Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine

L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to alpha-ketobutyrate, methanethiol, and ammonia with pyridoxal 5'-phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration gamma-replacement in the presence of L-methionine at 37 degrees C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine gamma-lyase activity.     

Role of tyrosine 114 of L-methionine gamma-lyase from Pseudomonas putida

L-Methionine gamma-lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of y-family pyridoxal 5'-phosphate dependent enzymes. A mutant form of L-methionine y-lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in kcat for alpha,gamma-elimination of L-methionine, while the Km remained the same as the wild type enzyme. The Y114F mutant had the reduced kcat by only 28- and 16-fold for substrates with an electron-withdrawing group at the gamma-position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of kcat for alpha,beta-elimination and deamination substrates. The hydrogen exchange reactions of substrate and the spectral changes of the substrate-enzyme complex catalyzed by the mutant enzyme suggested that gamma-elimination process for L-methionine is the rate-limiting determination step in alpha,gamma-elimination overall reaction of the Y114F mutant. These results indicate that Tyr114 of L-methionine gamma-lyase is important in y-elimination of the substrate.     

Suicide inactivation of bacterial cystathionine gamma-synthase and methionine gamma-lyase during processing of L-propargylglycine.

L-Propargylglycine, a naturally occurring gamma, delta-acetylenic alpha-amino acid, induces mechanism-based inactivation of two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine gamma-synthease, which catalyzes a gamma-replacement reaction in methionine biosynthesis, and (2) methionine gamma-lyase, which catalyzes a gamma-elimination reaction in methionine breakdown. Biphasic pseudo-first-order inactivation kinetics were observed for both enzymes. Complete inactivation is achieved with a minimum molar ratio ([propargylglycine]/[enzyme monomer]) of 4:1 for cystathionine gamma-synthase and of 8:1 for methionine gamma-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [alpha-2H]Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [alpha-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of four and six turnovers per monomer inactivated are indicated for cystathionine gamma-synthase and methionine gamma-lyase, respectively. The inactivation stoichiometry, using [alpha-14C]-propargylglycine, is four labels per tetramer of cystathionine gamma-synthase but usually only two labels per tetramer of methionine gamma-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are alpha 2 beta 2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine gamma-synthase tetramer becomes modified by propargylglycine, only one alpha and one beta subunit may be labeled in an inactive alpha 2 beta 2 tetramer of methionine gamma-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine gamma-synthase may reprotonate C2 of the enzyme--inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine gamma-lyase has lambda max values at 460 and 495 nm, which may represent conjugated pyridoximine paraquinoid that does not reprotonate at C2 of the bound adduct. Either species could arise from Michael-type addition of an enzymic nucleophile to an electrophilic 3,4-allenic paraquinoid intermediate, generated initially by propargylic rearrangement upon a 4,5-acetylenic pyridoximine structure, as originally proposed for propargylglycine inactivation of gamma-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124]. It is reasonable that cystathionine gamma-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine.     

Crystal structure of the pyridoxal 5'-phosphate dependent L-methionine gamma-lyase from Pseudomonas putida

L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 A resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli.     

Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions

Cystathionine gamma-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a gamma-replacement reaction. In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, gamma-elimination reaction to form succinate, alpha-ketobutyrate, and ammonia. Since elimination of the gamma-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate. Previously, this partitioning intermediate has been assigned to a highly conjugated alpha-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the gamma-replacement and gamma-elimination reactions of cystathionine gamma-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The gamma-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region. A 485-nm species subsequently accumulates in a much slower relaxation. The gamma-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (approximately 3 ms). This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region. Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine gamma-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species. In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allylglycine undergoes a gamma-replacement reaction to form a new amino acid, gamma-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.     

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.     

Purification and characterization of mouse hepatic enzyme that converts selenomethionine to methylselenol by its alpha,gamma-elimination

