691

Biochem. J. (1992) 288, 691-695 (Printed in Great Britain)

Escherichia coli dihydrodipicolinate synthase Identification of the active site and crystallization Bernd LABER,*t Franz-Xaver GOMIS-RUTH,* Maria Joao ROMAO*t and Robert HUBER*§ * Max-Planck-Institute fur Biochemie, D-8033 Martinsried, Federal Republic of Germany

Escherichia coli dihydrodipicolinate synthase (DHDPS) (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis, catalyses the condensation of pyruvate and aspartate /J-semialdehyde (ASA) by a ping-pong mechanism. Pyruvate binds first to the enzyme, forming a Schiff base with the c-amino group of Lys-161, followed by binding of ASA. Km values of 0.57 and 0.55 mm were determined for pyruvate and DL-ASA respectively. 3-Bromopyruvate inhibits DHDPS with a Ki of 1.6 mM. DHDPS is 50% inhibited by 1.0 mM-L-lysine, 1.2 mM-sodium dipicolinate or 4.6 mM-S-2-aminoethyl-Lcysteine. Crystals of DHDPS diffracting to beyond a resolution of 0.24 nm (2.4 A) were obtained under several experimental conditions. Diffraction patterns were compatible with trigonal space groups P3121 or P3221, with unit-cell parameters a = b = 12.26 nm and c = 11.19 nm. The density of the crystals indicates the presence of a dimer of DHDPS subunits per asymmetric unit.

INTRODUCTION Lysine biosynthesis via the diaminopimelate pathway of prokaryotes, some Phycomycetes and higher plants starts from aspartate and shares the first two reactions with the synthesis of methionine, threonine and isoleucine [1]. The first specific step of lysine biosynthesis, the condensation of aspartate p-semialdehyde (ASA) and pyruvate to dehydrodipicolinate, is catalysed by dihydrodipicolinate synthase (DHDPS) (Fig. 1). The complete amino-acid sequences of DHDPS from Escherichia coli [2], Corynebacterium glutamicum [3], wheat [4] and maize [5] have been communicated and it has been shown that these enzymes exist as homotetramers. DHDPS isolated from higher plants catalyses the condensation reaction by means of a ping-pong mechanism in which pyruvate forms a Schiff base with a lysine residue in the active site of the enzyme [6,7]. All higher-plant enzymes are strongly feedbackinhibited by low concentrations of lysine in an allosteric manner [6-9], indicating that lysine regulates the metabolite flux through its biosynthetic pathway. Additional evidence for DHDPS as the main regulatory point in higher-plant lysine biosynthesis is provided by the fact that a tobacco mutant with desensitized DHDPS overproduces lysine [101, while all plant mutants with aspartate kinases desensitized to lysine inhibition overproduce threonine [11]. The E. coli DHDPS is also inhibited by lysine [12], but to a much lesser extent. However, the physiological role of this feedback inhibition is questionable, since mutants wit-h desensitized aspartate kinase III excrete lysine but not threonine, an unexpected property for cells with efficient regulation of the branch-point enzyme [13]. Anyway, DHDPS must catalyse the rate-limiting step in E. coli lysine biosynthesis after aspartate kinase III, because increasing the dapA gene (encoding DHDPS) copy number in a lysC mutant with desensitized aspartate kinase III results in a parallel increase of DHDPS activity and lysine excretion [14]. In addition to its central role in lysine biosynthesis in higher plants and bacteria, DHDPS also delivers the direct precursor of

dipicolinic acid, which plays a key role in the bacterial sporulation process [15]. Consequently DHDPS has attracted interest as a suitable target for new herbicidal or antimicrobial compounds. Therefore, the full understanding of the reaction mechanism of this enzyme and of its kinetic and regulatory properties is highly desirable and best met by the availability of its three-dimensional structure as provided by X-ray crystallography. In the present paper -we report an improved purification procedure for DHDPS from an overproducing E. coli strain, the identification of the active site, and also describe the crystallization and preliminary crystallographic data of the enzyme.

MATERIALS AND METHODS

Materials E. coli RDA8/pDA3, carrying the dapA gene on a multicopy plasmid, was a gift from Dr. C. Richaud (Institut Pasteur, Paris, France). DL-ASA was synthesized by ozonolysis of DL-allylglycine and purified by ion-exchange chromatography [16]. N-Tosyl-Lphenylalanylchloromethane ('TPCK')-treated trypsin was purchased from Sigma.

