Proc. Nail. Acad. Sci. USA Vol. 88, pp. 4015-4019, May 1991 Switching preference of thermophilic from D-xylose to D- by redesigning the substrate binding pocket (glucose isomerase/site-directed mutagenesis/ /protein engineering/catalytic efficiency) MENGHSIAO MENG*t, CHANYONG LEEt§, MICHAEL BAGDASARIAN*t, AND J. GREGORY ZEIKUS*t¶II *Michigan Biotechnology Institute, Lansing, MI 48909; tDepartment of Microbiology, Michigan State University, East Lansing, MI 48824; Department of Biochemistry, Michigan State University, East Lansing, MI 48824; and *Department of Pharmaceutical Chemistry and Biochemistry/Biophysics, University of California, San Francisco, CA 94143 Communicated by T. Kent Kirk, February 15, 1991 (receivedfor review December 12, 1990)

ABSTRACT The substrate specificity of thermophilic xy- (D-xylose ketol-isomerase; EC 5.3.1.5) lose isomerase from Clostridium thenmosulfurogenes was ex- converts D-xylose to D- during in amined by using predictions from the known crystal structure various microorganisms (12). This enzyme also catalyzes the oftheArthrobacter enzyme and site-directed mutagenesis ofthe conversion of D-glucose to D- in vitro and has been thermophilexylA gene. The orientation ofglucose as a substrate used as an industrial biocatalyst for production of high in the active site of the thermophilic enzyme was modeled to fructose (13). Xylose isomerase displays lower position the C-6 end of hexose toward His-101 in the substrate- kcat and higher Km values for glucose than those for xylose, binding pocket. The locations ofMet-87, Thr-89, Val-134, and and it requires different metal for enzyme on Glu-180, which contact the C-6 -OH group of the substrate these substrates (i.e., Mn2+ for xylose and Co2+ for glucose) in the sorbitol-bound xylose isomerase from Arthrobacter [Col- (14-16). lyer, C. A., Henrick, K. & Blow, D. M. (1990) J. Mol. Biol. The catalytic mechanism for xylose isomerase was orig- 212, 211-235], are equivalent to those of Trp-139, Thr-141, inally believed to involve -directed general Val-186, and Glu-232 in the thermophilic enzyme. Replace- catalysis (17). Currently, an alternative mechanism of catal- ment ofTrp-139 with Phe reduced the K. and enhanced the kt ysis has been proposed based on results of x-ray crystallo- of the mutant thermophilic enzyme toward glucose, whereas graphic studies on Arthrobacter or Streptomyces this substitution reversed the effect toward xylose. Replace- (18-21) and biochemical properties exhibited by thermophilic ment of Val-186 with Thr also enhanced the catalytic efficiency enzymes obtained by site-directed mutagenesis of the xylA of the enzyme toward glucose. Double mutants with replace- gene from Clostridium thermosulfurogenes (22). ments Trp-139 -* Phe/Val-186 - Thr and Trp-139 The enzymatic interconversion of to by Phe/Val-186 -* Ser had a higher catalytic efficiency (kCt/Kl,) xylose involves binding of the substrate in the for glucose than the wild-type enzyme of 5- and 2-fold, respec- ring form, substrate ring opening, isomerization of the linear tively. They also exhibited 1.5- and 3-fold higher catalytic intermediate, intermediate ring closure, and release of the efficiency for D-glucose than for D-xylose, respectively. These product. The isomerization step is proposed to proceed by a results provide evidence that alteration in substrate specificity metal -assisted hydride shift mechanism (18-22), and this of factitious thermophilic xylose isomerases can be achieved by step, rather than ring opening, is rate determining (22). designing reduced steric constraints and enhanced hydrogen- D-xylose and D-glucose have identical atomic configuration, bonding capacity for glucose in the substrate-binding pocket of except for the presence of an additional -CH2OH group at the active site. the C-6 position in the glucose molecule. This extra hy- droxymethyl group must therefore be responsible for the Specificity of enzymes toward their substrates is determined differences in the catalytic efficiency exhibited by xylose in part by molecular residues that provide for binding