residues in these loop regions shows that the channel through which the steroid substrate might, enter the cavity is blocked by the side-chains of the loop residues. The high temperat’ure factors observed for these loops, however, suggest,that there may be some movement allowing the steroid to enter the cavity. The hydrophobic nature of this channel corresponds to the necessity for an apolar environment for the steroid molecule to bind. The FAD molecule is positioned at the floor of the cavity with N-5 of the flavin ring system exposed to the ordered lattice of 13 water molecules. An inspection of the side-chains of the protein that are directed into the active-site cavity shows a marked hydrophobic character on one side of the cavity, nearer the benzene ring of the FAD, and a more hydrophilic arrangement of residues on the opposite side. adjacent, to the pyrimidine moiety. This

arrangement of residues around the active site suggestshow the steroid substrate might bind. The ring system of the steroid substrate is very hydrophobic with the only polar region being the hydroxyl group at C-3 (seeFig. 1). This is the site of oxidation on the substrate molecule and would probably be positioned near N-5 of the FAD. Thus the polar end of the steroid would be positioned at the end of the active-site cavity that is lined by the hydrophilic residues, Glu361, His447. Asn485, Asn332 and Asn321. In order to fill the cavity, the hydrophobic ring system of the steroid would extend back towards the hydrophobic end of the active site. The length of the solvent’-filled cavity is 11 a, comparing closely with a distance of l@O a between the hydroxyl oxygen atom, O-l’ and C-17 on the structure of cholesterol (Craven, 1979). The cavity is adequate to accommodate the whole of the steroid ring system. The long hydrophobic “tail” on C-17 of cholesterol may pack amongst t,he hydrophobic loop region at the entrance to the active site. (i)

Comparison

to

p-hydroxybenzoate

hydroxylase

The structure of cholesterol oxidase has a number of interesting similarities to the structure of p-hydroxybenzoate hydroxylase. The topology of the FAD-binding domain on both these proteins is identical (Fig. 19). In both structures the FAD-binding domain is made up of three noncontinuous segments of sequence. There are two long chain excursions away from the FAD-binding domain between strands A2 and H7 (H6 in p-hydroxybenzoate hydroxylase) and between strands H8 (H7 in p-hydroxybenzoate hydroxylase) and A5 to make up the substrate-binding domain. These chain excursions are geometrically very different but they re-enter the FAD-binding domain at the same elements of secondary structure. A superposition between the FAD-binding domains reveals that although the topology is identical, the domains are

A. Vrielink

et al.

(a)

(b) Figure 18. A stereo view of (a) the C” backbone of the steroid-binding domain showing the proposed entrance to the active site. The FAD molecule is shown in thick lines. The mobile loops believed to be situated near the proposed

entrance are alsoshownin thick lines. (b) The side-chainscontainedin the loopsthat makeup the proposedentrance to the active site. The flavin ring of FAD is shown as open bonds and the protein

not completely superimposable. Only the strands Al, A2, A3 and A4 and helix Hl agree closely, giving r.m.s. separation between C” atoms of less than 1.0 A for a total of 47 residues. The most striking similarity is observed for the ADP-binding jc+fold made up of strand Al, helix Hl and strand A2. The r.m.s. C” separation for the 31 residues making up this motif is 0.79 A. This similarity has been reported between the FAD-binding domains of glutathione reductase and p-hydroxybenzoate hydroxylase (Wierenga et al., 1983). Together with the occurrences of conserved glycine residues at positions 18 to 23, this suggests the importance of this structural unit for FAD-binding proteins. A comparison of the substrate-binding domain of p-hydroxybenzoate hydroxylase and cholesterol oxidase shows topological similarities. In particular, these domains both contain a large b-sheet structure

atoms are shown as filled bonds.

