Identification of catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryotes by site-directed mutagenesis.

Pepstatin-insensitive carboxyl proteinases from Pseudomonas sp. (PCP) and Xanthomonas sp. (XCP) have no conserved catalytic residue sequences, -Asp*-Thr-Gly- (Asp is the catalytic residue) for aspartic proteinases. To identify the catalytic residues of PCP and XCP, we selected presumed catalytic residues based on their high sequence similarity, assuming that such significant sites as catalytic residues will be generally conserved. Several Ala mutants of Asp or Glu residues were constructed and analyzed. The D170A, E222A, and D328A mutants for PCP and XD79A, XD169A, and XD348A mutants for XCP were not converted to mature protein after activation, and no catalytic activity could be detected in these mutants. The specificity constants toward chromogenic substrate of the other PCP and XCP mutants, except for the D84A mutant of PCP, were similar to that of wild-type PCP or XCP. Coupled with the result of chemical modification (Ito, M., Narutaki, S., Uchida, K., and Oda, K. (1999) J. Biochem. (Tokyo) 125, 210-216), a pair of Asp residues (170 and 328) for PCP and a pair of Asp residues (169 and 348) for XCP were elucidated to be their catalytic residues, respectively. The Glu(222) residue in PCP or Asp(79) residue in XCP was excluded from the candidates as catalytic residues, since the corresponding mutant retained its original activity.     

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170?kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50?kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl₂, and HgCl₂ functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl₂ was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.     

Identification of the catalytic residues of carboxylesterase from Arthrobacter globiformis by diisopropyl fluorophosphate-labeling and site-directed mutagenesis

The role of amino acid residues in the enzymatic activity of carboxylesterase from Arthrobacter globiformis was analyzed by diisopropyl fluorophosphate (DFP) labeling and site-directed mutagenesis. The electrospray ionization mass spectrometric (ESI-MS) analysis of the esterase, covalently labeled by DFP, showed stoichiometric incorporation of the inhibitor into the enzyme. The further comparison of endopeptidase-digested fragments between native and DFP-labeled esterase by fast atom bombardment mass spectrometric (FAB-MS) analysis as well as site-directed mutagenesis indicated that Ser59 in the consensus sequence Ser-X-X-Lys, which is conserved exclusively in penicillin-binding proteins and some esterases, served as a catalytic nucleophile. In addition, the results obtained from analysis of the mutants at position 62 suggested the importance of the basic amino acid side chain at this position, and suggested the significance of this residue acting directly as a general base rather than its involvement in the maintenance of the optimum hydrogen-bonding network at the active site.     

Identification of catalytic residues of Ca2+-independent 1,2-alpha-D-mannosidase from Aspergillus saitoi by site-directed mutagenesis

The roles of six conserved active carboxylic acids in the catalytic mechanism of Aspergillus saitoi 1,2-alpha-d-mannosidase were studied by site-directed mutagenesis and kinetic analyses. We estimate that Glu-124 is a catalytic residue based on the drastic decrease of kcat values of the E124Q and E124D mutant enzyme. Glu-124 may work as an acid catalyst, since the pH dependence of its mutants affected the basic limb. D269N and E411Q were catalytically inactive, while D269E and E411D showed considerable activity. This indicated that the negative charges at these points are essential for the enzymatic activity and that none of these residues can be a base catalyst in the normal sense. Km values of E273D, E414D, and E474D mutants were greatly increased to 17-31-fold wild type enzyme, and the kcat values were decreased, suggesting that each of them is a binding site of the substrate. Ca2+, essential for the mammalian and yeast enzymes, is not required for the enzymatic activity of A. saitoi 1,2-alpha-d-mannosidase. EDTA inhibits the Ca2+-free 1,2-alpha-d-mannosidase as a competitive inhibitor, not as a chelator. We deduce that the Glu-124 residue of A. saitoi 1,2-alpha-d-mannosidase is directly involved in the catalytic mechanism as an acid catalyst, whereas no usual catalytic base is directly involved. Ca2+ is not essential for the activity. The catalytic mechanism of 1,2-alpha-d-mannosidase may deviate from that typical glycosyl hydrolase.     

Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis

The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66-->Asn and Asp-66-->Glu mutation remarkably decreased kinetic parameters such as Vmax and kcat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49-->Ser/Tyr and Cys-72-->Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211-->Ser/Tyr mutant enzyme was less than 10 min at 80 degrees C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211-->Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.     