Enzyme assay The assay mixture contained 100 mM-imidazole buffer, pH 7.4, 10 mM-K2HPO4, 5 mM-pyruvate, 2 mM-DL-ASA and enzyme in a volume of I ml. The reaction was started by the addition of enzyme and the increase in absorbance at 270 nm was measured at 37 'C against a blank without enzyme [16a], giving a linear

COOH H2N-CH

COOH + C=O

CH2

CH3

C'H

HOOC'Q

"COOH

Fig. 1. Reaction catalysed by DHDPS

Abbreviations used: DHDPS, dihydrodipicolinate synthase(s); ASA, aspartate fl-semialdehyde; OG, N-octyl-fl-D-glucopyranoside. t Present address: Schering AG, Agrochemical Research, D-1000 Berlin, Germany. t Permanent address: Centro de Tecnologia Quimica e Biol6gica (CTQB), Apart. 127, 2780 Oeiras, Portugal.§ To whom correspondence should be addressed.

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reaction rate after a short lag phase. One unit of DHDPS gave an increase in absorbance of 1.0/min (corresponding to about 5 nmol of tetrahydrodipicolinate formed/s). Enzyme purification Crude extract. Bacteria cultured in 5 1 of Luria-Bertani medium were harvested by centrifugation (approx. 30 g wet wt) at 10000 g for 10 min, resuspended in 20 mM-Tris, 10 mMpotassium pyruvate, 100 mM-KCl, pH 8.0, (approx. 3 ml/g wet wt) and disrupted by sonification (25 x 30 s). After centrifugation (15 min, 15000 g), the supernatant was collected and the sonification procedure repeated twice. Heat treatment. The combined supernatants were transferred to test tubes (approx. 14 mm i.d.) which were immersed for 2 min in a water bath at 70 °C and then immediately cooled on ice. Precipitated proteins were removed by centrifugation (15 min, 15000 g). DEAE-Sepharose chromatography. The supernatant was applied to a DEAE-Sepharose column (5 cm x 13 cm) which had been equilibrated with 20 mM-Tris/100 mM-KCl, pH 8.0. After extensive washing the column was eluted with a linear gradient of 0.1-0.5 M-KCI in 20 mM-Tris, pH 8.0. Phenyl-Sepharose chromatography. Active fractions were pooled, adjusted to a concentration of 0.5 M-ammonium sulphate by the addition of solid ammonium sulphate and applied to a phenyl-Sepharose column (2.4 cm x 17 cm, equilibrated with 0.5 M-ammonium sulphate in 20 mM-Tris, pH 8.0). After washing with equilibration buffer, the enzyme was eluted with 20 mmTris, pH 6.0. Fractions containing enzyme activity were pooled and dialysed against 20 mM-Tris, pH 8.0. F.p.l.c. on Q-Sepharose. F.p.l.c. was done with Pharmacia equipment and a Pharmacia Q-Sepharose fast-flow column. About 50 mg of protein (a third of the total sample) was applied to a Q-Sepharose column (HR 16/10, 1.6 cm x 10 cm) equilibrated with 20 mM-Tris, pH 8.0, and was eluted with 235 ml of a linear gradient from 0 to 0.5 M-KCI in 20 mM-Tris, pH 8.0. DHDPS purified in three successive runs was pooled, concentrated by ultrafiltration (Amicon PM 10 membranes) and, after the addition of 0.57 vol. of glycerol, stored at -20 °C for several months without loss of activity. A summary of the purification procedure is presented in Table 1. Molecular mass estimations The molecular mass of the enzyme was estimated by SDS/PAGE [17] and by gel filtration using a Pharmacia Ultropac TSK-G3000-SW f.p.l.c. column equilibrated with 20 mmTris/100 mM-KCl, pH 8.0. Carbonic anhydrase and monomers and dimers of BSA and ovalbumin were used to calibrate the column.

Protein concentration determination Protein concentrations were determined by the method of Bradford [18] using BSA as the standard. The concentration of homogeneous enzyme was calculated based on an absorption coefficient at 280 nm of 0.412 l mg- cm-.