of the isomerase toward glucose versus xylose. substrate and that maintain substrate steric configuration in We have cloned and overexpressed a gene encoding the the active site. A variety of factors influence enzyme- thermophilic xylose isomerase of C. thermosulfurogenes in substrate complementarity and catalytic efficiency including the mesophilic host, Escherichia coli, which enables very steric fit, charge interactions, hydrogen bonding, and hydro- simple purification of preparative amounts of homogenous phobic interactions (1). Until recently, the main strategy to gene product (16). We have identified the active site histidine reveal and study the molecular basis of these factors was to residue of the enzyme and the rate-limiting step in the determine the tertiary structure of the enzyme-substrate isomerization reaction (22). The crystal structure of the complexes by x-ray crystallography. Redesigning proteins by Arthrobacter xylose isomerase has been determined at 2.3 A engineering of their genes is now a viable approach that resolution (18, 21). Several amino residues, revealed by complements structural studies and enables determination of this structure, could constitute potential steric hindrance for substitution effects on mutant enzyme function. the binding ofa six-carbon substrate to the active site pocket Thus, substrate specificity has been altered by redesigning of the enzyme. In this work we have substituted several key the structural frame of an enzyme (1-4), its electrostatic residues other than histidine (adjacent to the C-6 -OH group network (5-8), or its hydrophobic interaction with the sub- of glucose) in the active site of the thermophilic xylose strate (9). Catalytic function of an enzyme can also be isomerase. By the analysis of kinetic properties of the re- changed and regulated by modifications of the physical microenvironment of its catalytic site (10, 11). §Present address: Department of Biochemical Process Research and Development, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065. The publication costs of this article were defrayed in part by page charge 'To whom reprint requests should be addressed at: Michigan Bio- payment. This article must therefore be hereby marked "advertisement" technology Institute, 3900 Collins Road, P.O. Box 27609, Lansing, in accordance with 18 U.S.C. §1734 solely to indicate this fact. MI 48909. 4015 Downloaded by guest on September 24, 2021 4016 Biochemistry: Meng et al. Proc. Natl. Acad. Sci. USA 88 (1991) sulting mutant enzymes, we found indication of extensive bation ofthe reaction mixture at 650C for 15 min, 0.5 ml of0.5 similarities between the structure of the active domain of M perchloric acid was added, and the products were deter- xylose isomerase from Arthrobacter and that from the ther- mined by the cysteine/carbazole/sulfuric acid method (28). mophilic Clostridium. By changing some of the key amino Km and VmS, were determined from Lineweaver-Burk and in the substrate-binding pocket of the active site, we from Eadie-Hofstee plots. kat (i.e., turnover number per have changed substrate kinetic specificity constants of the active site of the enzyme) was determined from the equation thermophilic xylose isomerase enzyme. Notable, some ofthe kcat[Elo = Vmax, where [Elo = total enzyme concentration designed, or factitious, enzymes display significantly higher (29). catalytic efficiency toward glucose (the industrial substrate) Computer-Aided Molecular Modeling. Atomic coordinates than xylose (the natural substrate). for the structure of xylose isomerase from Arthrobacter strain B3728 at 2.3 A resolution were kindly provided by D. M. Blow (21). The structure of the active site containing MATERIALS AND METHODS the six-carbon substrate analogue sorbitol was visualized on Strains, Plasmids, and Chemicals. E. coli strain HB101 [F- the IRIS-4D25 computer (Silicon Graphics Computer Sys- hsdS20 ara-l recA13 proA12 lacYl galK2 rpsL20 mtl-l xyl-5] tem, Mountain View, CA) with the aid of the INSIGHT II (23) was used for expression of the C. thermosulfurogenes graphic