Identification by mutagenesis of conserved arginine and tryptophan residues in rat liver carnitine palmitoyltransferase I important for catalytic activity

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80-98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K(m) values for carnitine or palmitoyl-CoA. However, these mutations decreased the V(max) values for both substrates by 10-40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four alpha-helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.     

Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.     

Identification of residues involved in v-Src substrate recognition by site-directed mutagenesis.

To study the role of the catalytic domain in v-Src substrate specificity, we engineered three site-directed mutants (Leu-472 to Tyr or Trp and Thr-429 to Met). The mutant forms of Src were expressed in Sf9 cells and purified. We analyzed the substrate specificities of wild-type v-Src and the mutants using two series of peptides that varied at residues C-terminal to tyrosine. The peptides contained either the YMTM motif found in insulin receptor substrate-1 (IRS-1) or the YGEF motif identified from peptide library experiments to be the optimal sequence for Src. Mutations at positions Leu-472 or Thr-429 caused changes in substrate specificity at positions P+1 and P+3 (i.e. one or three residues C-terminal to tyrosine). This was particularly evident in the case of the L-472W mutant, which had pronounced alterations in its preferences at the P+1 position. The results suggest that residue Leu-472 plays a role in P+1 substrate recognition by Src. We discuss the results in the light of recent work on the roles of the SH2, SH3 and catalytic domains of Src in substrate specificity.     

Site-directed mutagenesis of catalytic active-site residues of Taka-amylase A.

The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no alpha-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.     

Identification of catalytic residues involved in iron uptake by L-chain ferritins

When either horse spleen apoferritin (containing more than 90% of L chains) or recombinant horse L apoferritin are modified with glycineamide or taurine in the presence of a water-soluble carbodiimide, a total of 11 to 12 carboxyl groups per subunit are modified, and iron incorporation is effectively abolished. In contrast, when horse spleen ferritin (containing on average 2500 atoms per molecule) is modified under similar conditions, seven to eight carboxyl groups are modified. When apoferritin is prepared from this modified ferritin, it retains full iron incorporation activity. Apoferritin in which seven to eight carboxyls per subunit have been modified by glycineamide can subsequently be modified by taurine; a total of three to four carboxyl groups are modified accompanied by total loss of iron incorporation. Additional studies confirm that three carboxyl groups per subunit are protected from modification by glycineamide by Cr(III) inhibition of iron incorporation. Using tandem mass spectroscopy we have looked for taurine-labelled peptides in tryptic digests of succinylated apoferritins after taurine modification. In the sample where the residues involved in iron uptake have been modified with taurine, we have identified the peptide: This corresponds to residues 53–59 of the L subunit, where it is part of a region of the B-helix which is directed towards the inside of the apoferritin protein shell. The same peptide was identified using classical protein sequencing techniques after (1,2-³H)-taurine modification. We conclude that in L-chain apoferritins the Glu residues at positions 53, 56 and 57 are involved in the mechanism of iron incorporation. Glu 53 and 56 are conserved in L but not in H ferritins, and are located in close proximity to each other within the three-dimensional structure. There is ample room for rotation of Glu 57 to join with the other two to form an iron-binding site. This may represent a site of iron incorporation (most probably involving nucleation) unique to L-chain ferritins, and may explain the predominant L-chain involvement in conditions of iron overload.     

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.     

Identification of Pyrococcus furiosus amylopullulanase catalytic residues

Pyrococcus furiosus amylopullulanase (PfAPU) belongs to glycosyl hydrolase family 57. Using sequence alignments of the known family 57 enzymes and site-directed mutagenesis, E291, D394, and E396 were identified as PfAPU putative catalytic residues. The apparent catalytic efficiencies (k_cat/K_m) of PfAPU mutants E291Q and D394N on pullulan were 123.0 and 24.4 times lower, respectively, than that of PfAPU. The activity of mutant E396Q on pullulan was too low to allow reliable determination of its catalytic efficiency. The apparent specific activities of these enzymes on starch also decreased 91.0 times (E291Q), 11.7 times (D394N), and 37.2 times (E396Q). The hydrolytic patterns for pullulan and starch were the same, while the hydrolysis rates differed as reported. Based on sequence alignment and a previous report, E291 is proposed as the catalytic nucleophile.     