that makes up the roof of the active site and contains residuesthat are thought to be involved in substrate binding and catalysis. Although it is not possible to superimpose the c” atoms of the sheet,. the connections between the strands are similar. Figure 20 shows a representation of the /?-sheetand the connections made between the strands for p-hydroxybenzoate cholesterol oxidase and hydroxylase. The sheet is made up of six strands in cholesterol oxidase and seven strands in p-hydroxybenzoate hydroxylase. Strands Bl and B2 in p-hydroxybenzoate hydroxylase and helix H5 and strand Bl in cholesterol oxidase come from the first chain meander from the FAD-binding domain. Likewise,

the

second

chain

meander

from

the

FAD-binding domain makes up the remainder of the sheet structure; the only topological difference is that strands Fl and F2 in cholesterol oxidase

A’tructure of Cholesterol Oxidase

Cholesterol

551

oxidase m

p-Hydroxybenzoate

N

Figure 19. A representation of the topology of the FAD-binding domain of cholesterol oxidase and p-hydroxybenzoate hydroxylase. The triangles represent B-strands with the apex pointing down if the strand is viewed from the N terminus or up if seen from the C terminus. The a-helices are represented by circles. The boxes with SHD correspond to chain excursions away from the FAD-binding domain into the substrate-binding domain. Both structures exhibit the same secondary structural elements with identical connections. The numbering scheme shown is that adopted for cholesterol oxidase. It is identical to that of p-hydroxybenzoate hydroxylase except for helices H7 and HS, which correspond to H6 and H7, respectively.

replace helix H8 in p-hydroxybenzoate hydroxylase. Ahhough the connections between the strands are identical, the loops in cholesterol oxidase tend to be much longer than in p-hydroxybenzoate hydroxylase.

(j) The catalytic mechanism Cholesterol oxidase carries out two catalytic steps, an oxidation of the 3-hydroxyl group at C-3 of the steroid ring nucleus and an isomerization of the double bond at C-5 to form a conjugated ketosteroid (see Fig. 1). The enzyme A5-3-ketosteroid isomerase from Pseudomonastestosteroni carries out the same isomerization step as is observed for cholesterol oxidase. It has been suggested that the isomerization mechanism involves a 4-6 cis diaxial proton transfer (Batzold et al., 1976) through either a concerted mechanism or a stepwise procedure (Kuliopulos et al., 1989). In order for the catalysis to occur by this mechanism, the active site would require hydrophobic residues to bind the apolar steroid substrate, an acidic residue to polarize the carbonyl group at C-3 of the steroid and a basic residue t,o extract the 48 proton and transfer it to the 6/? position. Nuclear magnetic resonance studies of the A’-3-ketosteroid isomerase-steroid inhibitor complex enabled the location of the bound steroid

hydroxylase

Figure 20. A representation of the topology of the p-sheet region of the substrate-binding domains of cholesterol oxidase and p-hydroxybenzoate hydroxylase. The triangles represent j-strands with the apex pointing down if the strand is viewed from the N terminus or up if seen from the C terminus. The a-helices are represented by circles.

to be determined and, together with chemical modification studies (Ogez et al., 1977) and affinity labelling studies (Pollack et al., 1986), identified Asp38 as the proton acceptor and Tyr14 as the proton donor. Site-directed mutagenesis experiments and kinetic analyses further confirmed these residues as important for catalysis (Kuliopulos et al., 1989). The structure of A5-3-ketosteroid isomerase shows an arrangement of these residues on opposite faces of the enzyme-bound steroid. Smith & Brooks (1977) have suggested that the isomerization activity of cholesterol oxidase proceeds via the same mechanism as has been proposed for A’-3-ketosteroid isomerase. This is supported by the observed structural arrangement of residues at the active site of cholesterol oxidase. One charged residue, Glu361, is found at the active site, extending into the cavity from the roof (strand B4) of the substrate-binding domain. The electron density for this residue is poor (Fig. 5), suggesting multiple conformations and a high degree of mobility for the side-chain. Although the amino acid sequence of the Hrevibacterium enzyme is not available, the

552

A. Vrielink

et al.

