Protein Engineering vol.5 no.3 pp.235-240. 1992
Fluorescent properties of the Escherichia coli D-xylose isomerase active site
Andrew C.Jamieson and Carl A.Batt1 Institute of Food Science. Cornell University. Ithaca. NY 14853. USA 'To whom correspondence should be addressed
Introduction D-xylose isomerase (E.C. 5.3.1.5) is an intracellular microbial enzyme which catalyzes the reversible isomerization of xylose to xylulose. The enzyme will also accept glucose as a substrate, and is utilized industrially for the production of high fructose corn syrups (Chen, 1980). Recently, high resolution X-ray crystal structures have become available for enzyme derived from Streptomyces rubiginosus and an Arthrobacter species, leading to differing interpretations of the enzyme mechanism based on the proposed orientations of substrate in the active site (Carrell et al., 1989; Collyer et al., 1990). Some authors have proposed that the enzyme isomerization occurs as a metal cation mediated hydride shift (Farber et al., 1989; Collyer et al., 1990; Whitlow et al., 1991), rather than by the classical mechanism for aldose — ketose isomerases, which involves base catalyzed intramolecular proton transfer and a cis ene-diol intermediate (Hanson and Rose, 1974; Carrell etal, 1989). Site-directed mutagenesis experiments performed in enzymes from other species have focused primarily on the role of a conserved active site histidine residue, in attempting to discriminate between its functional role as a catalytic base in either substrate ring-opening, which precedes isomerization, and/or in the isomerization reaction itself (Batt et al., 1990; Lee et al., 1990). Another conserved feature of D-xylose isomerase revealed by X-ray crystallography is the presence of two tryptophan residues which sandwich both the pyranose and open-chain forms of bound substrate at the enzyme active site (Carrell et al., 1989; Collyer et al., 1990). The arrangement is reminiscent of the binding pocket of arabinose binding protein (ABP), a periplasmic protein involved in transport and chemotaxis in Escherichia coli, in which a tryptophan and a phenylalanine residue appear to mediate a © Oxford University Press
Materials and methods Strains and plasmids Escherichia coli JM109 [recA\ supEAA endAX hsdRXl gyrA96 relAl thi A(lac-proAB) F'(traD36proAB+ laclq ZAM15)] was used for the production of wild-type enzyme from a cloned xylA (D-xylose isomerase) construct, while HB101 (supEM hsdS20(r-B m~B) recA\3 ara-14 proAl lacYX galKl rpsLlO xyl-5 mtl-l) which is deficient for D-xylose isomerase activity, was used for the production of mutant proteins. Escherichia coli JM2r~ [hsdRM mcrAB A(lac-proAB) F'(traD36proAB+ laclq ZAM15)] was used as a recipient for site-directed mutagenesis. The plasmid pKK223, which carries a tac promoter and is regulated by the chromosomal lacP gene in E.coli JM109, was used as an expression vector. M13mpl9 was used for mutagenesis and nucleotide sequencing. DNA manipulations Restriction enzymes were purchased from New England Biolabs or US Biochemical Corporation and used according to the manufacturer's instuctions. Modifying enzymes utilized in sitedirected mutagenesis were purchased solely from US Biochemical Corporation. Subcloning of mutant xylA genes was facilitated by the observation that the overexpression of the xylA gene in trans in a Xyl+ background, represses the expression of the chromosomal operon. This phenomenon, can be screened using MacConkey's medium containing 2% xylose as the sole carbon source (Batt et al., 1986). Site-directed mutagenesis Mutagenesis was performed in M13mpl9 using synthetic oligodeoxynucleotide primers containing the desired substitutions. Selection of mutants was facilitated by enzymatic removal of the parental strand as described by Vandeyar et al. (1988). Enzyme purification Enzyme purification was initiated from cultures carrying the appropriate expression vector, induced with 2 mM isopropyl-/3D-thiogalactopyranoside (IPTG) for 3 h. Yields of up to 28% total soluble protein were routinely managed with this expression system. The purification involves a heat treatment of sonicated cell extracts for 10 min at 55°C in the presence of 70 mM MnCl2, and has been described elsewhere (Yamanaka, 1968; Batt et al., 1990). Final yields were between 50 and 100 mg enzyme per liter of culture. An estimate of protein concentration was made from a total amino acid analysis of the sample 235
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The consequences of active site mutations of the Escherichia coli D-xylose isomerase (E.C. 5.3.1.5) on substrate binding were examined by fluorescence spectroscopy. Site-directed mutagenesis of conserved tryptophan residues in the E.coli enzyme (Trp49 and Trpl88) reveals that fluorescence quenching of these residues occurs during the binding of xylose by the wild-type enzyme. The fluorescent properties of additional active site substitutions at HislOl were also examined. Substitutions of HislOl which inactivate the enzyme were shown to have altered spectral characteristics, which preclude detection of substrate binding. In the case of H101S, a mutant protein with measurable isomerizing activity, substrate binding with novel fluorescent properties was observed, possibly the bound pyranose form of xylose under steady-state conditions. Key words: D-xylose isomerase/fluorescence/site-directed mutagenesis
hydrophobic interaction with the aliphatic hydrogens of the bound arabinose sugar ring (Quiocho and Vyas, 1984). An additional feature of this protein -ligand interaction is revealed by fluorescence spectrometry, in which the fluorescence emission spectrum of ABP becomes quneched upon the addition of arabinose (Miller et al., 1983). In this paper we examine the fluorescent properties of the active site tryptophans in the E. coli D-xylose isomerase, and attempt to resolve the consequences of active site mutations on sugar binding.