Identification of residues essential for progesterone binding to uteroglobin by site-directed mutagenesis

In order to identify amino acids directly involved in progesterone binding to rabbit uteroglobin we have mutated Phe 6, Tyr 21 and Thr 60 by site-directed mutagenesis of the uteroglobin cDNA. These residues have been postulated previously to participate in progesterone binding. High-level expression of the mutated uteroglobin cDNAs in Escherichia coli yields recombinant protein mutants that, like natural uteroglobin, form stable dimers, suggesting that the tertiary structure of the protein has not been altered. Substitution of Phe 6 by Ser or Ala does not change the progesterone binding characteristics. In contrast, replacement of Tyr 21 by Phe or Ala, drastically decreases progesterone binding. In addition, replacement of Thr 60 by Ala reduces the affinity for progesterone by a factor of three. These data suggest a direct interaction of progesterone with these two amino acids and support the idea of direct hydrogen bonding of the carbonyl (C3 and C20) of progesterone with the hydroxyl groups of Tyr 21 and Thr 60, respectively.     

Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues.

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/sub-unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/Km values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.     

Identification of critical residues of the mycobacterial dephosphocoenzyme a kinase by site-directed mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the γ-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg(2+) for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.     

Characteristics of mutagenesis by glyoxal in Salmonella typhimurium: contribution of singlet oxygen

The characteristics of mutagenesis by glyoxal in Salmonella tester strains TA100 and TA104, and particularly a possible role of active oxygen species, were investigated. Glyoxal was converted into a non-mutagenic chemical with glutathione (GSH) by glyoxalase I, and the mutagenic activity was enhanced by the depletion of intracellular GSH. Glyoxal caused the reduction of nitro blue tetrazolium, which was suppressed by the addition of 2,5-diphenylfuran, superoxide dismutase (SOD) and catalase (CAT), scavengers of singlet oxygen (1O2), superoxide radical (O2-) and hydrogen peroxide (H2O2), respectively. However, only the 1O2 scavenger almost completely suppressed the mutagenic activity of glyoxal. Mutagenicity assays using strains pretreated with N,N-diethyldithiocarbamate of a SOD inhibitor and strains with low levels of SOD and CAT indicated that the mutagenesis by glyoxal was independent of intracellular levels of SOD and CAT, though glyoxal itself repressed them. Therefore, all the results suggest that 1O2 formed from glyoxal is related to its mutagenesis, but that neither O2- nor H2O2 is intracellularly predominantly related to it. The action of glyoxal against SOD and CAT, and the formation of glyoxal adducts with amino acids as their components are also discussed.     

Identification of the catalytic triad in tripeptidyl-peptidase II through site-directed mutagenesis

Tripeptidyl-peptidase II (TPP II) is a 138-kDa subtilisin-like serine peptidase forming high molecular mass oligomers of >1000 kDa. The enzyme participates in general protein turnover and apoptotic pathways, and also has specific substrates such as neuropeptides. Here we report the site-directed mutagenesis of amino acids predicted to be involved in catalysis. The amino acids forming the putative catalytic triad (Asp-44, His-264, Ser-449) as well as the conserved Asn-362, potentially stabilizing the transition state, were replaced by alanine and the mutated cDNAs were transfected into human embryonic kidney (HEK) 293 cells. In clones stably expressing the mutant proteins, TPP II activity did not exceed the endogenous activity, thus confirming the essential role of the above amino acids in catalysis. Mutant and wild-type TPP II subunits co-eluted from a gel filtration column, suggesting that the subunits associate and that the native subunit conformation was retained in the mutants. Interestingly, the S449A and a H264A mutant enzyme affected the quaternary structure of the endogenously expressed TPP II, resulting in formation of an active, larger complex of >10,000 kDa.     

Identification of important residues in diketoreductase from Acinetobacter baylyi by molecular modeling and site-directed mutagenesis

Diketoreductase (DKR) from Acinetobacter baylyi exhibits a unique property of double reduction of a β, δ-diketo ester with excellent stereoselectivity, which can serve as an efficient biocatalyst for the preparation of an important chiral intermediate for cholesterol lowering statin drugs. Taken the advantage of high homology between DKR and human heart 3-hydroxyacyl-CoA dehydrogenase (HAD), a molecular model was created to compare the tertiary structures of DKR and HAD. In addition to the possible participation of His-143 in the enzyme catalysis by pH profile, three key amino acid residues, Ser-122, His-143 and Glu-155, were identified and mutated to explore the possibility of involving in the catalytic process. The catalytic activities for mutants S122A/C, H143A/K and E155Q were below detectable level, while their binding affinities to the diketo ester substrate and cofactor NADH did not change obviously. The experimental results were further supported by molecular docking, suggesting that Ser-122 and His-143 were essential for the proton transfer to the carbonyl functional groups of the substrate. Moreover, Glu-155 was crucial for maintaining the proper orientation and protonation of the imidazole ring of His-143 for efficient catalysis.