Protein engineering of CYP105s for their industrial uses

Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D₃ 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.     

Improvement of oxidative and thermostability of N-carbamyl-d-amino Acid amidohydrolase by directed evolution

N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), which is currently employed in the industrial production of unnatural D-amino acid in conjunction with D-hydantoinase, has low oxidative and thermostability. We attempted the simultaneous improvement of the oxidative and thermostability of N-carbamoylase from Agrobacterium tumefaciens NRRL B11291 by directed evolution using DNA shuffling. In a second generation of evolution, the best mutant 2S3 with improved oxidative and thermostability was selected, purified and characterized. The temperature at which 50% of the initial activity remains after incubation for 30 min was 73 degrees C for 2S3, whereas it was 61 degrees C for wild-type enzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxide for 30 min at 25 degrees C resulted in a complete loss of activity, but 2S3 retained about 79% of the initial activity under the same conditions. The K(m) value of 2S3 was estimated to be similar to that of wild-type enzyme; however k(cat) was decreased, leading to a slightly reduced value of k(cat)/K(m), compared with wild-type enzyme. DNA sequence analysis revealed that six amino acid residues were changed in 2S3 and substitutions included Q23L, V40A, H58Y, G75S, M184L and T262A. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Q23L, H58Y, M184L and T262A were found to enhance both oxidative and thermostability of the enzyme and of them, T262A showed the most significant effect. V40A and G75S gave rise to an increase only in oxidative stability. The positions of the mutated amino acid residues were identified in the structure of N-carbamoylase from Agrobacterium sp. KNK 712 and structural analysis of the stabilizing effects of each amino acid substitution was also carried out.     

Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance

The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75 degrees C, which was 15 degrees C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9 degrees C. The half-life of ThMA-DM was 172 min at 80 degrees C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.     

Directed Evolution of Thermus Maltogenic Amylase toward Enhanced Thermal Resistance

The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.     

Directed evolution of <Emphasis Type="Italic">Streptomyces lividans</Emphasis> xylanase B toward enhanced thermal and alkaline pH stability

The thermal and alkaline pH stability of Streptomyces lividans xylanase B was improved greatly by random mutagenesis using DNA shuffling. Positive clones with improved thermal stability in an alkaline buffer were screened on a solid agar plate containing RBB-xylan (blue). Three rounds of directed evolution resulted in the best mutant enzyme 3SlxB6 with a significantly improved stability. The recombinant enzyme exhibited significant thermostability at 70°C for 360 min, while the wild-type lost 50% of its activity after only 3 min. In addition, mutant enzyme 3SlxB6 shows increased stability to treatment with pH 9.0 alkaline buffer. The K _m value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however k _cat was slightly decreased, leading to a slightly reduced value of k _cat/K _m, compared with wild-type enzyme. DNA sequence analysis revealed that eight amino acid residues were changed in 3SlxB6 and substitutions included V3A, T6S, S23A, Q24P, M31L, S33P, G65A, and N93S. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Our results suggest that DNA shuffling is an effective approach for simultaneous improvement of thermal and alkaline pH stability of Streptomyces lividans xylanase B even without structural information.     

Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain

The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.     

Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase from Bacillus megaterium IWG3 on Stabilization of Its Oligomeric State

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-Å resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.     

Construction of a catalytically inactive cholesterol oxidase mutant: investigation of the interplay between active site-residues glutamate 361 and histidine 447

Cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-5-en-3-one and its subsequent isomerization into cholest-4-en-3-one. Two active-site residues, His447 and Glu361, are important for catalyzing the oxidation and isomerization reactions, respectively. Double-mutants were constructed to test the interplay between these residues in catalysis. We observed that the k(cat) of oxidation for the H447Q/E361Q mutant was 3-fold less than that for H447Q and that the k(cat) of oxidation for the H447E/E361Q mutant was 10-fold slower than that for H447E. Because both doubles-mutants do not have a carboxylate at position 361, they do not catalyze isomerization of the reaction intermediate cholest-5-en-3-one to cholest-4-en-3-one. These results suggest that Glu361 can compensate for the loss of histidine at position 447 by acting as a general base catalyst for oxidation of cholesterol. Importantly, the construction of the double-mutant H447E/E361Q yields an enzyme that is 31,000-fold slower than wild type in k(cat) for oxidation. The H447E/E361Q mutant is folded like native enzyme and still associates with model membranes. Thus, this mutant may be used to study the effects of membrane binding in the absence of catalytic activity. It is demonstrated that in assays with caveolae membrane fractions, the wild-type enzyme uncouples platelet-derived growth factor receptor beta (PDGFRbeta) autophosphorylation from tyrosine phosphorylation of neighboring proteins, and the H447E/E361Q mutant does not. Thus maintenance of membrane structure by cholesterol is important for PDGFRbeta-mediated signaling. The cholesterol oxidase mutant probe described will be generally useful for investigating the role of membrane structure in signal transduction pathways in addition to the PDGFRbeta-dependent pathway tested.     

Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues

We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.     

Directed evolution of N-carbamyl-D-amino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability

Directed evolution of N-carbamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens NRRL B11291 was attempted in order to simultaneously improve oxidative and thermal stability. A mutant library was generated by DNA shuffling, and positive clones with improved oxidative and thermal stability were screened on the basis of the activity staining method on a solid agar plate containing pH indicator (phenol red) and substrate (N-carbamyl-D-p-hydroxyphenylglycine). Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly improved stability. Oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than that of the wild type, and it also showed an 8-fold increased thermostability. The K(m) value of 2S3 was comparable to that of wild-type enzyme, but k(cat) was slightly decreased. DNA sequence analysis revealed that six amino acid residues (Q23L, V40A, H58Y, G75S, M184L, and T262A) were substituted in 2S3. From the mutational analysis, four mutations (Q23L, H58Y, M184L, and T262A) were found to lead to an improvement of both oxidative and thermal stability. Of them, T262A had the most significant effect, and V40A and G75S only increased the oxidative stability.     

Cooperative effect of two surface amino acid mutations (Q252L and E170K) in glucose dehydrogenase from Bacillus megaterium IWG3 on stabilization of its oligomeric state

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-angstroms resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.     

Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.     

Catalytic cysteine of thymidylate synthase is activated upon substrate binding

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.     

Interconversion of E and S isoenzymes of horse liver alcohol dehydrogenase. Several residues contribute indirectly to catalysis.

The E and S isoenzymes of horse liver alcohol dehydrogenase differ by 10 amino acid residues, but only the S isoenzyme is active on 3 beta-hydroxysteroids. This functional difference was correlated to the differences in structures of the isoenzymes by characterizing a series of chimeric enzymes, which could represent intermediates in the evolution of catalytic activity. Deletion of Asp-115 from the E isoenzyme created the E/D115 delta enzyme that is active on steroids. The deletion alters the substrate binding pocket by moving Leu-116, which sterically hinders binding of steroids in the E isoenzyme. A chimeric enzyme (ESE) that has four changes in or near the substrate binding pocket (T94I/R101S/F110L/D115 delta) was 15-30-fold more catalytically efficient (V/Km) on uncharged steroids than was the E/D115 delta enzyme. Molecular modeling suggests that the substitutions at residues 94 and 110 indirectly affect the activity on steroids. ESE enzyme was 6-fold more active than the S isoenzyme on neutral steroids, due to substitutions not in the substrate binding pocket. The K366E and the Q17E/A43T/A59T substitutions in the S isoenzyme gave 2-fold increases in V/Km on steroids, which together can account for the changes observed with the ESE enzyme. The enzymes that are active on steroids did not bind 2,2,2-trifluoroethanol as tightly and were catalytically less efficient than the E isoenzyme with small alcohols. However, these enzymes were two to three and four to five orders of magnitude more efficient with 1-hexanol and 5 beta-androstane-3 beta,17 beta-diol, respectively, than with ethanol. These results demonstrate that several residues not directly participating in substrate binding or chemical catalysis contribute to catalytic efficiency.     

A structural role of histidine 15 in human glutathione transferase M1-1, an amino acid residue conserved in class Mu enzymes.