A.C.Jamieson and C.A.Batt
Results Site-directed mutagenesis A number of alignments of the homologous amino acid sequences of D-xylose isomerases from various species are currently available. In all cases, only two out of the 16 tryptophan residues in the E.coli sequence (Trp49 and Trpl88) are conserved throughout. The homologues of these residues are found at the active site of D-xylose isomerase from both 5. rubiginosus and Arthrobacter as idenified by X-ray crystallography studies (Carrell etai, 1989; Collyer etal., 1990). The following mutations were introduced at these sites: W49A, W188A, WI88F
and the double mutation, W49AW188F. We also constructed substitutions at HislOl, H101S and H101Q, in addition to those previously described at this locus (H101R and H101Y) (Batt et al, 1990). The HislOl residue in E.coli corresponds to the active site residue identified in crystal structures as the catalytic base involved in either substrate ring-opening and/or in the isomerization reaction (see Introduction). Fluorescence spectrometry Figure 1 demonstrates that quenching of the E.coli D-xylose isomerase fluorescence emission spectrum occurs upon the addition of xylose. Quenching of the emission spectrum is shown in the presence of 50 mM xylose, with a slight blue-shift in peak emission from 337 to 335 nm. The quench is titratable with A"app = 3 mM, somewhat lower than the Km calculated by the coupled enzyme assay (10 mM). The blue-shift in the peakemission wavelength has been attributed to the hydrophobic interaction between the sugar and the imidazole ring in ABP (Miller et al., 1983), although a more likely interpretation is that it arises from the background fluorescence of the remaining tryptophans in the E.coli. The quench is not observed with the addition of the open-chain substrate analogue, xylitol, at concentrations up to 100 mM. A disparity arose, however, when we attempted to demonstrate directly that the ring form of the substrate was responsible for the quench, by analogy with ABP: neither glucose nor the ring analogue of glucose, 5-thio-D-glucose, was able to quench the fluorescence spectrum when added at concentrations up to 100 mM (Figure 2). The reason for this disparity may lie in the high Km that the E.coli enzyme exhibits towards glucose (0.5 M), as has previously been reported (Wovcha et al., 1983). Attempts to use concentrations of glucose > 100 mM yielded problems with the fluorescence spectrum due to the high viscosity of the solution. wild type + 50 mM xylose
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Emission Wavelength (nm) Kig. 1. Fluorescence spectrum of the E.ntli D-xylosc isomerase Quenching of the emission spectrum is shown in the presence of 50 mM xylose, with a slight blue-shift in peak emission from 337 to 335 nm. The sample buffer contained 10 mM triethanolanine ipH 7.2). 10 mM MnCU and I mM DTT. The enzyme subunit concentration was estimated at 5 /imol. Spectra were recorded at room temperature, with 290 nm excitation light.
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and an extinction coefficient calculated at 280 nm. Mutant proteins were purified by the same procedures. Enzyme assays Qualitative assays of D-xylose isomerizing activity were performed by the cysteine-carbazole method (Dische, 1949), using induced E.coli cells permeabilized by toluene. An HPLC assay was used for detecting isomerizing activity in D-xylose isomerase mutant proteins as previously described (Ban et al., 1990). Kinetic measurements were made using a coupled assay linked to sorbitol dehydrogenase (SDH) (Kersters-Hilderson et al., 1987). The reaction mixture contained 10 U SDH (Boehringer Mannheim), 10 mM MnCl2, 5 mM NADH (Sigma) and mutarotated D-xylose in 10 mM triethanolamine buffer (pH 7.2). Fluorescence spectroscopy Fluorescence spectra were recorded on an SLM model 8000 fluorescence spectrometer (SLM Instruments, Inc). Protein samples (5—lO^mol subunit concentration) were in 10 mM triethanolamine buffer (pH 7.2), 10 mM MnCl2 and 1 mM DTT. The emission spectra (295-500 nm) were recorded with 290 nm excitation light at room temperature.
E.coli xylose isomera.se
Fluorescence properties of D-xylose isomerase mutant proteins In order to demonstrate that substrate quenching involves the active site tryptophans, the fluorescence spectra of the W49A, W188A and W49AW188A mutant proteins were examined. Figure 3 is a set of comparative spectra showing the effects of substitution of Trp49 and Trpl88 on fluorescent light emission. The spectra are essentially additive and demonstrate that Trp49
and Tip 188 together account for - 3 0 % of the light emitted by the wild-type enzyme. Mutant protein spectra were all insensitive to the addition of substrate (50 mM xylose), demonstrating that both residues are required to exhibit the quenching effect. An unusual correlation was found for the W49A spectrum, which is almost superimposable with the quenched wild-type spectrum obtained in the presence of 50 mM xylose. The correlation
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+
/
50mMxyUto.\
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1
1
Emission Wavelength (ran) Fig. 2. Fluorescence spectra of the E.coli D-xylose isomerase insensitive to quenching. The spectra were recorded in the absence of substrate, or in the presence of 5-thio-D-glucose (100 mM) or xylitol (50 mM) under the conditions described in the legend to Figure I.
