Switching substrate preference of thermophilic xylose isomerase from D-xylose to D-glucose by redesigning the substrate binding pocket.

Abstract

The substrate specificity of thermophilic xylose isomerase from Clostridium thenmosulfurogenes was examined by using predictions from the known crystal structure of the Arthrobacter enzyme and site-directed mutagenesis of the thermophilexylA gene. The orientation ofglucose as a substrate in the active site of the thermophilic enzyme was modeled to position the C-6 end of hexose toward His-101 in the substratebinding pocket. The locations of which (kCt/Kl,) for glucose than the wild-type enzyme of 5-and 2-fold, respectively. They also exhibited 1.5-and 3-fold higher catalytic efficiency for D-glucose than for D-xylose, respectively. These results provide evidence that alteration in substrate specificity of factitious thermophilic xylose isomerases can be achieved by designing reduced steric constraints and enhanced hydrogenbonding capacity for glucose in the substrate-binding pocket of the active site.Specificity of enzymes toward their substrates is determined in part by molecular residues that provide for binding of the substrate and that maintain substrate steric configuration in the active site. A variety of factors influence enzymesubstrate complementarity and catalytic efficiency including steric fit, charge interactions, hydrogen bonding, and hydrophobic interactions (1). Until recently, the main strategy to reveal and study the molecular basis of these factors was to determine the tertiary structure of the enzyme-substrate complexes by x-ray crystallography. Redesigning proteins by engineering of their genes is now a viable approach that complements structural studies and enables determination of amino acid substitution effects on mutant enzyme function. Thus, substrate specificity has been altered by redesigning the structural frame of an enzyme (1-4), its electrostatic network (5-8), or its hydrophobic interaction with the substrate (9). Catalytic function of an enzyme can also be changed and regulated by modifications of the physical microenvironment of its catalytic site (10, 11).Xylose isomerase (D-xylose ketol-isomerase; EC 5.3.1.5) converts D-xylose to D-xylulose during xylose metabolism in various microorganisms (12). This enzyme also catalyzes the conversion of D-glucose to D-fructose in vitro and has been used as an industrial biocatalyst for production of high fructose corn syrup (13). Xylose isomerase displays lower kcat and higher Km values for glucose than those for xylose, and it requires different metal ions for enzyme catalysis on these substrates (i.e., Mn2+ for xylose and Co2+ for glucose) (14-16).The catalytic mechanism for xylose isomerase was originally believed to involve histidine-directed general base catalysis (17). Currently, an alternative mechanism of catalysis has been proposed based on results of x-ray crystallographic studies on Arthrobacter or Streptomyces enzymes (18-21) and biochemical properties exhibited by thermophilic enzymes obtained by site-directed mutagenesis of the xylA gene from Clostridium thermosulfurogenes (22).The enzymatic interconversion of...

Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone

“…The importance of hydrogen bonding for protein function has been shown in many cases. For example, in phenol 2-monooxygenase from Trichosporon cutaneum, Y289 was reported to play an important role in leading to ortho attack of the substrate by forming a hydrogen bond with phenol substrate (23,44), and the thermophilic xylose isomerase from Clostridium thermosulfurogenes increased the k cat for glucose by 38% with an additional hydrogen bond to the C 6 -OH group of the substrate upon the mutation V186T (22). In addition, disruption of hydrogen bonds may cause the important amino acids in the catalytic site to lose their catalytic activity (2,19).…”

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

“…Despite their optimal activity at elevated temperatures (95 to 100°C) and their attractive, high catalytic efficiency at 90°C, the T. maritima and T. neapolitana xylose isomerase are active only at neutral pH and are only marginally active (10 to 15%) between 60 and 70°C (350). Based on structural information, Meng et al (245) have mutagenized the Thermoanaerobacterium thermosulfurigenes xylose isomerase and increased its catalytic efficiency on glucose (Table 11). Introducing the same substitutions in the T. neapolitana xylose isomerase also significantly increased this enzyme's catalytic efficiency on glucose (Table 11).…”

Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

“…Beyond the assembly of xylose catabolic pathways, xylose isomerase is an important enzyme for the food industry, especially in the production of high-fructose corn syrup. For these applications, xylose isomerase has been extensively studied (5) to improve the thermal stability (30,42), pH optimum (23), and substrate preference (31). However, these studies were mainly focused on obtaining a xylose isomerase that (i) has an optimum temperature and a pH range (60 to 80°C and pH 7.0 to 9.0, respectively) (44) different from those of conventional ethanol fermentation, (ii) is expressed in Escherichia coli rather than S. cerevisiae (2), and (iii) is found to be unsuccessfully expressed (40) or to be inactive at mesophilic temperature (46) in S. cerevisiae, mainly due to protein misfolding (12).…”