Peptide mapping Either 150 ,u of 100 mM-pyruvate or 150 ,1 of water were added to 1 ml samples of purified DHDPS (18 mg/ml in 20 mmTris, pH 8.0). After 10 min at room temperature, 30 ,umol of NaBH4 was added to both samples. After 4 h on ice the reaction mixtures were desalted on Pharmacia PD-10 columns (equilibrated with 50 mM-NH4HCO3) and freeze-dried. Freezedried DHDPS was denatured, mercaptolysed and then alkylated with 4-vinylpyridine [19]. After digestion with trypsin [20]

Table 1. Purification of DHDPS from E. coli

Purification step Crude extract Heat treatment DEAE-Sepharose Phenyl-Sepharose Q-Sepharose

Volume

(ml) 163 140 350 61 41

Specific Protein activity Yield (mg) (units/mg) (%) 4091 545 246 148 100

11 71 142 162 270

100 89 81 54 62

Purification (fold) I 6.7 13.3 15.1 25.2

fragments were separated on a Macherey-Nagel ET 250/8/4 Nucleosil 300-10 C-18 h.p.l.c. column in an elution gradient from 0 (solvent A) to 80% acetonitrile (solvent B) in 0.1 % trifluoroacetic acid. Peptides were further separated by rechromatography on the C-18 h.p.l.c. column in an elution gradient from 0 (solvent A) to 50 % acetonitrile (solvent B) in 50 mMpotassium phosphate buffer, pH 6.25. Amino-acid and sequence analysis Amino-acid analyses were carried out on a Biotronic LC 600 amino-acid analyser after hydrolysis with 6 M-HCI at 1 10 °C for 24 h. Peptides were sequenced by Edman degradation in a prototype spinning-cup sequenator [21,22].

Crystallization Purified DHDPS was passed through a Pharmacia PD-10 gelfiltration column, the buffer being exchanged against 2 mM-Mes, pH 6.8. Crystallization experiments were performed with and without effectors at room temperature and at 4 °C using sittingdrop vapour diffusion. Ammonium sulphate (1.25-3.0 M), sodium/potassium phosphate (1.25-3.0 M), sodium citrate (0.8-1.6 M) and phosphate (0.2-1.5 M)/10% (w/v) PEG 6000 as precipitating agents, were assayed in the pH range from 4 to 10. It has been shown [23] that DHDPS loses its enzymic activity irreversibly when treated with NaBH4 in the presence of sodium pyruvate. For crystallization experiments the enzyme. was inactivated as follows: 990 ,l of DHDPS solution (activity 160.8 units/ml) was incubated with 25 1l of 0.1 M-sodium pyruvate in 0.1 M sodium/potassium phosphate buffer, pH 7.0, for 10 min at 20 'C. Aliquots (25 gcl) of 0.2 M-NaBH4 solution in the same buffer were added under cooling in an ice bath. After 2 h, the reaction mixture was chromatographed using a Pharmacia NAP5 column. The assay for enzymic activity yielded a value of 1.3 + 0.5 % residual activity.

Crystal density determination The density of the crystals was determined by the Bode-Schirmer Ficoll density-gradient method [24]. Data collection X-ray intensity data from trigonal crystals grown at basic pH in the presence or absence of inhibitors and of a mercury methyl chloride derivative were collected using a FAST television area diffractometer (Enraf-Nonius, Delft, Holland) with CuK. Xradiation from a Rigaku Denki generator operated at 5.4 kW. A 0.24 nm (2.4 A) native data set was collected and processed using MADNES software [25], applying specifications described in [26]. Additionally, diffraction results were collected on film with a rotation-precession camera (Huber, Rimsting, Germany) at -4 'C (because of radiation sensitivity of the crystals). Film data were evaluated with the FILME program ([27] modified by S. Bennett). 1992

Dihydrodipicolinate synthase from Escherichia coli

693 Table 2. Amino acid compositions of tryptic peptides P-13, P-14, P-23 and P-X in comparison with those deduced from the corresponding DHDPS sequence 121 Values in parentheses are deduced from the corresponding DHDPS sequence.