program (Biosym Technologies, San Diego, CA). xylose isomerase gene present in the plasmid pCMG11-3 (22); E. coli strain TG1 [thi supE hisDS A(lac-proAB)/F' traD36 proA+B+ lacIq lacZAM15] in conjunction with bacteriophage RESULTS M13mp19 (24) was used for oligonucleotide-directed muta- Putative Structure of the Active Site. Assuming that con- genesis and nucleotide sequence determination as described servation of the primary sequence in the active site between (22). All chemicals were of reagent grade. xylose isomerases of different origins (Fig. 1) reflects simi- DNA Manipulation. Restriction and other larities in tertiary structure of the binding-site domain (Fig. enzymes for. DNA manipulation were from Bethesda Re- 2), the following residues in the thermophilic xylose search Laboratories or from New England Biolabs. The isomerase might constitute steric hindrance for effective oligonucleotide-directed mutagenesis was from Amer- binding of D-glucose: Trp-139, Thr-141, Val-186, and Glu- sham. The following oligonucleotides (obtained from Geno- 232. In the Arthrobacter enzyme, the structure ofthe enzyme sys, Woodlands, TX) were used for generation of site- complex with the six-carbon competitive inhibitor sorbitol directed mutants: 5'-ACGAAAGTYTTGNNNGGTACTGC- indicated that the C-6 hydroxymethyl group of the substrate GAAT-3', where NNN = TTT for Trp-139 -- Phe and TAT is oriented toward the bottom ofthe substrate-binding pocket for Trp-139 -* Tyr substitution; 5'-ACGAAAGTTT- and is adjacent to the residues Met-87, Thr-89, and Val-134. TGTGGGGTNNNGCGAATCTTTTCTCC-3', where NNN The distances to these atoms from the C-6 -OH group of = TCT for Thr-141 -) Ser substitution; 5'-GGCGAAAAC- sorbitol is ofthe order of3.4-3.7 A (21). The residues Thr-89, TAC NNlTTCTGGGGTGGA-3', where NNN = ACA for Val-134, and Glu-180 from the Arthrobacter enzyme, are Val-186--+ Thr, TCA for Val-186--* Ser, and GCA for Val-186 highly conserved among different sequences from divergent Ala substitution. Synthesis of mutant genes were per- origins and correspond to Thr-141, Val-186, and Glu-232, formed by the method of Sayers et al. (25), and their respectively, in the Clostridium enzyme. Met-87 is conserved nucleotide sequences were confirmed by the dideoxy chain aknong the enzymes of the Arthrobacter type, but it is termination method (26). The 1.4-kilobase EcoRI/BamHI replaced and conserved by Trp-139 in the Clostridium- and fragments containing the mutant genes were excised from the Bacillus-type isomerases (see Fig. 1). To prove that the M13mp19 double-stranded replicative form DNA, inserted residues discussed above are indeed part of the substrate- into the vector pMMB67EH (27), and introduced into E. coli binding pocket of the thermophilic Clostridium isomerase, strain HB101. we have replaced each of these residues with smaller amino Enzyme Purification and Assays. Wild-type and mutant acids. Catalytic properties of the mutant enzymes, resulting xylose isomerases, expressed by E. coli HB101, were puri- from the substitution of Trp-139, Thr-141, and Val-186, are fied as described previously through the DEAE-Sepharose described below. The role of Glu-232 will be the subject of a step, which gave enzymes homogeneous on SDS/PAGE (16). separate communication. For determination ofglucose isomerase activity, the reaction Properties of the Mutant Enzymes. Table 1 presents cata- mixtures (1.0 ml) contained 50 mM Mops (pH 7.0), 10 mM lytic constants of the site-directed mutants constructed to MgSO4, 1 mM CoCl2, D-glucose (Km = 0.3-2.0), and enzyme understand the relationship between specific amino acids in (3-5 ,g). For xylose isomerase activity, reaction mixtures the active-site pocket and enzyme-substrate interactions. (1.0 ml) contained, in the same buffer, 10 mM MnSO4, Replacement of Trp-139 with Phe, a smaller residue, pro- D-xylose (Km = 0.3-2.0), and enzyme (3-5 ,ug). After incu- duced an enzyme that had a higher catalytic efficiency for 85 131 179 A. r. VPMVTTNLFSHPVF ------ETFVMWGGREG ------LZPKPNEP -- A.m. VPNVTTNLFTHPVF ------KTLVLWGGREG ------IZPKPNEP -- S.v. VPKATTNLFTHPVF ------KTYVAWGGREG ------IZPKPNEP -- S. o. VPNATTNLFTHPVF ------KTYVAWGGREG ------IZPKPNQP -- S. g. VPNATTNLFTHPVF ------KTYVAWGGREG ------IZPKPNEP --