B.000
wild type
400 Emission Wavelength (nm) Fig. 3. A comparison between the normalized emission spectra of the wild-type D-xylose isomerase and the W49A, W188A and W49AWI88A mutant proteins. All spectra were recorded under the standard conditions described in Materials and methods. Subunit concentrations were in the range 5-10/imol.
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Relat ive Intensity
^ \ '—v
-
\ wild type
/
\
\ \ HIOIY\ \
\ /
0.000
1
Emission Wavelength (nm) Fig. 4. Fluorescence spectrum of the H101Y mutant protein compared with the wild-type enzyme. The spectra were recorded under standard conditions described in Materials and methods. The subunit concentration of the H101Y was estimated at 7 /unol prior to normalization.
implies that the single residue Trp49 is quenched in its entirety upon substrate binding, although this would be highly unusual. The fluorescent properties of substitutions at HislOl were examined to resolve the effects of active site mutations on substrate binding. Figure 4 shows what we expect to be the general case for substitution of HislOl in the E.coli D-xylose isomerase, as demonstrated by the H101Y mutant protein. The fluorescence spectrum of the mutant protein is partially quenched and blue-shifted with respect to the wild-type spectrum and is once again insensitive to the addition of substrate. We subsequently found the spectra derived from the H101Y, H101R and H101Q to be superimposable (data not shown). All three of these mutant proteins are also completely inactive (Table I; Batt et ai, 1990). Only in the case of the H101S mutant protein were we able to demonstrate an essentially unaltered fluorescence spectrum when compared with the wild-type enzyme in the absence of substrate. Addition of xylose to this mutant protein, however, resulted in an altered spectrum with novel properties. Substrate quenching does not occur in this example, but substrate can be detected with the rise of a new fluorescent peak at 335 nm as shown in Figure 5. The peak is titratable with a Km of 30 mM. This mutant protein is also distinct from all other HislOl substitutions we studied, in that it exhibits detectable isomerizing activity (Table I). Kinetic analyses A summary of the kinetic properties of the D-xylose isomerase and associated mutant proteins is presented in Table I. The substitution W49A inactivated the enzyme, whereas replacing Trpl88 with either alanine or phenylalanine resulted in reduction of £cal by two or three orders of magnitude. At this very low level of activity, Km measurements by the coupled spectrometric assay are only approximate. HislOl substitutions, with the exception of HI01S. completely 238
Table I. Kinetic parameters of D-xylose isomerase muiam proteins Substitution Wild-type W49A W188A W188F H101Q H101S
Km (mM xylose)
k,..,, (s active site )
10 NA 2O-3Ob 20b NA 70
5 NA 0.005 0.05 NA 0.05
K.app m (mM xylose)
3 ND ND ND ND 30
a
As measured by fluorescence titration. Estimated value. NA. no activity. ND. not determined.
b
inactivated the enzyme. The kc.d( for the H101S mutant protein was determined to be reduced by two orders of magnitude and the Km measured as 70 mM. In this case, as with the wild-type enzyme, the measured value is noticeably higher than the value obtained from titration of the fluorescence emission spectrum in the presence of substrate. Inactivation of the H101R and H101Y mutant proteins was reported previously (Batt et ai, 1990). Discussion Despite the apparent chemical simplicity of the isomerization reaction, three kinds of proton translocation are catalyzed by Dxylose isomerase. The first of these occurs during ring-opening of the pyranose form of the substrate, where only the or anomeric form of the sugar is accepted by the enzyme (Feather et al., 1970). During the isomerization reaction per se, proton movement which occurs between the hydroxyl and carbonyl groups is also accompanied by an additional intramolecular transfer of hydrogen between the Cl and C2 carbon centers. In
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E.coli xylose isomerase
Fig. 5. Fluorescence spectra of the partially active H101S mutant protein alone and after the addition of 50 mM xylose. Both spectra were recorded under standard conditions at 6 fimol subunit concentration.
D-xylose isomerase, the intramolecular transfer occurs completely without solvent exchange (Rose et al., 1969). X-ray crystal data on the S. rubiginosus and Arthrobacter D-xylose isomerases have led to differing interpretations of the role of the active site residue corresponding to His 101 in the E.coli enzyme. Carrell has proposed that this residue mediates the isomerization reaction by proton abstraction between carbon centers (Carrell et al., 1989), whereas Collyer has relegated the role of this residue to performing the ring-opening reaction, favoring a hydride shift for the isomerization, mediated by one of the two metal cations present in the active site (Collyer et al., 1990). Recently, this interpretation has been supported by sitedirected mutagenesis experiments in the Clostridium thermosulfogenes enzyme, on the basis that substitution of the residue does not render the enzyme completely inactive. This latter study further relegated the role of the histidine to serving solely as a hydrogen bond donor for the substrate (Lee et al., 1990). A first intepretation of our data appears to support the proposed ring-opening role for HislOl in E.coli. Firstly, where our substitutions at HislOl inactivate the enzyme, it appears that proximal changes are introduced at the active site, on the basis of altered fluorescence spectra. In crystal structures, this active site histidine is found at the base of a small single-turn interior helix, and is held in a specific tautomeric form by hydrogen bonding from a conserved aspartate residue (Asp 104 in E.coli) (Carrell et al., 1989). Substitution of this histidine may potentially relieve the helix configuration and transmit structural defects to the active site. If the inactivity were to be explained solely in terms of HislOl catalyzing intramolecular transfer of hydrogen during isomerization, substrate quenching should be observed, since either the ring or open-chain form of substrate, whichever induces the quench, would be free to accumulate under steadystate conditions prior to the isomerization reaction.