I

0.05-

Amino acid

P-13

P-14

P-23

P-X

Asp/Asn Thr Glu/Gln Gly Ala Val Ile Leu Lys Arg

1.1 (1)

1.0 (1) 1.9 (2) 1. 1(1) 0.9 (1) 1. 1(1) 1.0 (1)

1.0 (1) 1.9 (2) 1. 1(1) 0.9 (1) 1. 1(1) 1.0 (1) 2.9 (3)

2.8 3.7 2.1 2.5 2.0 1.1 1.5 4.0

1.0 (1) 1.7 (3) -

0.9 (1)

-

-

-

-

1.0 (1)

1.0 (1)

2.1

- 1 oo

P-23

0.1I0- (a)

/

Fig. 2. Reversed-phase h.p.l.c. separation of tryptic peptides of DHDPS (a) and of DHDPS reduced with NaBH4 in the presence of pyruvate (b)

7 r5

o.a m

8

RESULTS AND DISCUSSION Purification and molecular mass of the enzyme In a typical purification procedure, summarized in Table 1, DHDPS was purified 25-fold to a specific activity of270 units/mg of protein with 62 % yield. Starting from 30 g of E. coli wet cell paste approx. 100 mg ofpurified DHDPS was obtained. Addition of 10 mM-pyruvate to the crude extract greatly increased the recovery of enzyme activity during the heat treatment. The purified DHDPS (native or pyruvate/NaBH4-inactivated) showed a single band upon SDS/PAGE corresponding to a molecular mass of 32 +2 kDa. Since the molecular mass of the native enzyme was determined to be 112 kDa by gel filtration, DHDPS is probably a tetramer composed of four subunits with identical molecular masses. An isoelectric point of approx. 4.5 was determined by isoelectric focusing for both native and inactivated enzyme.

Identification of the active site Since the DHDPS reaction is known to proceed via Schiff base formation between the carbonyl group of pyruvate and an eamino group of a lysine residue [23], experiments were carried out to identify this lysine residue in the polypeptide chain. Treatment of DHDPS for 4 h with NaBH4 in the presence of pyruvate resulted in a 98.7 % loss of enzyme activity (pyruvateinactivated enzyme), while DHDPS treated with NaBH4 in the absence of pyruvate (control) had lost only 1.8 % of its activity. Control and pyruvate-inactivated DHDPS were denatured, reduced with mercaptoethanol and alkylated with 4vinylpyridine. After digestion with trypsin the resulting peptide (P-1-P-27) were separated by reversed-phase h.p.l.c. and identified by amino-acid analysis. Comparison of peptide maps obtained from control and pyruvate-inactivated DHDPS (Fig. 2) revealed three major differences. In the map of pyruvateinactivated enzyme (Fig. 2b) peaks corresponding to P-14 (Glu162-Arg-169) and P-13 (Asn-156-Lys-161) were missing, while the peak containing P-23 (Glu-258-Arg-268) in the control (Fig. 2a) had increased in size after pyruvate inactivation (P-X). The amino-acid composition of P-X was in agreement with the Vol. 288

M5 ° Iml) V5 10 0~~~~~~~301 Volume (ml)

2m4

4) 0

Cln

Volume (ml)

Fig. 3. Rechromatography at neutral pHon areversed-phaseh.p.l.c. column of tryptic peptides P-23 of DHDPS (a) and P-X of DHDPS reduced with NaBH4 in the presence of pyruvate (b)

sum of P- 13, P- 14 and P-23 with the exception of the single lysine (Lys-161) of P-13. This residue could not be identified; instead a peak of ninhydrin-positive material eluted from the analyser with a retention time different from any of the 20 protein amino acids (Table 2). Upon rechromatography with a mobile phase of nearly neutral pH (Fig. 3), material from P-23 eluted as a single peak (Fig. 3a), but material from P-X produced two fragments (P-X1 and P-X2) (Fig. 3b). Amino-acid analysis revealed that the composition of P-Xl was in agreement with the composition expected for P-23. The composition of P-X2 agreed with the composition expected

694

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Table 3. Amino-acid composition of peptides P-X1 and P-X2 in comparison with those of P-23 and P-13 plus P-14 deduced from the corresponding DHDPS sequence 121

Amino acid

P-X1 (P-23)

P-X2

(P-13+P-14)

(1) (2) (1) (1) (1) (1)

1.7 1.7 1.0 1.8 1.0

(2) (2) (1) (2) (1)

2.8 (3)

1.2 1.0

(3) (1)

-

(1)

1.0 (1)

1.0

(1)