137 183 . 231 C.t. --- VLWGTANLFSNPRF ------ENYVFWGGREG ------IZPKPKEP -- B. s. --- LLWNTANMFTNPRF ------ENYVFWGGREG ------IZPKPKEP -- E.c. --- LLNGTANCFTNPRY ------ENYVLWGGREG ------IIPKPQEP --

FIG. 1. Alignment of amino acid sequences of the substrate-binding region from different xylose isomerases. Boldfaced letters indicate residues changed in this work. A.r., Arthrobacter strain B3726; A.m., Ampullariella strain 3876; S.v., Streptomyces violaceoniger; S.o., Streptomyces olivochromogenes; S.g., Streptomyces griseofuscus; C.t., C. thermosulfurogenes; B.s., Bacillus subtilis; E.c., E. coli (see ref. 22). Downloaded by guest on September 24, 2021 Biochemistry: Meng et al. Proc. Natl. Acad. Sci. USA 88 (1991) 4017

FIG. 2. Stereo analysis ofthe active-site region ofxylose isomerase from Arthrobacter strain B3728. The carbon backbone of sorbitol (green), as a glucose analog, was positioned to be perpendicular to the plane of the paper by positioning C-1 in front and C-6 in back, which is the bottom ofthe active-site pocket. The hydroxyl groups at C-2 and C4 are coordinated with a Mg2+ metal ion (red), which are surrounded by the negatively charged amino acid residues (blue). Van der Waals dot surfaces are shown for the atoms in the side chains of Met-87, Thr-89, Val-134, and Glu-180 (yellow), which are adjacent (within 3.9 A distance) to the C-6-OH atoms ofthe sorbitol (green). The active-site histidine that has been proposed to form a to the C-5-OH of the substrate during the reaction is shown in red (see refs. 20-22).

glucose than the wild-type enzyme (Fig. 3). This property This is consistent with the view that Thr-141 hydrogen bonds resulted from a decrease of the Km and an increase in kcat. It to the substrate but does not strictly hinder the binding of should be noted that substitutions at this position increased glucose. the Km for xylose and decreased the catalytic efficiency for this substrate under the assay conditions employed. Thus, DISCUSSION Trp-139 constitutes a steric hindrance for binding of the substrate with the larger molecule. We speculate that mutant This work is relevant to both the scientific understanding of isomerases with either Phe or Tyr at position 139 exhibit xylose (glucose) isomerase catalysis and to the applied use of lower catalytic efficiencies toward xylose because the bind- this enzyme. First, the mechanism of catalysis for xylose ing pocket is more spacious and this increases the freedom of isomerase has become controversial in view of the x-ray movement of the xylose molecule, decreasing its binding crystallographic data and recent interpretations that question efficiency. The enlargement of the binding pocket also de- the previously proposed biochemical reaction mechanisms creases binding energy between the enzyme and the transi- for this enzyme (19, 20). Our analysis of site-specific mutant tion state resulting in the decrease of kcat. xylose isomerases, performed in this work and reported Mutant enzymes in which Val-186 was replaced by Thr, a previously (22), when taken together with structural data polar residue, had a slightly lower Km and a higher kcat for obtained at higher resolution (21), provides additional insight glucose. Placement of a Ser residue, which has a smaller side into the xylose isomerase catalysis mechanism. Second, a chain but otherwise is equivalent to Thr, in this position did molecular strategy has been delineated to improve on the not improve the catalytic efficiency for glucose. Likewise, industrially important catalytic reaction of the enzyme (i.e., replacement with Ala did not significantly change either Km fructose manufacture from glucose)-namely, to design an in or kcat. Thus it would appear that Val-186 does not strictly vitro function via protein engineering based on understanding hinder glucose binding, but the effect of placing a Thr in this of the structure and the catalytic