Secondly, the kinetic data on the Trp49 and Tip 188 substitutions can also be explained if the specific orientation of the substrate ring analogue, 5-thio-D-glucose, reported in the Arthrobacter enzyme, is assumed to represent xylose binding (Collyer et al., 1990). In this orientation, the anomeric hydroxyl of the a pyranose form of D-xylose projects towards the imidazole nitrogen of the tryptophan corresponding to Trp49 in the E.coli enzyme, which could additionally serve to stabilize the developing carbonyl during thering-openingreaction. Hence, although both tryptophans play an important role in efficient catalysis, presumably in stabilizing the transition state during ringopening, substitution of Trp49 inactivates the enzyme while residual activity remains for substitutions of Trpl88. A consequence of this orientation is that the residue corresponding to HislOl might catalyze the intramolecular transfer of a proton from the anomeric hydroxyl to the ring oxygen of the substrate during the ring-opening reaction. If this line of reasoning is to be followed, then the fluorescence characteristics of the H101S mutant protein observed in the presence of xylose (Figure 5) are best interpreted as an accumulation of the pyranose form of substrate, with the corollary that fluorescence quenching in the wild-type enzyme is due to the normal predomination of the open-chain form of the substrate under steady state conditions. In this case, HislOl would serve directly in mediating an intramolecular proton transfer during ring-opening. Although the open-chain is also observed to accumulate in Dxylose isomerase crystals under equilibrium conditions, the situation is no longer analogous to arabinose binding by ABP, where the pyranose form of the sugar clearly induces the quench. In conclusion we note that the fluorescent properties of the H10 IS mutant protein may also represent the formation of an adventitious binding mode in which isomerization proceeds by a different mechanism altogether. Thus, in the absence of further structural
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490> Emission Wavelength (nm)
A.C.Jamieson and C.A.Batt
data, mutant protein activity may be insufficient evidence to exclude this residue from a role in direct isomerization as has been previously suggested (Lee et ai, 1990). Acknowledgements We would like to thank H.L.Carrell and J.P.Glusker for providing the X-ray crystal coordinates of the S.rubiginosus enzyme and many useful discussions. This work was supported by the Cornell Biotechnology Program which is sponsored by the New York State Science and Technology foundation, a consortium of industries, the US Army Research Office and the National Science Foundation. In addition, support from New York Hatch Funds is also acknowledged.
References
258, 13665-13672. Quiocho.F.A. and Vyas.N.K. (1984) Nature, 310, 381-386. Rose,I.A., O'Connel.E.A. and Mortlock.R.P. (1969) Biochim. Biophvs. Ada, 178, 376-379. Vandeyar.M.A., Weiner,M.P., Hutton.C.J. and Batt.C.A. (1988) Gene, 65, 129-133. Whitlow,M., Howard,A.J.. Finzel.B.C. Poulos,T.L., Winbourne.E. and Gilliland.G.L. (1991) Proteins, 9, 153-173. Wovcha.M.G., Steuerwald.D.L. and Brooks,K.E. (1983) Appl. Environ. Microbiol, 45, 1402-1404. Yamanaka.K. (1968) Biochim. Biophys. Ada, 151, 670-680. Received on September 15, 1991; revised and accepted on January 15, 1992
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Batt.C.A., O'Neill.E., Novak,S.R., Ko,J. and Sinskey.A. (1986) Biotech. Progress, 2, 140-144. Batt.C.A., Jamieson.A.C. and Vandeyar.M.A. (1990) Proc. Nail. Acad. Sci. USA. 87, 618-622. Carrell.H.L., dusker,J.P., Burger.V., Manfre.F., Tritsch.D. and Bielmann.J.-F. (1989) Proc. Nail. Acad. Sci. USA, 86, 4440-4444. Chen,W.-P. (1980) Process Biochem., 15, 3 0 - 4 1 . Collyer.C.A., Henrick.K. and Blow,D.M. (1990)/ Mol Biol.. 212, 211 -235. Dische.Z. (1949)7. Biol. Chem., 166, 379-392. Farber,G.K., Glasfield.A., Tiraby.G., Ringe.D. and Petsko.G.A. (1989) Biochemistry, 28, 7289-7297. Feather.M.S., Deshpande.V. and Lybyer.M.J. (1970) Biochem. Biophys. Res. Commun., 38, 859-863. Hanson,K.R. and Rose,I.A. (1974) Accounts Chem. Res., 8, 1-10. Kersters-Hilderson.H., Callens,M., van Opstal.O., Vangrysperre,W. and DeBruyne.C.K. (1987) Enzyme Microb. Technol., 9, 145-148. Lee.C, Meng.M.. Bagdasarian,M. and Zeikus.J.G. (1990)7. Biol. Chem., 265. 19082-19090. MUler.D.M., OlsonJ.S., PflugrathJ.W. andQuiocho.F.A. (1983)7. Biol. Chem.,
Fluorescent properties of the Escherichia coli D-xylose isomerase active site
Andrew C.Jamieson and Carl A.Batt1 Institute of Food Science. Cornell University. Ithaca. NY 14853. USA 'To whom correspondence should be addressed
