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Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis.

ABSTRACT: Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors.

SUBMITTER: Matsuura K

PROVIDER: S-EPMC1218315 | biostudies-other | 1997 Apr

REPOSITORIES: biostudies-other

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Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits.

Project description:The aroma of peach fruit is predominantly determined by the accumulation of ?-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for ?-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of ?-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of ?-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of ?-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

| S-EPMC7847995 | biostudies-literature

Enhanced Activity and Substrate Specificity by Site-Directed Mutagenesis for the P450 119 Peroxygenase Catalyzed Sulfoxidation of Thioanisole.

Project description:P450 119 peroxygenase was found to catalyze the sulfoxidation of thioanisole and the sulfonation of sulfoxide in the presence of tert-butyl hydroperoxide (TBHP) for the first time with turnover rates of 1549 min-1 and 196 min-1 respectively. Several mutants were designed to improve the peroxygenation activity and thioanisole specificity by site-directed mutagenesis. The F153G/T213G mutant gave an increase of sulfoxide yield and a decrease of sulfone yield. Moreover the S148P/I161T/K199E/T214V mutant and the K199E mutant with acidic Glu residue contributed to improving the product ratio of sulfoxide to sulfone. Addition of short-alkyl-chain organic acids to the P450 119 peroxygenase-catalyzed sulfur oxidation of thioanisole was investigated. Octanoic acid was found to induce a preferred sulfoxidation of thioanisole catalyzed by the F153G/T213G mutant to give approximately 2.4-fold increase in turnover rate with a k cat value of 3687 min-1 relative to that of the wild-type, and by the F153G mutant to give the R-sulfoxide up to 30 % ee. The experimental control and the proposed mechanism for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole in the presence of octanoic acid suggested that octanoic acid could partially occupy the substrate pocket; meanwhile the F153G mutation could enhance the substrate specificity, which could lead to efficiently regulate the spatial orientation of thioanisole and facilitate the formation of Compound I. This is the most effective catalytic system for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole.

| S-EPMC6682931 | biostudies-literature

Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity.

Project description:BACKGROUND: Endonuclease G (EndoG), a member of DNA/RNA nonspecific betabetaalpha-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG. METHODS: To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies. RESULTS: Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified. CONCLUSION: Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena.

| S-EPMC2653514 | biostudies-literature

Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis.

Project description:RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys(138), His(270) and Asn(303) of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr(224) is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr(224) by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala(305) mutants and identification of their products showed that the substitution of Ala(305) by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr(224) (important for starter substrate selection) and Ala(305) (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.

| S-EPMC1222926 | biostudies-other

Alteration of chain length substrate specificity of Aeromonas caviae R-enantiomer-specific enoyl-coenzyme A hydratase through site-directed mutagenesis.

Project description:Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ(Ac)) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid beta-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ(Ac) by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJ(Ac) revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C(8)) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C(12)) was examined by using the mutated PhaJ(Ac) as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1(Ps)). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

| S-EPMC169158 | biostudies-literature

Probing the essential catalytic residues and substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-arabinose isomerase by site-directed mutagenesis.

Project description:The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.

| S-EPMC1855884 | biostudies-literature

Directed evolution of the substrate specificity of dialkylglycine decarboxylase.

Project description:Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show ~2.5-fold decrease in KM and ~2-fold increase in kcat, giving a modest ~5-fold increase in catalytic efficiency for AC6C. Additional rounds of directed evolution did not improve catalytic activity toward AC6C. Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in kcat/KM for AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle.

| S-EPMC4334570 | biostudies-literature

Site-directed mutagenesis of catalytic residues in N(5)-carboxyaminoimidazole ribonucleotide synthetase.

Project description:N(5)-CAIR synthetase, an essential enzyme in microorganisms, converts 5-aminoimidazole ribonucleotide (AIR) and bicarbonate to N(5)-CAIR with the aid of ATP. Previous X-ray crystallographic analyses of Aspergillus clavatus N(5)-CAIR synthetase postulated that R271, H273, and K353 were important for bicarbonate binding and for catalysis. As reported here, site-directed mutagenesis of these residues revealed that R271 and H273 are, indeed, critical for bicarbonate binding and catalysis whereas all K353 mutations, even ones conservative in nature, are inactive. Studies on the R271K mutant protein revealed cooperative substrate inhibition for ATP with a Ki of 1.2 mM. Kinetic investigation of the H273A mutant protein indicated that it was cooperative with respect to AIR; however, this effect was not seen in either the wild-type or any of the other mutant proteins. Cooperative ATP-dependent inhibition of wild-type N(5)-CAIR synthetase was also detected with ATP displaying a Ki of 3.3 mM. Taken together, these results indicate that N(5)-CAIR synthetase operates maximally within a narrow concentration of ATP.

| S-EPMC3820426 | biostudies-literature

Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin.

Project description:Redox active cysteine residues including ?Cys93 are part of hemoglobin's "oxidation hotspot". Irreversible oxidation of ?Cys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active ?Cys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of ?Cys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at ?19 on the exposed surface of the ?-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.

| S-EPMC6126208 | biostudies-literature

Purification and characterization of an arene cis-dihydrodiol dehydrogenase endowed with broad substrate specificity toward polycyclic aromatic hydrocarbon dihydrodiols.

Project description:Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent Mr of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyrene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with Km values in the range of 1.4 to 7.1 microM, and exhibited a much higher Michaelis constant for NAD+ (Km of 160 microM). At pH 7.0, the specificity constant ranged from (1.3 +/- 0.1) x 10(6) M(-1) s(-1) with benz[a]anthracene 1,2-dihydrodiol to (20.0 +/- 0.8) x 10(6) M(-1) s(-1) with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.

| S-EPMC1489356 | biostudies-literature

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