His15 is a conserved amino acid residue in all known class Mu glutathione transferases. This His residue in human glutathione transferase M1-1 has been mutated into 17 different amino acid residues by means of site-directed random mutagenesis to determine if any substitutions are compatible with catalytic activity. The majority of the mutant proteins appeared unstable and could not be isolated in reasonable quantities by heterologous expression in Escherichia coli. Five mutant enzymes, H15C, H15K, H15N, H15Q and H15S were purified and more extensively characterized. The mutant proteins shared the same size as that of the wild-type enzyme but could be separated from the parental enzyme by reverse phase HPLC. For all the mutant forms except H15N, the sp. act. with 1-chloro-2,4-dinitrobenzene was less than 3% of the wild-type value--the H15N mutant enzyme displayed 29% of the wild-type activity. None of the catalytically active mutant enzymes showed any major alteration of the binding affinity for the substrate analog S-hexylglutathione, suggesting that His15 is not part of the active site of the enzyme. The high activity of the mutant H15N, also reflected in the kcat/Km, V and S0.5 values, rules out the possibility that His15 in the native enzyme contributes to catalysis by serving as a base. The role of His15, largely replaceable by Asn in the same position, appears to be structural, probably involving hydrogen bonds that maintain the protein in a stable and catalytically active conformation. A critical structural role of His15 in a buried position may explain the evolutionary conservation of this residue in the class Mu glutathione transferases.     

Improvement of 2'-hydroxybiphenyl-2-sulfinate desulfinase, an enzyme involved in the dibenzothiophene desulfurization pathway, from Rhodococcus erythropolis KA2-5-1 by site-directed mutagenesis

In the microbial dibenzothiophene desulfurization pathway, 2'-hydroxybiphenyl-2-sulfinate is converted to 2-hydroxybiphenyl and sulfinate by desulfinase (DszB) at the last step, and this reaction is rate-limiting for the whole pathway. The catalytic activity and thermostability of DszB were enhanced by the two amino acid substitutions. Based on information on the 3-D structure of DszB and a comparison of amino acid sequences between DszB and reported thermophilic and thermostable homologs (TdsB and BdsB), two amino acid residues, Tyr63 and Gln65, were selected as targets to mutate and improve DszB. These two residues were replaced by several amino acids, and the promising mutant enzymes were purified and their properties were examined. Among the wild-type and mutant enzymes, Y63F had higher catalytic activity but similar thermostability, and Q65H showed higher thermostability but less catalytic activity and affinity for the substrate. To compensate for these drawbacks, the double mutant enzyme Y63F-Q65H was purified and its properties were investigated. This mutant enzyme showed higher thermostability without loss of catalytic activity or affinity for the substrate. These superior properties of the mutant enzyme have also been confirmed with resting cells harboring the mutant gene.     

Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution

We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45 degrees C, whereas the wild-type enzyme was not stable above 37 degrees C. The K(m) values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.     

Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase

In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli beta-galactosidase and beta-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more beta-glucuronidase activity than wild-type beta-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high beta-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.     

Characterization of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme involved in isopentenyl diphosphate biosynthesis, and identification of its catalytic amino acid residues

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase, which simultaneously catalyzes the intramolecular rearrangement and reduction of DXP to form 2-C-methyl-d-erythritol 4-phosphate, constitutes a key enzyme of an alternative mevalonate-independent pathway for isopentenyl diphosphate biosynthesis. The dxr gene encoding this enzyme from Escherichia coli was overexpressed as a histidine-tagged protein and characterized in detail. DNA sequencing analysis of the dxr genes from 10 E. coli dxr-deficient mutants revealed base substitution mutations at four points: two nonsense mutations and two amino acid substitutions (Gly(14) to Asp(14) and Glu(231) to Lys(231)). Diethyl pyrocarbonate treatment inactivated DXP reductoisomerase, and subsequent hydroxylamine treatment restored the activity of the diethyl pyrocarbonate-treated enzyme. To characterize these defects, we overexpressed the mutant enzymes G14D, E231K, H153Q, H209Q, and H257Q. All of these mutant enzymes except for G14D were obtained as soluble proteins. Although the purified enzyme E231K had wild-type K(m) values for DXP and NADPH, the mutant enzyme had less than a 0.24% wild-type k(cat) value. K(m) values of H153Q, H209Q, and H257Q for DXP increased to 3.5-, 7.6-, and 19-fold the wild-type value, respectively. These results indicate that Glu(231) of E. coli DXP reductoisomerase plays an important role(s) in the conversion of DXP to 2-C-methyl-d-erythritol 4-phosphate, and that His(153), His(209), and His(257), in part, associate with DXP binding in the enzyme molecule.