Introduction D-xylose isomerase (E.C. 5.3.1.5) is an intracellular microbial enzyme which catalyzes the reversible isomerization of xylose to xylulose. The enzyme will also accept glucose as a substrate, and is utilized industrially for the production of high fructose corn syrups (Chen, 1980). Recently, high resolution X-ray crystal structures have become available for enzyme derived from Streptomyces rubiginosus and an Arthrobacter species, leading to differing interpretations of the enzyme mechanism based on the proposed orientations of substrate in the active site (Carrell et al., 1989; Collyer et al., 1990). Some authors have proposed that the enzyme isomerization occurs as a metal cation mediated hydride shift (Farber et al., 1989; Collyer et al., 1990; Whitlow et al., 1991), rather than by the classical mechanism for aldose — ketose isomerases, which involves base catalyzed intramolecular proton transfer and a cis ene-diol intermediate (Hanson and Rose, 1974; Carrell etal, 1989). Site-directed mutagenesis experiments performed in enzymes from other species have focused primarily on the role of a conserved active site histidine residue, in attempting to discriminate between its functional role as a catalytic base in either substrate ring-opening, which precedes isomerization, and/or in the isomerization reaction itself (Batt et al., 1990; Lee et al., 1990). Another conserved feature of D-xylose isomerase revealed by X-ray crystallography is the presence of two tryptophan residues which sandwich both the pyranose and open-chain forms of bound substrate at the enzyme active site (Carrell et al., 1989; Collyer et al., 1990). The arrangement is reminiscent of the binding pocket of arabinose binding protein (ABP), a periplasmic protein involved in transport and chemotaxis in Escherichia coli, in which a tryptophan and a phenylalanine residue appear to mediate a © Oxford University Press
Materials and methods Strains and plasmids Escherichia coli JM109 [recA\ supEAA endAX hsdRXl gyrA96 relAl thi A(lac-proAB) F'(traD36proAB+ laclq ZAM15)] was used for the production of wild-type enzyme from a cloned xylA (D-xylose isomerase) construct, while HB101 (supEM hsdS20(r-B m~B) recA\3 ara-14 proAl lacYX galKl rpsLlO xyl-5 mtl-l) which is deficient for D-xylose isomerase activity, was used for the production of mutant proteins. Escherichia coli JM2r~ [hsdRM mcrAB A(lac-proAB) F'(traD36proAB+ laclq ZAM15)] was used as a recipient for site-directed mutagenesis. The plasmid pKK223, which carries a tac promoter and is regulated by the chromosomal lacP gene in E.coli JM109, was used as an expression vector. M13mpl9 was used for mutagenesis and nucleotide sequencing. DNA manipulations Restriction enzymes were purchased from New England Biolabs or US Biochemical Corporation and used according to the manufacturer's instuctions. Modifying enzymes utilized in sitedirected mutagenesis were purchased solely from US Biochemical Corporation. Subcloning of mutant xylA genes was facilitated by the observation that the overexpression of the xylA gene in trans in a Xyl+ background, represses the expression of the chromosomal operon. This phenomenon, can be screened using MacConkey's medium containing 2% xylose as the sole carbon source (Batt et al., 1986). Site-directed mutagenesis Mutagenesis was performed in M13mpl9 using synthetic oligodeoxynucleotide primers containing the desired substitutions. Selection of mutants was facilitated by enzymatic removal of the parental strand as described by Vandeyar et al. (1988). Enzyme purification Enzyme purification was initiated from cultures carrying the appropriate expression vector, induced with 2 mM isopropyl-/3D-thiogalactopyranoside (IPTG) for 3 h. Yields of up to 28% total soluble protein were routinely managed with this expression system. The purification involves a heat treatment of sonicated cell extracts for 10 min at 55°C in the presence of 70 mM MnCl2, and has been described elsewhere (Yamanaka, 1968; Batt et al., 1990). Final yields were between 50 and 100 mg enzyme per liter of culture. An estimate of protein concentration was made from a total amino acid analysis of the sample 235
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The consequences of active site mutations of the Escherichia coli D-xylose isomerase (E.C. 5.3.1.5) on substrate binding were examined by fluorescence spectroscopy. Site-directed mutagenesis of conserved tryptophan residues in the E.coli enzyme (Trp49 and Trpl88) reveals that fluorescence quenching of these residues occurs during the binding of xylose by the wild-type enzyme. The fluorescent properties of additional active site substitutions at HislOl were also examined. Substitutions of HislOl which inactivate the enzyme were shown to have altered spectral characteristics, which preclude detection of substrate binding. In the case of H101S, a mutant protein with measurable isomerizing activity, substrate binding with novel fluorescent properties was observed, possibly the bound pyranose form of xylose under steady-state conditions. Key words: D-xylose isomerase/fluorescence/site-directed mutagenesis
hydrophobic interaction with the aliphatic hydrogens of the bound arabinose sugar ring (Quiocho and Vyas, 1984). An additional feature of this protein -ligand interaction is revealed by fluorescence spectrometry, in which the fluorescence emission spectrum of ABP becomes quneched upon the addition of arabinose (Miller et al., 1983). In this paper we examine the fluorescent properties of the active site tryptophans in the E. coli D-xylose isomerase, and attempt to resolve the consequences of active site mutations on sugar binding.