Figure 21. A stereo view of the active-site cavity showing the flavin ring and ribityl chain of FAD and the amino acid residues that line this pocket. The FAD molecule is shown as open bonds and the amino acid residues are shown as filled bonds. The water molecules in the active site have also been included as double circles but are not labelled.

sequence alignment (Fig. 3) shows the analogous residue in the sequence of the Streptomyces enzyme to be glutamic acid. Since this residue is located at the active site, it is very probable that it is highly conserved amongst sequences of the same enzyme from different species. Indeed, of the residues that line the active site, only two are not completely conserved between the X-ray sequence from Hievibacterium and the sequence from Streptomyces; Va1375 and Gln75 from Rrevibacterium correspond to Leu370 and Ala69 respectively from Streptomyces. In both of these cases the electron density allows an unambiguous assignment. The mechanism suggested for A5-3-ketosteroid isomerase involves a tyrosine residue as the proton donor positioned on the opposite side of the steroid ring to the proton acceptor, Asp38. A tyrosine residue, Tyr446, is also present in the active site of cholesterol oxidase lying on the floor of the cavity. Figure 21 shows the arrangement of these residues in the active-site cavity of the enzyme. Although no detailed model of steroid binding is available, the position of the steroid hydroxyl group can be assumed to lie very close to the flavin ring ,system, in particular to N-5 of FAD. Although the orientation of Tyr446 places the phenolic group away from the expected position of the steroid hydroxyl group, further speculation on the role of these active-site residues will be possible only when an X-ray structure of the enzyme-steroid complex is available. We thank the Tokyo Research Laboratories of Kyowa Hakko Kogyo Company, especially Takayuki Uwajima and Noriaki Hirayama, for providing the protein. We are grateful to John Akins for the protein purification and to Yoshikatsu Murooka for the early communication of the

sequence of cholesterol oxidase from Streptomyces sp. strain SA-COO. We thank Peter Brick and Alan Wonacott for providing many helpful discussions. This project was supported by the Medical Research Council of the United Kingdom. A.\‘. was a recipient of a Royal Commission for the Exhibition of 1851 Scholarship.

References Baker, E. N. & Hubbard, R. E. (1984). Hydrogen bonding in globular proteins. Progr. Biophys. Mol. Biol. 44. 97-179. Batzold, F. H., Benson, A. M., Covey, 1). F., Robinson. C. H. & Talalay, P. (1976). The A5-3-ketosteroid isomerase reaction: catalytic mechanism. specificity and inhibition. Advan. Enzyme Regul. 14, 243-267. Benisek, W. F., Ogez, J. R. & Smith, S. B. (1980). Recent studies on steroid isomerases from Pseudomonas testosteroni and Pseudomonas putida. Ann. II’. Y. Acad. Sci. 346, 115130. Blow, D. M. & Crick, F. H. C. (1959). The treatment of errors in the isomorphous replacement method. Acta Crystallogr.

12,

794-802.

Bricogne, G. (1976). Methods and programs for direct space exploitation of geometric redundancies. ilcto Crystallogr. sect. A, 32, 832-847. Brunger, A. T. (1988). Crystallographic refinement by simulated annealing. Application to a 2.8 A resolution structure of aspartate aminotransferase. .I. Mol. Biol. 203, 803-816. Briinger, A. T., Kuriyan, J. & Karplus. M. ( 1987). Crystallographic R factor refinement by molecular dynamics. Science, 235> 458-460. Chou, P. Y. & Fasman, G. I). (1977). P-Turns in proteins. J. Mol. Biol. 115, 135-175. Craven, B. M. (1979). Pseudosymmetry in cholesterol monohydrate. Acta Crystallogr. sect. H. 35. 1123-1128.