mechanism of the enzyme. position may be to improve the catalytic efficiency for Although the overall homology between the isomerase of glucose by providing additional hydrogen bonding, presum- Arthrobacter and that of Clostridium is only 26% (22), it was ably to the C-6 -OH group of the substrate. Replacement of possible to predict the probable location of several essential Thr-141 with Ser increased the Km for both xylose and amino acid residues in the Clostridium isomerase on the basis glucose and resulted in Lower catalytic efficiency for glucose. of sequence homology and the tertiary structure of the Table 1. Catalytic properties of wild-type and mutant enzymes obtained by substitution of amino acids in the active center of C. thermosulfurogenes xylose isomerase Glucose Xylose kcat/Km, kcat/Kmg Mutant enzyme changes Ki, mM kat min-1 min-I-mM-1 Km, mM kcat, min-1 min-'mM-1 wt 110 ± 7.6 640 ± 85 5.8 12 ± 2.2 1100 ± 106 97.2 Trp-139 --Phe 65 ± 7.4 970 ± 40 15 46 ± 1.1 620 ± 25 13.6 Trp-139 Tyr 91 ± 12 540 ± 25 6.0 110 ± 13.2 360 ± 25 3.2 Val-186 Thr 91 ± 7 880 ± 86 9.7 13 ± 1.7 740 ± 40 55.4 Val-186 Ser 140 ± 7 790 ± 15 5.7 49 ± 8.7 780 ± 96 15.9 Val-186 Ala 100 ± 11 540 ± 56 5.3 27 ± 0.7 1000 ± 15 36.4 Trp-139 Phe/Val-186 Thr* 29 ± 3.7 950 ± 106 32.9 36 ± 2.4 780 ± 66 21.6 Trp-139 Phe/Val-186 Ser* 58 ± 3.9 720 ± 20 12.4 63 ± 0.4 250 ± 5 4.0 Thr-141 Ser 160 ± 19 470 ± 31 2.9 68 ± 12 1500 ± 162 21.7 wt, wild type. *Double mutant. Downloaded by guest on September 24, 2021 4018 Biochemistry: Meng et al. Proc. Natl. Acad. Sci. USA 88 (1991) by the properties of the mutant enzymes with Val-186 -* Ser and, particularly, Val-186 -+ Ala substitutions. The results of crystallographic studies on xylose isomerases bound to different substrate analogs were inter- preted to point to two alternative orientations ofthe substrate in the active site of the enzyme (17, 20, 21). Results of this work provide functional evidence supporting the orientation in which the C-5 atom of glucose is located near the His-101 residue at the bottom ofthe active site pocket and the C-1 and i2 C-2 hydroxyls can form coordination bonds with the metal J ions. The results presented here do not give a comprehensive 0) picture of all molecular interactions between the substrate 0 and the enzyme in the of xylose isomerase. They merely provide indications that require further direct enzyme structural measurements on the thermophilic Clostridium xylose isomerase molecule. Biochemical analysis of addi- tional site-directed mutant xylose isomerases is required to understand the function of metal centers in the and stability. 0 0- amt LL> This work resulted from an equal contribution by the first two .> > > 2 authors. We thank D. M. Blow for providing structural coordinates ofArthrobacter isomerase before submission to the data bank, C. S. 0 0) Craik for reading the manuscript, R. J. Fletterick for the use of computer modeling equipment, J. H. McKerrow for the use of his laboratory facilities, and C. Bystroff for helpful discussions. This Enzymes work was supported by grants from the U.S. Department of Agri- FIG. 3. Diagram illustrating amino acid changes of substrate culture (89-01053 to Michigan Biotechnology Institute), Center for preference from xylose (Xyl) to glucose (Glc) associated with the Microbial Ecology, a National Science Foundation Science and amino acid substitutions in the substrate-binding pocket of xylose Technology Center at Michigan State University, the Research Excellence Fund from Michigan State University, and by isomerase. The ratios of catalytic efficiency (katt/Km) of enzymes National with xylose versus that with glucose, shown in Table 1, are expressed Science Foundation Grant BCS-8897179 (Dr. C. S. Craik as the in a logarithmic scale. The negative values shown by factitious principal investigator) for C.L. enzymes indicate more favored enzyme specificity toward glucose 1. Craik, C. S., Largman, C., Fletcher, T., Roczniak, S., Barr, than xylose, which is required of "true" glucose isomerase. Amino P. J., Fletterick, R. & Rutter, W. (1985) Science 228, 291-297. acids are indicated by the single-letter code. 2. Wilks, H. M., Hart, K. W., Feeney, R., Dunn, C. R., Muir- head, H., Chia, W. N., Barstow, D. A., Atkinson, T., Clarke, Arthrobacter isomerase resolved by x-ray diffraction (18, 20, A. R. & Holbrook, J. J. (1988) Science 242, 1541-1544. 