A.C.Jamieson and C.A.Batt
Results Site-directed mutagenesis A number of alignments of the homologous amino acid sequences of D-xylose isomerases from various species are currently available. In all cases, only two out of the 16 tryptophan residues in the E.coli sequence (Trp49 and Trpl88) are conserved throughout. The homologues of these residues are found at the active site of D-xylose isomerase from both 5. rubiginosus and Arthrobacter as idenified by X-ray crystallography studies (Carrell etai, 1989; Collyer etal., 1990). The following mutations were introduced at these sites: W49A, W188A, WI88F
and the double mutation, W49AW188F. We also constructed substitutions at HislOl, H101S and H101Q, in addition to those previously described at this locus (H101R and H101Y) (Batt et al, 1990). The HislOl residue in E.coli corresponds to the active site residue identified in crystal structures as the catalytic base involved in either substrate ring-opening and/or in the isomerization reaction (see Introduction). Fluorescence spectrometry Figure 1 demonstrates that quenching of the E.coli D-xylose isomerase fluorescence emission spectrum occurs upon the addition of xylose. Quenching of the emission spectrum is shown in the presence of 50 mM xylose, with a slight blue-shift in peak emission from 337 to 335 nm. The quench is titratable with A"app = 3 mM, somewhat lower than the Km calculated by the coupled enzyme assay (10 mM). The blue-shift in the peakemission wavelength has been attributed to the hydrophobic interaction between the sugar and the imidazole ring in ABP (Miller et al., 1983), although a more likely interpretation is that it arises from the background fluorescence of the remaining tryptophans in the E.coli. The quench is not observed with the addition of the open-chain substrate analogue, xylitol, at concentrations up to 100 mM. A disparity arose, however, when we attempted to demonstrate directly that the ring form of the substrate was responsible for the quench, by analogy with ABP: neither glucose nor the ring analogue of glucose, 5-thio-D-glucose, was able to quench the fluorescence spectrum when added at concentrations up to 100 mM (Figure 2). The reason for this disparity may lie in the high Km that the E.coli enzyme exhibits towards glucose (0.5 M), as has previously been reported (Wovcha et al., 1983). Attempts to use concentrations of glucose > 100 mM yielded problems with the fluorescence spectrum due to the high viscosity of the solution. wild type + 50 mM xylose
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Emission Wavelength (nm) Kig. 1. Fluorescence spectrum of the E.ntli D-xylosc isomerase Quenching of the emission spectrum is shown in the presence of 50 mM xylose, with a slight blue-shift in peak emission from 337 to 335 nm. The sample buffer contained 10 mM triethanolanine ipH 7.2). 10 mM MnCU and I mM DTT. The enzyme subunit concentration was estimated at 5 /imol. Spectra were recorded at room temperature, with 290 nm excitation light.
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and an extinction coefficient calculated at 280 nm. Mutant proteins were purified by the same procedures. Enzyme assays Qualitative assays of D-xylose isomerizing activity were performed by the cysteine-carbazole method (Dische, 1949), using induced E.coli cells permeabilized by toluene. An HPLC assay was used for detecting isomerizing activity in D-xylose isomerase mutant proteins as previously described (Ban et al., 1990). Kinetic measurements were made using a coupled assay linked to sorbitol dehydrogenase (SDH) (Kersters-Hilderson et al., 1987). The reaction mixture contained 10 U SDH (Boehringer Mannheim), 10 mM MnCl2, 5 mM NADH (Sigma) and mutarotated D-xylose in 10 mM triethanolamine buffer (pH 7.2). Fluorescence spectroscopy Fluorescence spectra were recorded on an SLM model 8000 fluorescence spectrometer (SLM Instruments, Inc). Protein samples (5—lO^mol subunit concentration) were in 10 mM triethanolamine buffer (pH 7.2), 10 mM MnCl2 and 1 mM DTT. The emission spectra (295-500 nm) were recorded with 290 nm excitation light at room temperature.