Structure

of Cholesterol

Crawford, J. L,., Lipscomb, W. N. & Schellman, C. G. (1973). The reverse turn as a polypeptide conformation in globular proteins. Proc. Nat. Acad. &i., U.S.A. 70, 5388542. Eklund, H., Samama, J. P. & Jones, T. A. (1984). Crystallographic investigation of nicotinamide adenine dinucleot,ide binding to horse liver alcohol dehydrogenase. Biochemistry, 23, 5982-5996. Fox, G. C. t Holmes, K. C. (1966). An alternative method of solving the layer scaling equations of Hamilton, Rollett and Sparks. Acta Crystallogr. sect. A, 20,

886-891. French, G. S. & Wilson, K. S.. (1978). On the treatment of negative intensity observations. Acta Crystallogr. sect. A, 34, 517-525. Fukuda, H., Kawakatni, Y. & PJakamura, S. (1973). A method to screen anti-cholesterol substances J’roduced by microbes and a new cholesterol oxidase produced by Streptomyces violascens. Chrm. Pharwl. Bull. 21. 2057-2060. Fukuyama, M. & Miyake, Y. (1979). Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin. J. Riochem. 85, 1183-l 193. Ghosh, I)., Punzi, J. S. & I)uax, W. L. (1986). Crystals of active tetramers of 3a,20/?-hydroxysteroid dehydrogenase. J. Biol. Chem. 261, 1306-1308. Greer. .J. (1985). Computer skeletonization and automatic electron density map analysis. Methods Enzymol. 115, 206.-224. Hendrickson. LV. A. & Konnert, tJ. (1981). Stereochemically restrained crystallographic leastsquares refinement, of macromolecule structures. In Biomolecular Structure, Function, Conformation and Evolution (Srinivasan. R., ed.). vol. 1. pp. 43347. Pergamon Press. Oxford. HOI, W. Q. J., van Duijnen, P. ‘I’. $ Berendsen, H. J. (‘. (1978). The u-helix dipole and the properties of pro teins. Xaturr (London), 273, 443-446. Inouye, Y.. Taguchi. K.. Fujii. A.. Ishimaru, K., Zjakamura, S. & h’omi. R. (1982). Purification and c.haracterizat,ion of extracellular 3/I-hy-droxysteroid oxidase produced by Streptoverticillium cholesterolicum. (‘hem. Pharm. Bull. 30, 951-958. Tshizaki. T.. Hirayama, N.. Shinkawa, H.. Mimi. 0. & Murooka. Y. (1989). xucleotide sequence of the gene for cholesterol oxidasr from a Streptomyces ~1). .I. Hactwiol. 171. 596 601. Janin. .J.. Wodak. S.. Levitt. M. & Maigret, B. (1978). (‘onformation of amino acid side-chains in proteins. .J. Mol. Biol. 125. 357-386. *Jones. T. A. (1978). A graphics model building and refinemrnt system for macromolecules. 1. Appl. (‘rystallogr. 11. 268-27”. Jones. T. A. &, Thirup. 8. (1986). Using known substrmt ures in protein model building and crystallography. ITMBC J. 5. 819-822. Kabsch, W’. & Sander. 8. (1983). Dictionary of protein secondary, structure: pattern recognition of hydrogen-bonded and geometrical feat.ures. Riopolymers, 22 2577-2637. Kamri.’ ‘I’.. Takipuchi. Y.. Suzuki, H., Matsuzaki, M. & Nakamura. S. (1978). Purification of 3/J-hydroxysteroid oxidasr of Streptomyces violascens origin by affinit) chromatography on cholesterol. Chem. Pharm. Bull. 26, 2799-2804. Karplus. P. A. & Schulz, G. E. (1987). Refined structure of’glutathione reductasr at 1.54 A resolution. J. Mol.

Bid.

195. 701 ~729.

