Replacement of methionine 208 in a truncated Bacillus sp. TS-23 alpha-amylase with oxidation-resistant leucine enhances its resistance to hydrogen peroxide

The methionine residues at positions 17, 104, 208, 214, 292, 315, 324, and 446 in the primary amino acid sequence of a truncated Bacillus sp. TS-23 alpha-amylase (His(6)-tagged BLADeltaNC) was changed to oxidative-resistant leucine by site-directed mutagenesis. The mutant enzymes with an apparent molecular mass of approximately 54 kDa were overexpressed in recombinant Escherichia coli. The specific activity for Met315Leu and Met446Leu was decreased by more than 76%, while Met17Leu, Met104Leu, Met208Leu, Met214Leu, Met292Leu, and Met324Leu showed 247, 128, 37, 260, 232, and 241%, respectively, higher activity than the wild-type enzyme. In comparison with wild-type enzyme, a lower K(m) value was observed for all mutant enzymes. The 3.2- and 4.5-fold increases in the catalytic efficiency (k(cat)/K(m)) for Met208Leu and Met324Leu, respectively, were partly contributed by a 68% and 38% decrease in K(m) values. Wild-type enzyme was sensitive to chemical oxidation, but Met208Leu was stable even in the presence of 500 mM H(2)O(2). Except for Met214Leu, which was quite sensitive to H(2)O(2), the other mutants showed a profile of oxidative inactivation similar to that of the wild-type enzyme. These observations indicate that the oxidative stability of His(6)-tagged BLADeltaNC can be improved by replacement of the critical methionine residue with leucine.     

Enhancing oxidative resistance of Agrobacterium radiobacter N-carbamoyl D-amino acid amidohydrolase by engineering solvent-accessible methionine residues

N-Carbamoyl D-amino acid amidohydrolase (D-NCAase) that catalyzes the stereospecific hydrolysis of N-carbamoyl D-amino acids to their corresponding D-amino acids is valuable in pharmaceutical industry. Agrobacterium radiobacter D-NCAase is sensitive to oxidative damage by hydrogen peroxide. To investigate the role of methionine residues in oxidative inactivation, each of the nine methionine residues in A. radiobacter D-NCAase was substituted with leucine, respectively, by site-directed mutagenesis. Except for two mutants (Met5Leu and Met31Leu) with similar activities, seven mutants (Met73Leu, Met167Leu/Met169Leu, Met184Leu, Met220Leu, Met239Leu, Met244Leu, and Met239Leu/Met244Leu) were found to have reduced activities. In the presence of H(2)O(2), three mutants (Met239Leu, Met244Leu, and Met239Leu/Met244Leu) with substitution of highly solvent-accessible methionines by leucines retained their activities. The other mutants were also considerably resistant to chemical oxidation than was the wild-type enzyme. Thus, substitution of solvent-accessible methionine residues with leucine to enhance oxidative stability of D-NCAase is practical but might be with compromised activity.     

Replacement of Methionine 208 in a Truncated <Emphasis Type="Italic">Bacillus</Emphasis> sp. TS-23 α-Amylase with Oxidation-Resistant Leucine Enhances Its Resistance to Hydrogen Peroxide

The methionine residues at positions 17, 104, 208, 214, 292, 315, 324, and 446 in the primary amino acid sequence of a truncated Bacillus sp. TS-23 α-amylase (His₆-tagged BLAΔNC) was changed to oxidative-resistant leucine by site-directed mutagenesis. The mutant enzymes with an apparent molecular mass of approximately 54 kDa were overexpressed in recombinant Escherichia coli. The specific activity for Met315Leu and Met446Leu was decreased by more than 76%, while Met17Leu, Met104Leu, Met208Leu, Met214Leu, Met292Leu, and Met324Leu showed 247, 128, 37, 260, 232, and 241%, respectively, higher activity than the wild-type enzyme. In comparison with wild-type enzyme, a lower K _m value was observed for all mutant enzymes. The 3.2- and 4.5-fold increases in the catalytic efficiency (k _cat/K _m) for Met208Leu and Met324Leu, respectively, were partly contributed by a 68% and 38% decrease in K _m values. Wild-type enzyme was sensitive to chemical oxidation, but Met208Leu was stable even in the presence of 500 mM H₂O₂. Except for Met214Leu, which was quite sensitive to H₂O₂, the other mutants showed a profile of oxidative inactivation similar to that of the wild-type enzyme. These observations indicate that the oxidative stability of His₆-tagged BLAΔNC can be improved by replacement of the critical methionine residue with leucine. Received: 12 April 2002 / Accepted: 8 June 2002     

The oxidative inactivation of plasminogen activator inhibitor type 1 results from a conformational change in the molecule and does not require the involvement of the P1' methionine.