0.9 1.7 1.0 1.1 0.9 1.1

Asp/Asn Thr Glu/Gln

Gly Ala Val Ile Leu Lys Arg

Precipitating agent Phosphate Ammonium sulphate

WhDHDPS17 AVTLDDYLPMRSTEVKNRTSTDGIKSLRLITAVKTPYLPD GRFDL EAYD S LINTQINGGAEGV WhDHDPS26 AITTDDYLPMRSTEVKNRTSVDGIKSLRLITAVKTPYLPD GRFDL EAYD S LINTQINGGAEGV MaDHDPS AITLDDYLPMRSTEVKNRTSTDDITRLRLITAVKTPYLPD GRFOL EAYD S LINMQIEGGAEGV CoDHDPS ----------MSTGLTAKTGVEHFGT--VGVAMVTPFTES GDIDI AAGR E VAAYLVDKGLDSL EcDHDPS ------------------MFTG--SIVAIVTPMDEK GNVCR ASLK K LIDYHVASGTSAI - -1-

---

-

P-1 64

-

P-2

----

--

I-

39

-

P-3 P-4

:N28

WhDHDPS17 IVGGTTGEGHLMSWDEHIMLIGHTVNCFGAN IKVIGNTGSNSTREAVHATEQ WhDHDPS26 IVGGTTGEGHLMSWDEHIMLIGHTVNCFGTN IKVIGNTGSNSTREAIHASEQ MaDHDPS IVGGTTGEGHLMSWDEHIMLIGHTVNCFGSR IKVIGNTGSNSTREAVHATEQ CoDHDPS VLAGTTGESPTTTAAEKLELLKAVREEVGDR AKLIAGVGTNNTRTSVELAEA EcDHDPS VSVGTTGESATLNHDEHADVVMMTLDLADGR IPVIAGTGANATAEAISLTQR

P-5

GFAVGMHAALHVN GFAVGMHAALHVN GFAVGMHAALHIN

-----

P-7

------------------

7F-

.

WhDHDPS17 ECVGH--- -ERVK CYTDKGISIWSGNDDECHDSRWKYGATGVISVASNLVPG WhDHDPS26 ECVGH--- -ERVK CYTDKGITIWSGNDDECHDSRWKYGATGVISVTSNLVPG MaDHDPS ECVGH--- -ERVK HYADKGITIWSGNDDECHDSKWKHGATGVISVTSNLVPG CoDHNDPS DAKGDL-- VAATS LIKETGLAWYSG-DDPLNLVWLALGGSGFISVIGHAAPT EATGNLTR VNQIK ELVSDDFVLLSG-DDASALDFMQLGGHGVISVTTNVAAR EcDHDPS

?-12

P-

LMHSLMF EGEN LMRSLMF EGEN LMHSLMY KGEN ALRELYT SFEE DMAQMCK LAAE

2

.4.9

P-18

P-l9

P-20

LFCEPNPIGLN TALA QLG-VVRPVFR LPYTPLPLEKRV LFSEPNPIGLN TALA QLG-VVRPVFR RPYAPLSLEKRT LFCQPNPIALN TALA QLG-VARPVFR LPYVPLPLEKRA L----GGVSLA KAAS RLQGINVGDPR LPIMAPN-EQEL LFVEPNPIPVK WACK ELGLVATDTLR LPMTPIT-DSGR P-2!

P-22

P-7.1

P-24

3 42

30 9

EcDHDPS

P-118 3

WhDHDPS17 AAL---- --NEK LLPLM---KW WhDHDPS26 AAL---- --NEK LLPLM---KW ATL---- --NEK LSPLM---KW MaDHDPS GDLVRAR EINAK LSPLVAAQGR CoDiiDPS EcDHDPS EHFAEAR VINQR LMPL---HNK

WhDHDPS17 WhDHDPS26 MaDHDPS CoDHDPS

P-171

P-.6

221

-

-

P_1 5~

P-14

EAIG RENFV GQKESRVLDSDDFVLISRY EAIG RFNFV GQKEVRVLDDDDFVLISRY ESIG RENFV GQKEARF----------EDMK KAGVL ------------------ETVR-- AALK HAGLL ------------------I. -----292 EFVRIV EFVRIV EFVRIV EALR--

i:-25

P-26

pH

(mg.ml)

1.5-1.75 2.5 1.75-2.25

9-10 9 8-10

22 22

4/22

10.4*t 15.0* 10.4*t

2.5-3.0 1.0-1.5

8-9 8-10

4/22 4/22

1.5 10 * Native protein. t Protein reduced with NaBH4.