Oxidase

553

Konnert. J. H. & Hendrickson, W. A. (1980). A restrained-parameter thermal-factor refinement procedure. Acta Crystallogr. sect. A, 36, 3444350. Kuliopulos, A., Westbrook, E. M., Talalay. P. & Mildvan, A. S. (1987). Positioning of a spin-labeled substrate analogue into the structure of A5-3-ketosteroid isomerase bp combined kinetic, magnetic resonance and X-ray diffraction methods. Biochemistry, 26, 3927-3937. Kuliopulos, A., Mildvan, A. S., Shortle, I). & Talalay, P. (1989). Kinetic and ultraviolet spectroscopic studies of act’ive-site mutants of As-3-ketosteroid isomerase. Biochemistry, 28, 149-157. Leslie. A. G. W. (1987a). Solvent flattening vs additional derivative data in the improvement of the M.I.R. phases. In Computational Aspects of Protein Crystal Data Analysis. (Helliwell, .J. R., Machin, P. A. & Papiz. M. Z., eds), pp. 39-55. SERC. Daresbury. Leslie. A. G. W. (1987b). A reciprocal-space method for calculating a molecular envelope using the algorithm of B. C. Wang. Acta Crystallogr. sect. A, 43, 134-136. Matthews, B. W. (1968). Solvent content of protein crystals. 1. Mol. Biol. 33, 491-497. McGregor, M. J., Islam, S. A. & Sternberg, M. J. E. (1987). Analysis of the relationship between sidechain conformation and secondary structure in globular proteins. J. Mol. Biol. 198, 295-310. Messerschmidt, A. & Pflugrath, J. W. (1987). Crystal orientation and X-ray pattern prediction routines for area-detector diffractometer systems in macromolecular crystallography. J. Appl. Crystallogr. 20,

306-315. Morize.

l., Surcouf, E., Vaney, M. C., Epelboin, Y., Buehner, M.. Frindlandsky, F.. Milgrom, E. & Mornon. J. P. (1987). Refinement of the C222,’ crystal form of oxidized uteroglobin at 1.34 A resolutilm. J. Mol. Biol. 194, 725-739. Mornon. J. I’., Frindlansky, F., Bally, R. & Milgrom, E. (1980). X-ray crystallographic analysis of a progesterone-binding protein. The C222, crystal form of oxidized uteroglobin at 2.2 A resolution. .J. Mol. Biol.

137, 415-429. Korrestam, R. & Stensland, B. (1972). Studies on flavin derivatives. The crystal and molecular structure of 3-met,hyl-lumoflavin. Acta Orystallogr. ,sect. B, 28. 440-447. Ogre, ,J. R.. Tivol, W. F. & Benisek, W. F. (1977). A novel chemical modification of A5-3-ketosteroid isomerase occurring during its 3-oxo-4-estren17b-yl acetatedependent photoinactivation. J. Biol. (‘hem. 252, 6151 6155. l’ollack. R. M., Bantia, S., Bounds, P. 1,. bi Koffman, B M. (1986). pH dependence of the kinetic parameters for 3-oxo-A5-steroid isomerase. Substrate catalysis and inhibition by (3S)-Spiro(Sa-androstane3,2’-oxiranj-17-one. Biochemistry, 25. 1905~1911. Priestlr, ,J. I’. (1988). RIBBON: A st.ereo cartoon drawing program for proteins. .J. Appl. Crystallogr. 21, 572 mFi76. Ramakrishman. C. & Ramachandran, U. S. (1965). Stereochemical criteria for polypeptide and protein chain conformations. IT. Allowed conformations for a pair of peptide units. Biophys. J. 5, 909-933. Richardson, rJ. S. (1981). The anatomy and taxonomy of protein structure. Advan. Protein Chem. 34, 167-363. Schirrbeek, B. (1988). The three-dimensional structure of liJ)oamide dehydrogenase from Azotobacter vinelandii. Ph.D. t,hesis, University of Groningen, The ~cthrrlands.

Schreuder, H. A., van der Laan, .J. M., HOI. W. G. ,J. & Drenth, J. (1988). Crystal structure of p-hydroxybenzoate hydroxylase complexed with its reaction product 3,4-dihydroxybenzoate. J. Mol. Biol. 199. 637-648. Schreuder. H. A., Prick, P. A. J., Wierenga, R. K.. Vriend, G., Wilson, K. S., Hol, W. G. J. & Drenth. J. (1989). Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. J. Mol. Biol. 208, 679-696. Schulz. G. E., Schirmer, R. H., Sachsenheimer, W. & Pai, E. F. (1978). The structure of the flavoenzyme reductase. Nature (London), 273, glutathione 120-124. Schulz, G. E., Schirmer, R. H. & Pai, E. F. (1982). FAD-binding site of glutathione reductase. J. Mol.