The objective of this study was to purify and characterize a mouse hepatic enzyme that directly generates CH3SeH from seleno-l-methionine (l-SeMet) by the alpha,gamma-elimination reaction. The l-SeMet alpha,gamma-elimination enzyme was ubiquitous in tissues from ICR mice and the activity was relatively high in the large intestine, brain, and muscle, as well as the liver. Aging and sex of the mice did not have any significant influence on the activity in the liver. The enzyme was purified from the mouse liver by ammonium sulfate precipitation and four kinds of column chromatography. These procedures yielded a homogeneous enzyme, which was purified approx 1000-fold relative to mouse liver extract. Overall recovery was approx 8%. The purified enzyme had a molecular mass of approx 160 kDa with four identical subunits. The Km value of the enzyme for the catalysis of l-SeMet was 15.5 mM, and the Vmax was 0.29 units/mg protein. Pyridoxal 5'-phosphate (pyridoxal-P) was required as a cofactor because the holoenzyme could be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of pyridoxal-P. The enzyme showed the optimum activity at around pH 8.0 and the highest activity at 50 degrees C; it catalyzed the alpha,gamma-elimination reactions of several analogs such as d,l-homocysteine and l-homoserine in addition to l-SeMet. This enzyme also catalyzed the alpha,beta-elimination reaction of Se-methylseleno-l-cysteine. However, l-methionine was inert. Therefore, the purified enzyme was different from the bacterial l-methionine gamma-lyase that metabolizes l-SeMet to CH3SeH, in terms of the substrate specificity. These results were the first identification of a mammalian enzyme that specifically catalyzes the alpha,gamma-elimination reaction of l-SeMet and immediately converts it to CH3SeH, an important metabolite of Se.     

The role of cysteine 116 in the active site of the antitumor enzyme L-methionine gamma-lyase from Pseudomonas putida

The cysteinyl residue at the active site of L-methionine gamma-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k(cat) toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the gamma-elimination reaction of L-methionine and in substrate recognition in the MGLs.     

Conversion of the aminocrotonate intermediate limits the rate of gamma-elimination reaction catalyzed by L-cystathionine gamma-lyase of the yeast Saccharomyces cerevisiae

L-Cystathionine gamma-lyase [EC 4.4.1.1] of Saccharomyces cerevisiae was shown to bind cofactor pyridoxal 5'-phosphate, up to 2 molecules/subunit. The association constants of the enzyme for the cofactor were estimated to be 3.67 x 10(5) M(-1) and 9.05 x 10(3) M(-1). However, the latter value was too small for the binding to play a catalytic role. Changes in the absorption spectra of the enzyme in gamma-elimination reaction mixtures with various amino acids as substrates were observed at 10 degrees C to elucidate the reaction mechanism of the enzyme. The enzyme formed a chromophore exhibiting absorption at approximately 480 nm, which is characteristic of an aminocrotonate intermediate with O-succinyl-L-homoserine, L-cystathionine, L-homoserine, or O-acetyl-L-homoserine, at rates in this order. The intermediate was consumed at much lower rates than those of formation. The order of the rates of consumption was the same as the order of the formation rates and the order of the gamma-elimination activity of the enzyme with the above-mentioned substrates. These results strongly suggested that the intermediate was essential for gamma-elimination and that the reaction was rate-limited by its conversion into the product alpha-ketobutyrate. L-Cysteine sensitively inhibited the alpha, gamma-elimination activity of the enzyme, and also retarded the formation of the chromophore when it was provided to the enzyme together with a substrate. The reason for these phenomena is discussed.     

Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway

Methionine gamma-lyase (MGL) catalyzes the degradation of L-methionine to alpha-ketobutyrate, methanethiol and ammonia. The Arabidopsis (Arabidopsis thaliana) genome includes a single gene (At1g64660) encoding a protein (AtMGL) with approximately 35% identity to bacterial and protozoan MGLs. When overexpressed in Escherichia coli, AtMGL allowed growth on L-methionine as sole nitrogen source and conferred a high rate of methanethiol emission. The purified recombinant protein exhibited a spectrum typical of pyridoxal 5'-phosphate enzymes, and had high activity toward l-methionine, L-ethionine, L-homocysteine and seleno-L-methionine, but not L-cysteine. Quantitation of mRNA showed that the AtMGL gene is expressed in aerial organs and roots, and that its expression in leaves was increased 2.5-fold by growth on low sulfate medium. Emission of methanethiol from Arabidopsis plants supplied with 10 mM L-methionine was undetectable (<0.5 nmol min(-1) g(-1) FW), suggesting that AtMGL is not an important source of volatile methanethiol. Knocking out the AtMGL gene significantly increased leaf methionine content (9.2-fold) and leaf and root S-methylmethionine content (4.7- and 7-fold, respectively) under conditions of sulfate starvation, indicating that AtMGL carries a significant flux in vivo. In Arabidopsis plantlets fed L-[(35)S]methionine on a low sulfate medium, label was incorporated into protein-bound cysteine as well as methionine, but incorporation into cysteine was significantly (30%) less in the knockout mutant. These data indicate that plants possess an alternative to the reverse trans-sulfuration pathway (methionine-->homocysteine-->cystathionine-->cysteine) in which methanethiol is an intermediate.     

YtjE from Lactococcus lactis IL1403 Is a C-S lyase with alpha, gamma-elimination activity toward methionine

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.     