22

Additive

lys+OG*t dpc + OG* lys + OG*t/ bpy ± OG* 4.4 15* dpc + OG* 10.4*t lys+OG*t/ bpy ± OG* 8.8-15* dpc + OG*

------

TIQDIPPP\'IEA LST YP NIAGVlK SAQDIPPEVILA ISG YT N.MAGVC SGIPIESDTMRR LSE LP TILAVK TGCDLLPETVGR LAK VK NIIGIK -- - ---I I-------

p-9

P-81

Protein concn.

Temp. (°C)

AASAGADGLLVVT

WhLEIPSP 17 PYYGK TSTEGLISHFK EVLPMG--PTIIYNVPSR TSQDIPPPVIEA LSS YS NMAGVX

SI

Citrate

Final drop concn. (M)

FNDSGIVGCLTVT

-----------__ ___{______----

---

WhDlIPS26 PYYGK TSTAGLISHFI EVLPMG--PTIIYNVPSR oaCHDPS IYYGK TSAEGMISHFE AVLPMG--PTIIYNVPSR -oCHDPS PYYSK PSQEGLLAHFG A'AAATEVPICLYDIPGR PYYNR PSQEGLYQHFK AIAEHDLPQTLYNVPSR EcrHDPS

Table 4. Crystallization conditions for co-crystallization of DHDPS with inhibitors Composition of drops: 1.5 ,ul of precipitating agent, 4 ,ul of protein solution and 3 ,ul of inhibitor solution (with or without OG added). Abbreviations used: dpc, sodium dipicolinate [100 mm in 2 mMMes, pH 6.3, with or without 50O0 (w/v) OG]; bpy, bromopyruvate; lys, lysine [both bpy and lys at 10 mm in 2 mM-Mes, pH 6.8, with or without 600 (w/v) OG].

P-27

Fig. 4. Sequence alignment between the five DHDPS sequences so far published, together with the tryptic peptides from the E. coli enzyme sequence analysis (P-1 to P-27) The active-site lysine residue (Lys-161) is shown in bold and is present in all the isoenzymes. The upper numeration corresponds to the longest possible sequence and the lower one to E. coli DHDPS. Abbreviations used are: WhDHDPS17 and WhDHDPS26, wheat DHDPS clones pDA17 and pDA26 respectively [4]; MaDHDPS, maize DHDPS [5]; CoDHDPS, the enzyme from C. glutamicum [3]; EcDHDPS, E. coli DHDPS [2].

Kinetics Measurements of the initial velocities of the condensation reaction were performed either with pyruvate as the variable substrate in the presence of various fixed concentrations of DLASA or vice versa. Double-reciprocal plots of initial velocities versus pyruvate concentration and versus DL-ASA concentration gave a family of parallel lines, consistent with a ping-pong mechanism [29]. Secondary intercept plots gave K., values of 0.57 and 0.55 mm for pyruvate and DL-ASA respectively (results not

shown). Altogether, in the DHDPS reaction pyruvate binds to the enzyme first by forming a Schiff base with the c-amino group of Lys-161. After release of the first water molecule, ASA binds to the active site and the condensation reaction to form 4-hydroxy2,3,4,5-tetrahydrodipicolinic acid takes place. Whether the removal of the second water molecule to form 2,3-dihydrodipicolinate is also catalysed by the enzyme or is the result of a spontaneous dehydration reaction, remains to be elucidated. Effect of inhibitors DHDPS was 50 0°0 inhibited by 1.0 mM-L-lysine, 1.2 mMsodium dipicolinate or 4.6 mM-S-2-aminoethyl-L-cysteine. However, inhibition by lysine was not complete, and a residual activity of about 20 00o was still present with 30 mM-L-lysine. No significant inhibition of DHDPS activity was observed in the presence of 3-fluoropyruvate, ax-oxobutyrate, oxaloacetate, ax-oxoglutarate, phenylpyruvate, 2,6-pipecolinate, aspartate or

asparagine. 3-Bromopyruvate competitively inhibited the condensation for P- 13 plus P- 14, again with the exception of the missing lysine (Lys- 161) and the appearance of an unidentified amino acid