Biol. 160, 287-308. Skariyhski, T., Moody, P. C. E. & Wonacott, A. ,J. (1987). Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution. J. Mol. Biol. 193, 171-187. Smith. A. G. & Brooks, C. J. W. (1975). Studies of the substrate specificity of cholesterol oxidase from Nocardia erythropolis in the oxidation of 3-hydroxy steroids. Biochem. Sot. Trans. 3, 675-677. Smith. A. G. & Brooks, C. ,J. W. (1977). The substrate specificity and stereochemistry, reversibility and inhibition of the 3-0~0 steroid A4-A5-isomerase component of cholesterol oxidase. Biochem. J. 167, 121-129. Stadtman. T. C.. Cherkes, A. & Anfinsen. (1. R. (1954). Studies on the microbiological degradation of cholesterol. J. Biol. Ghem. 206, 511-523. Takenaka, A., Kizawa, K., Hata, T., Sate, S., Misaka. IL. Tamuram, C. & Sasada, Y. (1988). X-ray study of baker’s yeast lipoamide dehydrogenase at 4.5 .& resolution by molecular replacement method. .J. Biochrm.

103. 463-469. Uwajima, T., Yagi. H., Nakamura, 8. Hr Terada, 0. (1973). Isolation and crystallization of extracellular SD-hydroxysteroid oxidase of Brevibacterium sterolicum nov. sp. Ayr. Biol. Chews. 37, 2345-2350. Edited

by

W. A.

Iiwajima, T., Yagi, H. & Terada, 0. (1974). Properties oxidase crystalline 3P-hydroxysteroid Brevibacterium sterolicum. Agr. Hiol. (‘hewt. 1149-l 156. Von Glehn. M. & Norrestam, R. (1972). The crystal molecular structure of 9-bromo-3,7,8,10-tetramethylisoalloxazine monohydrate. Acta Chem. Stand.

of of 38. and 26,

1490&1502. B. C. (1985). Resolution of phase ambiguity in macromolecular crystallography. Methods Enzyrnol. 115. 90-l 12. Westbrook, E. M., Piro, 0. E. & Sigler, P. B. (1984). The 6 A crystal structure of A*-3-ketosteroid isomrrase. ,I. Biol. Chem. 259. 9096-9103. Wierenga. R. K.. DeJong, R. J.. Kalk, K. H., HOI. W. G. .J. & Drenth, ,J. (1979). Crystal structure of p-hydroxybenzoate hydroxylase. .I. Mol. Biol. 13 1. 55.-73. Wierenga, R. K., Drenth. *I. & Schulz. G. E. (1983). Comparison of the three-dimensional protein and nucleotide structure of the FAD-binding domain of p-hydroxybenzoate hydroxylase with the FAD MS well as NADPH-binding domains of glutathionr reductase. J. Mol. Biol. 167, 725-739. Wierenga. R. K., De Maeyer. M. C. H. & HOI, \Y. (:. ,J. (1985). Interaction of pyrophosphate moieties witch a-helixes in dinucleotide binding proteins. Riochemistry, 24, 1346.-1357. Wierenga. R. K.. Terpstra, P. & HOI, W. (2. ,J. (1986). Prediction of the occurrence of the Al)l’-hinding pc@-fold in proteins using an amino acid xeyurnc~r fingerprint. J. Mol. Riol. 187. lOl- 107. Wilson, A. J. (:. (1950). Largest likely values for the reliability index. Acta Crystallogr. 3, 397-398. Zhao, H., Simard, ,J.. Labrir, (1.. Breton. X.. Rhreume. E., I,uu-The, V. bt Labrie, F. (1989). Molrc*ular cloning, cDKA structure and predicted a,mino acid sequence of bovine 3/J-hydroxy-5-me st.rroid dehydrogenase/A5-A4 isomerase. FEBS Letters. 259. 153-157. Wang,

Hendrickson)