Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans

An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 °C and withstanding incubation at temperatures up to 70 °C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 Å. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those factors most important for maintaining the structure and function of the enzyme.     

Rationally selected single-site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates

Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via site-directed mutagenesis. Using a rational mutagenesis approach based on structural bioinformatics and evolutionary analysis, two positions (F16S and Y95F) were identified as priority sites for mutagenesis. The mutant and parent enzymes were expressed and secreted from Pichia pastoris and the single site mutants F16S and Y95F showed 1.7- and 4.0-fold increases in k(cat) and 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, respectively, while maintaining thermostability. Similar to the parent enzyme, the two variants were active between pH 4.0 and 8.0 and showed optimal activity at temperature 70°C at pH 5.0. The purified enzymes were active at 50°C for over 12 h and retained at least 80% of initial activity for 2 h at 70°C. In contrast to the improved hydrolysis seen with the single mutation enzymes, no improvement was observed with a third variant carrying a combination of both mutations, which instead showed a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.     

Manipulation of the active site loops of D-hydantoinase, a (beta/alpha)8-barrel protein, for modulation of the substrate specificity

We previously proposed that the stereochemistry gate loops (SGLs) constituting the substrate binding pocket of D-hydantoinase, a (beta/alpha)(8)-barrel enzyme, might be major structural determinants of the substrate specificity [Cheon, Y. H., et al. (2002) Biochemistry 41, 9410-9417]. To construct a mutant D-hydantoinase with favorable substrate specificity for the synthesis of commercially important non-natural amino acids, the SGL loops of the enzyme were rationally manipulated on the basis of the structural analysis and sequence alignment of three hydantoinases with distinct substrate specificities. In the SGLs of D-hydantoinase from Bacillus stearothermophilus SD1, mutations of hydrophobic and bulky residues Met 63, Leu 65, Phe 152, and Phe 159, which interact with the exocyclic substituent of the substrate, induced remarkable changes in the substrate specificities. In particular, the substrate specificity of mutant F159A toward aromatic substrate hydroxyphenylhydantoin (HPH) was enhanced by approximately 200-fold compared with that of the wild-type enzyme. Saturation mutagenesis at position 159 revealed that k(cat) for aromatic substrates increased gradually as the size of the amino acid side chain decreased, and this seems to be due to reduced steric hindrance between the bulky exocyclic group of the substrate and the amino acid side chains. When site-directed random mutagenesis of residues 63 and 65 was conducted with the wild type and mutant F159A, the selected enzymes (M63F/L65V and L65F/F159A) exhibited approximately 10-fold higher k(cat) values for HPH than the wild-type counterpart, which is likely to result from reorganization of the active site for efficient turnover. These results indicate that the amino acid residues of SGLs forming the substrate binding pocket are critical for the substrate specificity of D-hydantoinase, and the results also imply that substrate specificities of cyclic amidohydrolase family enzymes can be modulated by rational design of these SGLs.     

Engineered amadoriase II exhibiting expanded substrate range

Amadori compounds are ubiquitous in vivo as well as in food and have been implicated in diabetic complications and aging. In recent years, fructosyl amine oxidases (FAOXs) which cleave Amadori products are gaining increasing attention. Until now, however, all FAOXs can only react with small glycated substrates (such as fructosyl amino acids or dipeptides), which has hindered the applications of this new class of enzymes in diagnosis, therapeutics, and detergents. In this study, Aspergillus fumigatus amadoriase II was engineered with the aim to expand its substrate range, using a heat-inducible autolytic vector and fructosyl–polylysine (3–13 lysines) as an intermediate-sized model substrate. After two rounds of directed evolution, a mutant (SII-82) was obtained that showed an 8.78-fold increase in the activity toward fructosyl–polylysine and which also performed several fold better than the wild-type on real gravy stains at concentrations of 10–100 μg/ml (parts per million). Mutational analyses revealed useful clues for altering the substrate-binding pocket. This study suggests that it is possible to manipulate fructosyl amine oxidases to accommodate larger substrates, and that mutant SII-82 might serve as a template for further engineering.     

Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8

Glycoside hydrolase family 77 (GH77) belongs to the alpha-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-alpha-glucanotransferases, involved in maltose metabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltase from the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesis of the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transition state stabilizer) shows that GH77 Tt AMase and GH13 enzymes share the same catalytic machinery. Quantification of the enzyme's transglycosylation and hydrolytic activities revealed that Tt AMase is among the most efficient 4-alpha-glucanotransferases in the alpha-amylase superfamily. The active site contains at least seven substrate binding sites, subsites -2 and +3 favoring substrate binding and subsites -3 and +2 not, in contrast to several GH13 enzymes in which subsite +2 contributes to oligosaccharide binding. A model of a maltoheptaose (G7) substrate bound to the enzyme was used to probe the details of the interactions of the substrate with the protein at acceptor subsites +2 and +3 by site-directed mutagenesis. Substitution of the fully conserved Asp249 with a Ser in subsite +2 reduced the activity 23-fold (for G7 as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of alpha-amylases only up to 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13 amylases and GH77 amylomaltases.     

Enhancement of thermostability of fungal deglycating enzymes by directed evolution

Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50°C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50°C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.     

Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates

The substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S and S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (S-KHPB). These mutations improve the value of k(cat)/K(M)(S-KHPB) by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of k(cat)(S-KHPB) for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximal value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate, a nonfunctionalized hydrophobic analogue. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate aldolases. Furthermore, targeting mutations to the active site provides a marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to those of naturally occurring enzymes.     

First crystal structure of an endo-inulinase,INU2, from Aspergillus ficuum: Discovery of an extra-pocket in the catalytic domain responsible for its endo-activity

Endo-inulinase is a member of glycosidase hydrolase family 32 (GH32) degrading fructans of the inulin type with an endo-cleavage mode and is an important class of industrial enzyme. In the present study, we report the first crystal structure of an endo-inulinase, INU2, from Aspergillus ficuum at 1.5 Å. It was solved by molecular replacement with the structure of exo-inulinase as search model. The 3D structure presents a bimodular arrangement common to other GH32 enzymes: a N-terminal 5-fold β-propeller catalytic domain with four β-sheets and a C-terminal β-sandwich domain organized in two β-sheets with five β-strands. The structural analysis and comparison with other GH32 enzymes reveal the presence of an extra pocket in the INU2 catalytic site, formed by two loops and the conserved motif W-M(I)-N-D(E)-P-N-G. This cavity would explain the endo-activity of the enzyme, the critical role of Trp40 and particularly the cleavage at the third unit of the inulin(-like) substrates. Crystal structure at 2.1 Å of INU2 complexed with fructosyl molecules, experimental digestion data and molecular modelling studies support these hypotheses.     

Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (k_cat/K_m) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.     

Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta-fucosidase from beta-galactosidase

Protein engineers use a variety of mutagenic strategies to adapt enzymes to novel substrates. Directed evolution techniques (random mutagenesis and high-throughput screening) offer a systematic approach to the management of protein complexity. This sub-discipline was galvanized by the invention of DNA shuffling, a procedure that randomly recombines point mutations in vitro. In one influential study, Escherichia coli beta-galactosidase (BGAL) variants with enhanced beta-fucosidase activity (tenfold increase in k(cat)/K(M) in reactions with the novel para-nitrophenyl-beta-d-fucopyranoside substrate; 39-fold decrease in reactivity with the "native"para-nitrophenyl-beta-d-galactopyranoside substrate) were evolved in seven rounds of DNA shuffling and screening. Here, we show that a single round of site-saturation mutagenesis and screening enabled the identification of beta-fucosidases that are significantly more active (180-fold increase in k(cat)/K(M) in reactions with the novel substrate) and specific (700,000-fold inversion of specificity) than the best variants in the previous study. Site-saturation mutagenesis thus proved faster, less resource-intensive and more effective than DNA shuffling for this particular evolutionary pathway.     

Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates

Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals.     

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase

Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.     

Changing the substrate specificity of creatine kinase from creatine to glycocyamine: evidence for a highly evolved active site

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.     