Plasminogen activator inhibitor 1 (PAI-1) is sensitive to oxidative inactivation, and it has been suggested that specific oxidation of a methionine residue, Met347, situated in the P1' position of the reactive center may be the cause of the inactivation. To test this hypothesis we have purified and biochemically characterized mutant proteins of PAI-1 in which Met347 and either of two other methionines, Met266 or Met354, has been replaced with oxidation-resistant valine residues. The mutant proteins were found to be equally sensitive to oxidation as wild-type PAI-1, suggesting that a specific oxidation of the P1' Met347 is not responsible for the inactivation. When PAI-1 was oxidized, circular dichroism analysis revealed a rapid conformational change that correlated to the loss of inhibitory activity. The oxidation sensitivity of PAI-1 was enhanced dramatically in the presence of 0.001% sodium dodecyl sulfate, and the circular dichroism spectrum was significantly different from that of untreated PAI-1, suggesting that the increased sensitivity to oxidation may be caused by a conformational change in the inhibitor molecule. Taken together, our data suggest that the oxidative inactivation of PAI-1 is not caused by the specific oxidation of the P1' methionine but results from a conformational change in the protein structure.     

Elimination of a reactive thiol group from the active site of chloramphenicol acetyltransferase.

1. The type III variant of chloramphenicol acetyltransferase (CATIII) is resistant to inactivation by ionizable modifying reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and iodoacetate, whereas it is sensitive to inhibition by similar but uncharged reagents, including 4,4'-dithiodipyridine, methyl methanethiolsulphonate (MMTS) and iodoacetamide. The target for these thiol-modifying reagents has been postulated to be Cys-31. This residue is situated within a part of the chloramphenicol-binding site formed largely from the side chains of hydrophobic amino acid residues, which might be expected to discriminate against the access of ionized ligands to Cys-31. 2. The substitution of Cys-31 by alanine, serine, threonine or methionine yields an enzyme that is resistant to inactivation by thiol-specific reagents. Replacement of Cys-31 by alanine, serine or threonine results in increased Km values for chloramphenicol with only small changes in kcat.. In contrast, the Cys-31----Met substitution mainly affects kcat. values. Although the kcat. for chloramphenicol acetylation is decreased 13-fold compared with wild-type CAT, the kcat. for the acetyl-CoA hydrolysis reaction, which occurs in the absence of chloramphenicol, is increased 2.7-fold. 3. MMTS modification of cysteine residues results in an adduct (-CH2-S-S-CH3) that is structurally similar to the side chain of a methionine residue (-CH2-CH2-S-CH3). The kinetic properties of MMTS-modified CATIII closely resemble those of [Met31]CAT.     

Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts calmodulin binding and calcineurin activation

Calcineurin is a Ca (2+)/calmodulin-activated Ser/Thr phosphatase important in cellular actions resulting in memory formation, cardiac hypertrophy, and T-cell activation. This enzyme is subject to oxidative inactivation by superoxide at low micromolar concentrations and by H 2O 2 at low millimolar concentrations. On the basis of the hypothesis that oxidation of Met residues in calmodulin-binding domains inhibits binding to calmodulin, purified calcineurin was used to study the susceptibility of Met residues to oxidation by H 2O 2. The rate for oxidation of Met 406 in the calmodulin-binding domain was determined to be 4.4 x 10 (-3) M (-1) s (-1), indicating a high susceptibility to oxidation. Functional repercussions of Met 406 oxidation were evaluated using native enzyme and a calcineurin mutant in which Met 406 was exchanged for Leu. Measurement of fluorescent calmodulin binding demonstrated that oxidation of Met 406 results in a 3.3-fold decrease in the affinity of calmodulin for calcineurin. Calcineurin activation exhibited a loss in cooperativity with respect to calmodulin following Met 406 oxidation as shown by a reduction in the Hill slope from 1.88 to 0.86. Maximum phosphatase activity was unaffected by Met oxidation. Changes in the calcineurin-calmodulin interaction were accompanied by a 40% loss in the ability of calmodulin to stimulate binding of immunophilin/immunosuppressant to calcineurin. All effects on calmodulin binding to the native enzyme by the treatment with H 2O 2 could be reversed by treating the enzyme with methionine sulfoxide reductase. These results indicate that the calmodulin-binding domain of calcineurin is susceptible to oxidation at Met 406 and that oxidation disrupts calmodulin binding and enzyme activation. Oxidation-dependent decreases in the affinity of calmodulin for calcineurin can potentially modulate calmodulin-dependent signaling and calmodulin distribution.     

Significance of the Conserved Tyr352 and Asp380 Residues in the Catalytic Activity of Bacillus stearothermophilus Aminopeptidase II as Evaluated by Site-directed Mutagenesis

The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.     

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.     

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.     

Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 M phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K _m and k _cat values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K _m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K _m and k _cat values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.     

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base

His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in the wild-type enzyme. The structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, provides a rationale for these effects. The orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. In addition, the active site residues Arg289, Asp 292, and Leu 286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.     

