Leishmania donovani methionine adenosyltransferase. Role of cysteine residues in the recombinant enzyme.

Methionine adenosyltransferase (MAT, EC 2.5.1.6)-mediated synthesis of S-adenosylmethionine (AdoMet) is a two-step process consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities, AdoMet synthesis and tripolyphosphate hydrolysis, can be measured separately is particularly useful when the site-directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. The present report describes the cloning and subsequent functional refolding, using a bacterial expression system, of the MAT gene (GenBank accession number AF179714) from Leishmania donovani, the etiological agent of visceral leishmaniasis. The absolute need to include a sulfhydryl-protection reagent in the refolding buffer for this protein, in conjunction with the rapid inactivation of the functionally refolded protein by N-ethylmaleimide, suggests the presence of crucial cysteine residues in the primary structure of the MAT protein. The seven cysteines in L. donovani MAT were mutated to their isosterical amino acid, serine. The C22S, C44S, C92S and C305S mutants showed a drastic loss of AdoMet synthesis activity compared to the wild type, and the C33S and C47S mutants retained a mere 12% of wild-type MAT activity. C106S mutant activity and kinetics remained unchanged with respect to the wild-type. Cysteine substitutions also modified PPPi cleavage and AdoMet induction. The C22S, C44S and C305S mutants lacked in tripolyphosphatase activity altogether, whereas C33S, C47S and C92S retained low but detectable activity. The behavior of the C92S mutant was notable: its inability to synthesize AdoMet combined with its retention of tripolyphosphatase activity appear to be indicative of the specific involvement of the respective residue in the first step of the MAT reaction.     

Hysteretic behavior of methionine adenosyltransferase III. Methionine switches between two conformations of the enzyme with different specific activity

Methionine adenosyltransferase III (MATIII) catalyzes S-adenosylmethionine (AdoMet) synthesis and, as part of its reaction mechanism, it also hydrolyzes tripolyphosphate. Tripolyphosphatase activity was linear over time and had a slightly sigmoidal behavior with an affinity in the low micromolar range. On the contrary, AdoMet synthetase activity showed a lag phase that was independent of protein concentration but decreased at increasing substrate concentrations. Tripolyphosphatase activity, which appeared to be slower than AdoMet synthesis, was stimulated by preincubation with ATP and methionine so that it matched AdoMet synthetase activity. This stimulation process, which is probably the origin of the lag phase, represents the slow transition between two conformations of the enzyme that could be distinguished by their different tripolyphosphatase activity and sensitivity to S-nitrosylation. Tripolyphosphatase activity appeared to be the rate-determining reaction in AdoMet synthesis and the one inhibited by S-nitrosylation. The methionine concentration necessary to obtain half-maximal stimulation was in the range of physiological methionine fluctuations. Moreover, stimulation of MAT activity by methionine was demonstrated in vivo. We propose that the hysteretic behavior of MATIII, in which methionine induces the transition to a higher specific activity conformation, can be considered as an adaptation to the specific functional requirements of the liver.     

Tripolyphosphatase from Methanobacterium thermoautotrophicum (strain ΔH)

Abstract A low-melecular-mass polyphosphatase (tripolyphosphatase, PPPi) from the archaeon Methanobacterium thermoautotrophicum (strain ΔH) was purified 340-fold and characterized. The tripolyphosphatase showed an optimal activity at pH 9.7 (at 60°C). Though the highest activities were measured with tripolyphosphate, tetrapolyphosphate (57%), phosphate glass type 5 (41%) and phosphate glass type 15 (20%) could also be used as substrates. However, tripolyphosphatase was unable to use pyrophosphate. The enzyme was dependent on the presence of Mg²⁺. In the presence of 2 mM PPPi, an optimal activity was found at 6 mM Mg²⁺. The K _m for PPPi was estimated at 0.37 mM. In addition, the enzyme was inhibited by KF (50% at 6 mM) and appeared to be very heat stable: after an incubation of 2 h at 85°C about 85% of the activity was still present.     

Tripolyphosphatase associated with S-adenosylmethionine synthetase isozymes from rat liver

S-Adenosylmethionine (AdoMet) synthetase alpha and beta were purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis from rat liver. When the purified enzymes were applied onto Sephacryl S-200, each synthetase was eluted together with a tripolyphosphatase. The activities of these isozymes in synthesizing AdoMet and in hydrolyzing tripolyphosphate decreased in parallel with increasing amounts of rabbit anti-(beta-form) IgG. The activity of the beta-form isozyme was markedly stimulated by the addition of tripolyphosphate, whereas that of the alpha-form isozyme was inhibited. The tripolyphosphatase activity of both the alpha- and the beta-form was markedly stimulated by the addition of AdoMet. The tripolyphosphatases of each isozyme showed some other similar properties.     