21). Thus, in a previous report, the function of His-101 was 3. Bone, R., Silen, J. L. & Agard, D. A. (1989) Nature (London) elucidated and found in agreement with the structural data 339, 191-195. (22). In the present work good evidence was obtained that 4. Scrutton, N. S., Berry, A. & Perham, R. N. (1990) Nature Thr-141, Trp-139, and Val-186 are part of the substrate- (London) 343, 3843. 5. Wilkinson, A. J., Fersht, A. R., Blow, D. M., Carter, P. & binding site and that they play roles in substrate binding and Winter, G. (1984) Nature (London) 307, 187-188. in delineating the borders of the active-site pocket, based on 6. Wells, J. A., Powers, D. B., Bott, R. R., Graycar, T. P. & a functional test (i.e., the change of kinetic properties of the Estell, D. A. (1987) Proc. Natl. Acad. Sci. USA 84, 1219-1223. mutant enzymes resulting from substitution of these amino 7. Dean, A. D. & Koshland, Jr., D. E. (1990) Science 249, 1044- acids). 1046. We do not know at present whether the enzymatic - 8. Evnin, L. B., Vdsquez, J. R. & Craik, C. S. (1990) Proc. Natl. ization of xylose and glucose proceeds via the formation of a Acad. Sci. USA 87, 6659-6663. 9. D. T. D. Michaelis-type enzyme-substrate re- Estell, A., Graycar, P., Miller, J. V., Powers, B., complex. In fact, the Burnier, J. P., Ng, P. G. & Wells, J. A. (1986) Science 233, sults of crystallographic studies of the enzyme bound to 659-663. D-xylose indicated that a non-Michaelis-type complex is 10. Hurley, J. H., Dean, A. M., Sohl, J. L., Koshland, Jr., D. E. formed (20). However, a lower apparent Km of the mutant & Stroud, R. M. (1990) Science 249, 1012-1016. enzymes in which Trp-139 was replaced by smaller residues 11. Higaki, J. N., Haymore, B. L., Chen, S., Fletterick, R. & indicates an increased affinity of the enzyme for glucose or Craik, C. S. (1990) Biochemistry 29, 8582-8586. for its reaction intermediates. We interpret it, therefore, as a 12. Chen, W. (1980) Process Biochem. 15, 30-35. result of the increased volume of the substrate-binding 13. Antrim, R. L., Colliala, W. & Schnyder, B. (1979) in Applied Biochemistry and Bioengineering, ed. Wangard, E. B. (Aca- pocket, which now accommodates more readily the extra demic, New York), pp. 97-155. hydroxymethyl group of the glucose molecule. The observed 14. Danno, G. (1970) Agric. Biol. Chem. 34, 1805-1814. changes in kcat are more difficult to explain on the basis of the 15. Danno, G. (1971) Agric. Biol. Chem. 35, 997-1006. available data. We can conclude that the stabilization of the 16. Lee, C. & Zeikus, J. G. (1991) Biochem. J. 273, 565-571. transition state has changed in the mutant enzymes as a result 17. Carrell, H. L., Glusker, J. P., Burger, V., Manfre, F., Tritsch, of structural changes in the binding site. Although it is not D. & Biellmann, J.-F. (1989) Proc. Natl. Acad. Sci. USA 86, surprising that the kcat has changed in these mutants, the 4440-4444. molecular basis of these changes must await further struc- 18. Henrick, K., Collyer, C. A. & Blow, D. M. (1989) J. Mol. Biol. tural studies. 208, 129-157. 19. Farber, G. K., Glasfeld, A., Tiraby, G., Ringe, D. & Petsko, The Val-186 residue does not seem to hinder substrate G. A. (1989) Biochemistry 28, 7289-7297. binding in the Clostridium xylose isomerase. The increased 20. Collyer, C. A. & Blow, D. M. (1990) Proc. NatI. Acad. Sci. affinity for glucose upon replacement of Val-186 with Thr USA 87, 1362-1366. seems to result from the ability of this residue to provide an 21. Collyer, C. A., Henrick, K. & Blow, D. M. (1990) J. Mol. Biol. additional hydrogen bond to the substrate. This is indicated 212, 211-235. Downloaded by guest on September 24, 2021 Biochemistry: Meng et al. Proc. Nadl. Acad. Sci. USA 88 (1991) 4019

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