Structural and functional role of the amino-terminal region of porcine cytosolic aspartate aminotransferase. Catalytic and structural properties of enzyme derivatives truncated on the amino-terminal side

In porcine cytosolic aspartate aminotransferase, a dimeric enzyme, the amino-terminal region anchoring onto the neighboring subunit is linked to the adjoining floppy peptide segment (residues 12-47), an integral part of the small domain whose facile movement upon substrate binding is a striking "induced fit" feature of this enzyme. To assess the contribution by the amino-terminal region to small domain movement and protein stability, a series of enzyme derivatives truncated on the amino-terminal side (residues 1-9) was prepared by using oligonucleotide-directed in vitro mutagenesis. Deletion of residues 1-3 showed no effect on catalytic activity and heat stability. Del 1-5 mutant enzyme with an extra methionine at position 5 showed only 43% of the kappa cat value (in the overall transamination) of the wild-type enzyme. Further deletion up to residue 9 resulted in a slight decrease in kappa cat values. Del 1-9 mutant enzyme still retained a kappa cat value of 33% that of wild-type enzyme. Km values for aspartate and 2-oxoglutarate increased sharply upon deletion of residues 1-9. Accordingly, Del 1-9 mutant enzyme showed a striking decrease in the kappa cat/Km value, to only 2% of that for the wild-type enzyme. Deletion of amino-terminal residues 1-9 resulted also in a large decrease in thermostability and in an enhanced susceptibility to limited proteolysis by protease 401, which is known to cleave at Leu20 of the wild-type enzyme. These findings indicate that an increase in the conformational freedom of the floppy segment (residues 12-47) would occur upon the loss of most of the anchorage region, thereby presenting an entropic barrier to conformational changes that facilitate substrate binding with high affinity.     

Analysis of oxidation sensitivity of maleate cis-trans isomerase from Serratia marcescens

The maleate cis-trans isomerase gene (maiA) from Serratia marcescens IFO3736 was cloned and sequenced. Serratia MaiA has 62.4% amino acid identity with Alcaligenes faecalis IFO13111 MaiA and 64.9% with Bacillus stearothermophilus MI-102 MaiA. All known ten amino acid sequences of MaiA had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. The maiA gene was expressed in Escherichia coli, and expressed products MaiA was purified and characterized. The purified enzyme of strain IFO3736 showed high activity at room temperature and high heat stability. It also showed higher activity in the presence of high concentration of aspartic acid than the enzyme of A. faecalis IFO13111, but it was also sensitive to chemical oxidation. By amino acid composition analysis, cysteine, methionine, and tyrosine residues were suggested to be oxidized to inactivate the enzyme by chemical oxidation. To investigate the mechanism of chemical oxidation of the enzyme, six methionine residues in the conserved regions of S. marcescens MaiA were replaced with cysteine residues by site-directed mutagenesis. The analysis of the constructed mutants suggested that the Met201 residue near the Cys198 residue is involved in the sensitivity of the enzyme to chemical oxidation.     

Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase

We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768]. Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain [Leon, O., & Schulman, L. H. (1987) Biochemistry 26, 5416-5422]. In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors. Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation. The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction. Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters. Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons. Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C. Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor and its implications for redox control and enzyme structure

Redox control of disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor was investigated using cysteine mutants expressed in Escherichia coli. The wild-type enzyme, but not a cysteine-less mutant, was reversibly inactivated by oxidation. To determine the residues involved in oxidative inactivation, different cysteine residues were replaced. Analysis with a cysteine-modifying reagent revealed that the formation of a disulfide bond between cysteines 253 and 621 was responsible for enzyme inactivation. This result suggests that residues in different cytoplasmic loops are close to each other in the tertiary structure. Both cysteine residues are conserved in K+-independent (type II) H+-pyrophosphatases.     

Characterization of Met95 mutants of a heme-regulated phosphodiesterase from Escherichia coli. Optical absorption, magnetic circular dichroism, circular dichroism, and redox potentials.

On the basis of amino acid sequences and crystal structures of similar enzymes, it is proposed that Met95 of the heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) acts as a heme axial ligand. In accordance with this proposal, the Soret and visible optical absorption and magnetic circular dichroism spectra of the Fe(II) complexes of the Met95Ala and Met95Leu mutant proteins indicate that these complexes are five-coordinated high-spin, suggesting that Met95 is an axial ligand for the Fe(II) complex. However, the Fe(III) complexes of these mutants are six-coordinated low-spin, like the wild-type enzyme. The latter spectral findings are inconsistent with the proposal that the axial ligand to the Fe(III) heme is Met95. To determine the possibility of a redox-dependent ligand switch in Ec DOS, we further analyzed Soret CD spectra and redox potentials, which provide direct evidence on the environmental structure of the heme protein. CD spectra of Fe(III) Met95 mutants were all different from those of the wild-type protein, suggesting indirect coordination of Met95 to the Fe(III) wild-type heme. The redox potentials of the Met95Leu, Met95Ala and Met95His mutants were considerably lower than that of the wild-type enzyme (+70 mV) at -1, -26, and -122 mV vs. SHE, respectively. Thus, it is reasonable to speculate that water (or hydroxy anion) interacting with Met95, rather than Met95 itself, is the axial ligand to the Fe(III) heme.     

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.