E.coli xylose isomera.se
Fluorescence properties of D-xylose isomerase mutant proteins In order to demonstrate that substrate quenching involves the active site tryptophans, the fluorescence spectra of the W49A, W188A and W49AW188A mutant proteins were examined. Figure 3 is a set of comparative spectra showing the effects of substitution of Trp49 and Trpl88 on fluorescent light emission. The spectra are essentially additive and demonstrate that Trp49
and Tip 188 together account for - 3 0 % of the light emitted by the wild-type enzyme. Mutant protein spectra were all insensitive to the addition of substrate (50 mM xylose), demonstrating that both residues are required to exhibit the quenching effect. An unusual correlation was found for the W49A spectrum, which is almost superimposable with the quenched wild-type spectrum obtained in the presence of 50 mM xylose. The correlation
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+
/
50mMxyUto.\
0.000
1
1
Emission Wavelength (ran) Fig. 2. Fluorescence spectra of the E.coli D-xylose isomerase insensitive to quenching. The spectra were recorded in the absence of substrate, or in the presence of 5-thio-D-glucose (100 mM) or xylitol (50 mM) under the conditions described in the legend to Figure I.
B.000
wild type
400 Emission Wavelength (nm) Fig. 3. A comparison between the normalized emission spectra of the wild-type D-xylose isomerase and the W49A, W188A and W49AWI88A mutant proteins. All spectra were recorded under the standard conditions described in Materials and methods. Subunit concentrations were in the range 5-10/imol.
237
A.C.Jamieson and C.A.Batt
6.000
Relat ive Intensity
^ \ '—v
-
\ wild type
/
\
\ \ HIOIY\ \
\ /
0.000
1
Emission Wavelength (nm) Fig. 4. Fluorescence spectrum of the H101Y mutant protein compared with the wild-type enzyme. The spectra were recorded under standard conditions described in Materials and methods. The subunit concentration of the H101Y was estimated at 7 /unol prior to normalization.
implies that the single residue Trp49 is quenched in its entirety upon substrate binding, although this would be highly unusual. The fluorescent properties of substitutions at HislOl were examined to resolve the effects of active site mutations on substrate binding. Figure 4 shows what we expect to be the general case for substitution of HislOl in the E.coli D-xylose isomerase, as demonstrated by the H101Y mutant protein. The fluorescence spectrum of the mutant protein is partially quenched and blue-shifted with respect to the wild-type spectrum and is once again insensitive to the addition of substrate. We subsequently found the spectra derived from the H101Y, H101R and H101Q to be superimposable (data not shown). All three of these mutant proteins are also completely inactive (Table I; Batt et ai, 1990). Only in the case of the H101S mutant protein were we able to demonstrate an essentially unaltered fluorescence spectrum when compared with the wild-type enzyme in the absence of substrate. Addition of xylose to this mutant protein, however, resulted in an altered spectrum with novel properties. Substrate quenching does not occur in this example, but substrate can be detected with the rise of a new fluorescent peak at 335 nm as shown in Figure 5. The peak is titratable with a Km of 30 mM. This mutant protein is also distinct from all other HislOl substitutions we studied, in that it exhibits detectable isomerizing activity (Table I). Kinetic analyses A summary of the kinetic properties of the D-xylose isomerase and associated mutant proteins is presented in Table I. The substitution W49A inactivated the enzyme, whereas replacing Trpl88 with either alanine or phenylalanine resulted in reduction of £cal by two or three orders of magnitude. At this very low level of activity, Km measurements by the coupled spectrometric assay are only approximate. HislOl substitutions, with the exception of HI01S. completely 238
Table I. Kinetic parameters of D-xylose isomerase muiam proteins Substitution Wild-type W49A W188A W188F H101Q H101S
Km (mM xylose)
k,..,, (s active site )
10 NA 2O-3Ob 20b NA 70
5 NA 0.005 0.05 NA 0.05
K.app m (mM xylose)
3 ND ND ND ND 30
a
As measured by fluorescence titration. Estimated value. NA. no activity. ND. not determined.
b
inactivated the enzyme. The kc.d( for the H101S mutant protein was determined to be reduced by two orders of magnitude and the Km measured as 70 mM. In this case, as with the wild-type enzyme, the measured value is noticeably higher than the value obtained from titration of the fluorescence emission spectrum in the presence of substrate. Inactivation of the H101R and H101Y mutant proteins was reported previously (Batt et ai, 1990). Discussion Despite the apparent chemical simplicity of the isomerization reaction, three kinds of proton translocation are catalyzed by Dxylose isomerase. The first of these occurs during ring-opening of the pyranose form of the substrate, where only the or anomeric form of the sugar is accepted by the enzyme (Feather et al., 1970). During the isomerization reaction per se, proton movement which occurs between the hydroxyl and carbonyl groups is also accompanied by an additional intramolecular transfer of hydrogen between the Cl and C2 carbon centers. In
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/
E.coli xylose isomerase
Fig. 5. Fluorescence spectra of the partially active H101S mutant protein alone and after the addition of 50 mM xylose. Both spectra were recorded under standard conditions at 6 fimol subunit concentration.