Biochemical basis for the dominant inheritance of hypermethioninemia associated with the R264H mutation of the MAT1A gene. A monomeric methionine adenosyltransferase with tripolyphosphatase activity

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main alkylating agent in living cells. Additionally, in the liver, MAT is also responsible for up to 50% of methionine catabolism. Humans with mutations in the gene MAT1A, the gene that encodes the catalytic subunit of MAT I and III, have decreased MAT activity in liver, which results in a persistent hypermethioninemia without homocystinuria. The hypermethioninemic phenotype associated with these mutations is inherited as an autosomal recessive trait. The only exception is the dominant mild hypermethioninemia associated with a G-A transition at nucleotide 791 of exon VII. This change yields a MAT1A-encoded subunit in which arginine 264 is replaced by histidine. Our results indicate that in the homologous rat enzyme, replacement of the equivalent arginine 265 by histidine (R265H) results in a monomeric MAT with only 0.37% of the AdoMet synthetic activity. However the tripolyphosphatase activity is similar to that found in the wild type (WT) MAT and is inhibited by PP(i). Our in vivo studies demonstrate that the R265H MAT I/III mutant associates with the WT subunit resulting in a dimeric R265H-WT MAT unable to synthesize AdoMet. Tripolyphosphatase activity is maintained in the hybrid MAT, but is not stimulated by methionine and ATP, indicating a deficient binding of the substrates. Our data indicate that the active site for tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant. Moreover, our results provide a molecular mechanism that might explain the dominant inheritance of the hypermethioninemia associated with the R264H mutation of human MAT I/III.     

Identification of the catalytic residues involved in the carboxyl transfer of pyruvate carboxylase

To clarify the mechanism of carboxyl transfer from carboxylbiotin to pyruvate, the following conserved amino acid residues present in the carboxyl transferase domain of Bacillus thermodenitrificans pyruvate carboxylase were converted to homologous amino acids: Asp543, Glu576, Glu592, Asp649, Lys712, Asp713, and Asp762. The carboxylase activity of the resulting mutants, D543E, E576D, E576Q, E592Q, D649N, K712R, K712Q, D713E, D713N, D762E, and D762N, was generally less than that of the wild type from mutation, but it decreased the most to 5% or even less than that of the wild type with D543E, D576Q, D649N, K712R, and K712Q. The decrease in activity observed for Asp543, Asp649, and Lys712 mutants was not for structural reasons because their structures seemed to remain intact as assessed by gel filtration and circular dichroism. On the basis of these data, a mechanism is proposed where Lys712 and Asp543 serve as the key acid and base catalyst, respectively.     

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.     

Chlorella virus RNA triphosphatase. Mutational analysis and mechanism of inhibition by tripolyphosphate

Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses. To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site. Twelve residues (Glu(24), Glu(26), Asp(64), Arg(76), Lys(90), Glu(112), Arg(127), Lys(129), Arg(131), Asp(142), Glu(163), and Glu(165)) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value. Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions. The mutational results suggest that the active site of cvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry. Nonetheless, there are several discordant mutational effects in cvRtp1 versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel. We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors of cvRtp1 (K(i) = 0.6 microm tripolyphosphate and 2.4 microm pyrophosphate, respectively), whereas phosphate had little effect. cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate.     

The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the smaller Mg(2+).     

S-Adenosylmethionine synthetase from Escherichia coli

Adenosylmethionine (AdoMet) synthetase has been purified to homogeneity from Escherichia coli. For this purification, a strain of E. coli which was derepressed for AdoMet synthetase and which harbors a plasmid containing the structural gene for AdoMet synthetase was constructed. This strain produces 80-fold more AdoMet synthetase than a wild type E. coli. AdoMet synthetase has a molecular weight of 180,000 and is composed of four identical subunits. In addition to the synthetase reaction, the purified enzyme catalyzes a tripolyphosphatase reaction that is stimulated by AdoMet. Both enzymatic activities require a divalent metal ion and are markedly stimulated by certain monovalent cations. AdoMet synthesis also takes place if adenyl-5'yl imidodiphosphate (AMP-PNP) is substituted for ATP. The imidotriphosphate (PPNP) formed is not hydrolyzed, permitting dissociation of AdoMet formation from tripolyphosphate cleavage. An enzyme complex is formed which contains one equivalent (per subunit) of adenosylmethionine, monovalent cation, imidotriphosphate, and presumably divalent cation(s). The rate of product dissociation from this complex is 3 orders of magnitude slower than the rate of AdoMet formation from ATP. Studies with the phosphorothioate derivatives of ATP (ATP alpha S and ATP beta S) in the presence of Mg2+, Mn2+, or Co2+ indicate that a divalent ion is bound to the nucleotide during the reaction and provide information on the stereochemistry of the metal-nucleotide binding site.     

Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation

The specific functions of the amino acid residues in the streptokinase (SK) gamma-domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK gamma-domain, SK(K278A,K279A,E281A,K282A), and SK(D360A,R363A) had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SK(D322A,R324A,D325A) and SK(R330A,D331A,K332A,K334A) had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SK(D328A,R330A) had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg(324), Asp(325), Lys(332), and Lys(334) play important roles in the formation of a HPlg.SK complex. On the other hand, amino acid residues Asp(322), Asp(328), and Arg(330) of SK are involved in the virgin enzyme induction. Potential contact between Lys(332) of SK and Glu(623) of human microplasmin and strong interactions between Asp(328) and Lys(330), Asp(331) and Lys(334), and Asp(322) and Lys(334) of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK gamma-domain, SK(Leu(314)-Ala(342)), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.     

Engineering PQQ glucose dehydrogenase with improved substrate specificity. Site-directed mutagenesis studies on the active center of PQQ glucose dehydrogenase

Site-directed mutagenesis was carried out on the active site of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to improve its substrate specificity. Amino acid substitution of His168 resulted in a drastic decrease in the enzyme's catalytic activity, consistent with its putative catalytic role. Substitutions were also carried out in neighboring residues, Lys166, Asp167, and Gln169, in an attempt to alter the enzyme's substrate binding site. Lys166 and Gln169 mutants showed only minor changes in substrate specificity profiles. In sharp contrast, mutants of Asp167 showed considerably altered specificity profiles. Of the numerous Asp167 mutants characterized, Asp167Glu showed the best substrate specificity profile, while retaining most of its catalytic activity for glucose and stability. We also investigated the cumulative effect of combining the Asp167Glu substitution with the previously reported Asn452Thr mutation. Interpretation of the effect of the replacement of Asp167 to Glu on the alteration of substrate specificity in relation with the predicted 3D model of PQQGDH-B is also discussed.     

The role of OXA-1 beta-lactamase Asp(66) in the stabilization of the active-site carbamate group and in substrate turnover

The OXA-1 beta-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser(67). In class A beta-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp(66) in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp(66) mutants expressed in Escherichia coli DH10B ranged from < or =8 microg/ml for cysteine, proline and the basic amino acids to > or =256 microg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys(70), that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria-Bertani-broth agar resulted in a > or =16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp(66) forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO2 bound to Lys(70) and thereby profoundly affects substrate turnover.     

Interleukin 8, neutrophil-activating peptide-2 and GRO-alpha bind to and elicit cell activation via specific and different amino acid residues of CXCR2

The objective of this investigation was to determine the amino acid residues of the human neutrophil CXC chemokine receptor-2 (CXCR2) that are critical for binding the ligands interleukin 8 (IL-8), neutrophil-activating peptide-2 (NAP-2), and growth-related protein alpha (GROalpha) and critical for receptor-mediated signal transduction. Charged residues of the amino terminus and the first extracellular loop of CXCR2 were targeted for point mutagenesis studies. Seven separate CXCR2 mutants (Glu7, Asp9, Glu12, Asp13, Lys108, Asn110, and Lys120, all to Ala) were generated. Based on the Scatchard analysis of radioligand binding studies, the following amino acids were deemed critical for ligand binding: (i) Asp9, Glu12, Lys108, and Lys120 for IL-8 and (ii) Glu7, Asp9, and Glu12 for GROalpha. Point mutations in the amino terminus domain (Asp9 and Glu12) and the first extracellular loop (Lys108, Asn110, and Lys120) of CXCR2 reduced cell activation to all three ligands as measured by changes in intracellular calcium concentration. In conclusion, high-affinity binding of IL-8, NAP-2, and GROalpha to CXCR2 involves interaction with specific and different amino acid residues of CXCR2. Furthermore, we propose that the CXCR2 amino acid residues required for cell activation are not necessarily the same residues required for ligand binding.     

Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.