Directed Evolution of the Actinomycete Cytochrome P450 MoxA (CYP105) for Enhanced Activity

Actinomycete cytochrome P450 from Nonomuraea recticatena NBRC 14525 (P450moxA) catalyzes the hydroxylation of a broad range of substrates, including fatty acids, steroids, and various aromatic compounds. Hence, the enzyme is potentially useful in medicinal applications, but the activity is insufficient for practical use. Here we applied directed evolution to enhance the activity. A random mutagenesis library was screened using 7-ethoxycoumarin as a substrate to retrieve 17 variants showing >2-fold activities. Twenty-five amino acid substitutions were found in the variants, of which five mutations were identified to have the largest effects (Q87W, T115A, H132L, R191W, and G294D). These mutations additively increased the activity; the quintet mutant had 20-times the activity of the wildtype. These five single mutations also increased in activity toward structurally distinct substrates (diclofenac and naringenin). Based on the three-dimensional structure of the enzyme, we discerned that mutations in the substrate recognition site improved the activity, which was substrate dependent; mutations apart from the active site improved the activity as well as the substrates did.     

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.     

Crystal structure of the deglycating enzyme fructosamine oxidase (amadoriase II)

Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6A resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12A deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.     

Directed evolution of a thermostable l-aminoacylase biocatalyst

Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution strategies to identify improved variants. Mutants P237S and F251Y were 2.4-fold more active towards N-benzoyl valine relative to the wild type at 65 °C. F251 mutations to basic residues resulted in 4.5-11-fold shifts in the substrate preference towards N-benzoyl phenylalanine relative to N-benzoyl valine. The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of k_cat values for N-benzoyl valine and N-benzoyl phenylalanine. Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance, whereas those improving activity towards N-benzoyl-valine occurred in the hinge region loops linking the dimerization and zinc-binding domains in each monomer. These observations support a previously proposed substrate induced conformational transition between open and closed forms of aminoacylases.     

Isoleucine 69 and valine 325 form a specificity pocket in human muscle creatine kinase

Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by ATP. From a structural perspective, the enzyme utilizes two flexible loop regions to sequester and position the substrates for catalysis. There has been debate over the specific roles of the flexible loops in substrate specificity and catalysis in CK and other related phosphagen kinases. In CK, two hydrophobic loop residues, I69 and V325, make contacts with the N-methyl group of creatine. In this study, we report the alteration of the substrate specificity of CK through the mutagenesis of V325. The V325 to glutamate mutation results in a more than 100-fold preference for glycocyamine, while mutation of V325 to alanine results in a slight preference of the enzyme for cyclocreatine (1-carboxymethyl-2-iminoimidazolidine). This study enhances our understanding of how the active sites of phosphagen kinases have evolved to recognize their respective substrates and catalyze their reactions.     

Changes in the Catalytic Properties of Pyrococcus furiosus Thermostable Amylase by Mutagenesis of the Substrate Binding Sites

Pyrococcus furiosus thermostable amylase (TA) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of four residues (His414, Gly415, Met439, and Asp440) in the function of P. furiosus TA by using site-directed mutagenesis and kinetic analysis. A variant form of P. furiosus TA containing two mutations (H414N and G415E) exhibited strongly enhanced α-(1,4)-transglycosylation activity, resulting in the production of a series of maltooligosaccharides that were longer than the initial substrates. In contrast, the variant enzymes with single mutations (H414N or G415E) showed a substrate preference similar to that of the wild-type enzyme. Other mutations (M439W and D440H) reversed the substrate preference of P. furiosus TA from CDs to maltooligosaccharides. Relative substrate preferences for maltoheptaose over β-CD, calculated by comparing k_cat/K_m ratios, of 1, 8, and 26 for wild-type P. furiosus TA, P. furiosus TA with D440H, and P. furiosus TA with M439W and D440H, respectively, were found. Our results suggest that His414, Gly415, Met439, and Asp440 play important roles in substrate recognition and transglycosylation. Therefore, this study provides information useful in engineering glycoside hydrolase family 13 enzymes.     

Mechanism of the family 1 beta-glucosidase from Streptomyces sp: catalytic residues and kinetic studies

The Streptomyces sp. beta-glucosidase (Bgl3) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl beta-D-glycosides revealed that the highest k(cat)/K(M) values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bgl3 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl beta-glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slope of beta(lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 beta-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the beta-glucosyl azide adduct (by (1)H NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K(M) on azide concentration. On the other hand, the E383A mutant with the same substrate gave the alpha-glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bgl3 beta-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.