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Switching Substrate Preference of Thermophilic Xylose Isomerase From Proc. Nail. Acad. Sci. USA Vol. 88, pp. 4015-4019, May 1991 Biochemistry Switching substrate preference of thermophilic xylose isomerase from D-xylose to D-glucose by redesigning the substrate binding pocket (glucose isomerase/site-directed mutagenesis/enzyme active site/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- Xylose isomerase (D-xylose ketol-isomerase; EC 5.3.1.5) lose isomerase from Clostridium thenmosulfurogenes was ex- converts D-xylose to D-xylulose during xylose metabolism 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-fructose 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 corn syrup (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 ions for enzyme catalysis 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 histidine-directed general base 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 enzymes 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 aldose to ketose by Phe/Val-186 -* Ser had a higher catalytic efficiency (kCt/Kl,) xylose isomerases 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 ion-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 acid residues, revealed by complements structural studies and enables determination of this structure, could constitute potential steric hindrance for amino acid 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 acids 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 endonucleases 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 kit 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
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