D-xylose isomerase, the intramolecular transfer occurs completely without solvent exchange (Rose et al., 1969). X-ray crystal data on the S. rubiginosus and Arthrobacter D-xylose isomerases have led to differing interpretations of the role of the active site residue corresponding to His 101 in the E.coli enzyme. Carrell has proposed that this residue mediates the isomerization reaction by proton abstraction between carbon centers (Carrell et al., 1989), whereas Collyer has relegated the role of this residue to performing the ring-opening reaction, favoring a hydride shift for the isomerization, mediated by one of the two metal cations present in the active site (Collyer et al., 1990). Recently, this interpretation has been supported by sitedirected mutagenesis experiments in the Clostridium thermosulfogenes enzyme, on the basis that substitution of the residue does not render the enzyme completely inactive. This latter study further relegated the role of the histidine to serving solely as a hydrogen bond donor for the substrate (Lee et al., 1990). A first intepretation of our data appears to support the proposed ring-opening role for HislOl in E.coli. Firstly, where our substitutions at HislOl inactivate the enzyme, it appears that proximal changes are introduced at the active site, on the basis of altered fluorescence spectra. In crystal structures, this active site histidine is found at the base of a small single-turn interior helix, and is held in a specific tautomeric form by hydrogen bonding from a conserved aspartate residue (Asp 104 in E.coli) (Carrell et al., 1989). Substitution of this histidine may potentially relieve the helix configuration and transmit structural defects to the active site. If the inactivity were to be explained solely in terms of HislOl catalyzing intramolecular transfer of hydrogen during isomerization, substrate quenching should be observed, since either the ring or open-chain form of substrate, whichever induces the quench, would be free to accumulate under steadystate conditions prior to the isomerization reaction.
Secondly, the kinetic data on the Trp49 and Tip 188 substitutions can also be explained if the specific orientation of the substrate ring analogue, 5-thio-D-glucose, reported in the Arthrobacter enzyme, is assumed to represent xylose binding (Collyer et al., 1990). In this orientation, the anomeric hydroxyl of the a pyranose form of D-xylose projects towards the imidazole nitrogen of the tryptophan corresponding to Trp49 in the E.coli enzyme, which could additionally serve to stabilize the developing carbonyl during thering-openingreaction. Hence, although both tryptophans play an important role in efficient catalysis, presumably in stabilizing the transition state during ringopening, substitution of Trp49 inactivates the enzyme while residual activity remains for substitutions of Trpl88. A consequence of this orientation is that the residue corresponding to HislOl might catalyze the intramolecular transfer of a proton from the anomeric hydroxyl to the ring oxygen of the substrate during the ring-opening reaction. If this line of reasoning is to be followed, then the fluorescence characteristics of the H101S mutant protein observed in the presence of xylose (Figure 5) are best interpreted as an accumulation of the pyranose form of substrate, with the corollary that fluorescence quenching in the wild-type enzyme is due to the normal predomination of the open-chain form of the substrate under steady state conditions. In this case, HislOl would serve directly in mediating an intramolecular proton transfer during ring-opening. Although the open-chain is also observed to accumulate in Dxylose isomerase crystals under equilibrium conditions, the situation is no longer analogous to arabinose binding by ABP, where the pyranose form of the sugar clearly induces the quench. In conclusion we note that the fluorescent properties of the H10 IS mutant protein may also represent the formation of an adventitious binding mode in which isomerization proceeds by a different mechanism altogether. Thus, in the absence of further structural
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A.C.Jamieson and C.A.Batt
data, mutant protein activity may be insufficient evidence to exclude this residue from a role in direct isomerization as has been previously suggested (Lee et ai, 1990). Acknowledgements We would like to thank H.L.Carrell and J.P.Glusker for providing the X-ray crystal coordinates of the S.rubiginosus enzyme and many useful discussions. This work was supported by the Cornell Biotechnology Program which is sponsored by the New York State Science and Technology foundation, a consortium of industries, the US Army Research Office and the National Science Foundation. In addition, support from New York Hatch Funds is also acknowledged.
References
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Batt.C.A., O'Neill.E., Novak,S.R., Ko,J. and Sinskey.A. (1986) Biotech. Progress, 2, 140-144. Batt.C.A., Jamieson.A.C. and Vandeyar.M.A. (1990) Proc. Nail. Acad. Sci. USA. 87, 618-622. Carrell.H.L., dusker,J.P., Burger.V., Manfre.F., Tritsch.D. and Bielmann.J.-F. (1989) Proc. Nail. Acad. Sci. USA, 86, 4440-4444. Chen,W.-P. (1980) Process Biochem., 15, 3 0 - 4 1 . Collyer.C.A., Henrick.K. and Blow,D.M. (1990)/ Mol Biol.. 212, 211 -235. Dische.Z. (1949)7. Biol. Chem., 166, 379-392. Farber,G.K., Glasfield.A., Tiraby.G., Ringe.D. and Petsko.G.A. (1989) Biochemistry, 28, 7289-7297. Feather.M.S., Deshpande.V. and Lybyer.M.J. (1970) Biochem. Biophys. Res. Commun., 38, 859-863. Hanson,K.R. and Rose,I.A. (1974) Accounts Chem. Res., 8, 1-10. Kersters-Hilderson.H., Callens,M., van Opstal.O., Vangrysperre,W. and DeBruyne.C.K. (1987) Enzyme Microb. Technol., 9, 145-148. Lee.C, Meng.M.. Bagdasarian,M. and Zeikus.J.G. (1990)7. Biol. Chem., 265. 19082-19090. MUler.D.M., OlsonJ.S., PflugrathJ.W. andQuiocho.F.A. (1983)7. Biol. Chem.,