(Table 3). Automatic sequence analysis of 12 residues of P-X2 gave NIIGIXEATGNL (X = unidentifiable residue), identical with Asn-156-Leu-167 of the DHDPS sequence. Thus, Lys-161 is the active-site lysine residue of DHDPS. Sequence alignment and comparison (performed with the GCG Sequence Analysis Software Package Version 7.0 [28]) revealed that Lys- 161 (Fig. 4) is the only lysine residue conserved in the five known DHDPS isoenzymes [E. coli, C. glutamicum, wheat (two isoenzymes) and maize].

reaction with respect to pyruvate with a K, value of 1.6 mM. When DHDPS was pre-incubated with 30 mM-3-bromopyruvate, the enzyme irreversibly lost about 35 %," of its original activity after 5 min. However, the loss of enzyme activity did not follow (pseudo) first-order kinetics and did not exceed 60 00 after 15 min of pre-incubation. The inactivation of DHDPS by 3bromopyruvate was not investigated further. None of the above-mentioned a-ketoacids replaced pyruvate as a substrate in the condensation reaction.

Crystallization and crystal characterization With 1 M-sodium/potassium phosphate buffer

at

pH

10.0

as

1992

Dihydrodipicolinate synthase from Escherichia coli the precipitating agent and 6 % (w/v) n-octyl-,8-D-glucopyranoside (OG), equilibrated against 1.8-2.0 M-sodium/potassium phosphate, well-formed trigonal crystals grew up to 0.8 mm in their largest dimension and up to 0x6 mm wide in about one week. These crystals were suitable for X-ray diffraction analysis, diffracting to beyond 0.24 nm (2.4 A) resolution. Diffraction patterns were compatible with trigonal space group P3121 or P3221, with unit-cell parameters a = b = 12.26 nm and c = 11.19 nm. The density of the crystals was determined as 1.105 + 0.004 g/cm3, which corresponds to a dimer of DHDPS subunits in the asymmetric unit. The tetrameric molecule is located on the twofold axis. The volume per unit-protein molecular mass, V., is 0.381 nm3/Da (3.81 A3/Da). The corresponding solvent content of 68 % is slightly higher than commonly observed in other protein crystals (27-65%) [30]. Trigonal crystals of the same space group and with similar cell constants were obtained when the crystallization experiments were performed in the presence of inhibitors (bromopyruvate, lysine or sodium dipicolinate) or with DHDPS which had been inactivated with pyruvate/NaBH4. Crystals usually grew in 3-5 days for a wide range of conditions, summarized in Table 4. A new crystal form was, however, obtained in 1.5-2.0 Mammonium sulphate, pH 4, 22 °C [4 ,ul of inactivated enzyme solution 2 mg/ml, 1.5 ,1 of precipitating agent and 3 ,ul of 5 % (w/v) OG in 2 mM-MES, pH 6.8}. These crystals have the shape of small hexagonal rods (< 0.1 mm). In similar conditions, using 1.75 M-sodium/potassium phosphate as precipitant (22 °C, pH 4), very small prisms (< 0.1 mm) were obtained. In 1.5 Mcitrate, pH 7.0 [10 mM-L-lysine and 5 % (w/v) OG in 2 mM-Mes, pH 6.8, as additives], after transferring the dish from 4 °C to 22 °C, and in 0.5 M-phosphate/10 0% (w/v) PEG 6000, pH 4.0, at 4 OC (with 10 mM-L-lysine) little prisms were also obtained (< 0.1 mm). M.J.R. was supported by an Alexander von Humboldt fellowship. F.-X. G.-R. thanks I. Mayr for introduction to crystallization techniques, 0. Epp for help with the FILME program system, and M. Bauer for help in gel chromatography studies. We thank Dr. A. Henschen, K. Krieglstein, and Dr. D. Georgopoulos for the amino-acid analyses.

REFERENCES 1. Herrmann, K. M. & Somerville, R. L. (eds.) (1983) Amino Acids, Biosynthesis and Genetic Regulation, Addison Wesley, London 2. Richaud, F., Richaud, C., Ratet, P. & Patte, J. C. (1986) J. Bacteriol. 166, 297-300

Received 16 April 1992/22 May 1992; accepted 2 June 1992

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