Why do proteins use selenocysteine instead of cysteine?

Selenocysteine is present in a variety of proteins and catalyzes the oxidation of thiols to disulfides and the reduction of disulfides to thiols. Here, we compare the kinetic and thermodynamic properties of cysteine with its selenium-containing analogon, selenocysteine. Reactions of simple selenols at pH 7 are up to four orders of magnitude faster than their sulfur analogs, depending on reaction type. In redox-related proteins, the use of selenium instead of sulfur can be used to tune electrode, or redox, potentials. Selenocysteine could also have a protective effect in proteins because its one-electron oxidized product, the selanyl radical, is not oxidizing enough to modify or destroy proteins, whereas the cysteine-thiyl radical can do this very rapidly.     

3-Chloro-DL-alanine resistance by L-methionine-alpha-deamino-gamma-mercaptomethane-lyase activity

The antibacterial agent 3-chloro-DL-alanine (3CA) is an inhibitor of peptidoglycan synthesis. Fusobacterium nucleatum and Porphyromonas gingivalis, the bacteria responsible for oral malodor, are shown to be resistant to 1 mM 3CA, whereas Streptococcus mutans and Escherichia coli are sensitive to this antibacterial agent at the same concentration. We isolated the 3CA resistance gene from F. nucleatum and showed that the gene encodes an L-methionine-alpha-deamino-gamma-mercaptomethane-lyase that catalyzes the alpha,gamma-elimination of L-methionine to produce methyl mercaptan. The enzyme also exhibits 3CA chloride-lyase (deaminating) activity. This antibacterial agent is expected to be useful for specific selection of malodorous oral bacteria producing high amounts of methyl mercaptan.     

1H NMR studies of substrate hydrogen exchange reactions catalyzed by L-methionine gamma-lyase

Hydrogen exchange reactions of various L-amino acids catalyzed by L-methionine gamma-lyase (EC 4.4.1.11) have been studied. The enzyme catalyzes the rapid exchange of the alpha- and beta-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium from the solvent. The rate of alpha-hydrogen exchange was about 40 times faster than that of the enzymatic elimination reaction of the sulfur-containing amino acids. The enzyme also catalyzes the exchange reaction of alpha- and beta-hydrogens of the following straight-chain L-amino acids which are not susceptible to elimination: norleucine, norvaline, alpha-aminobutyrate, and alanine. The exchange rates of the alpha-hydrogen and the total beta-hydrogens of L-alanine and L-alpha-aminobutyrate with deuterium followed first-order kinetics. For L-norvaline, L-norleucine, S-methyl-L-cysteine, and L-methionine, the rate of alpha-hydrogen exchange followed first-order kinetics, but the rate of total beta-hydrogen exchange decreased due to a primary isotope effect at the alpha-position. One beta-hydrogen of S-methyl-L-cysteine was exchanged faster than the other, although both the beta-hydrogens were exchanged completely with deuterium ultimately. L-Phenylalanine and L-tryptophan slowly underwent alpha-hydrogen exchange. The pro-R hydrogen of glycine was deuterated stereospecifically. None of the following amino acids were susceptible to the enzymatic hydrogen exchange: D isomers of the above amino acids, branched chain L-amino acids, acidic L-amino acids, and basic L-amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

Enzymatic assay for total plasma Cys

Patients with vascular disease and end-stage renal disease have significantly higher concentrations of plasma total Cys (tCys) than do healthy individuals. Described here is a nonradioactive, precise, rapid and sensitive enzymatic colorimetric assay for tCys in plasma samples and is homogeneous in that it avoids separation methods. The tCys assay uses only the recombinant enzymes methionine alpha,gamma-lyase (rMETase) and S-adenosylhomocysteine hydrolase (rSAHH) cloned from Pseudomonas putida and Trichomonas vaginalis, respectively. The rSAHH traps homocysteine and the rMETase thereby produces H(2)S exclusively from Cys. The reaction product, H(2)S, is measured colorimetrically following its reaction with N,N-dibutylphenylenediamine (DBPDA). The procedure takes 3-4 h to complete.     

Selenium in chemistry and biochemistry in comparison to sulfur

What makes selenoenzymes--seen from a chemist's view--so special that they cannot be substituted by just more analogous or adapted sulfur proteins? This review compiles and compares physicochemical properties of selenium and sulfur, synthetic routes to selenocysteine (Sec) and its peptides, and comparative studies of relevant thiols and selenols and their (mixed) dichalcogens, required to understand the special role of selenium in selenoproteins on the atomic molecular level. The biochemically most relevant differences are the higher polarizability of Se- and the lower pKa of SeH. The latter has a strikingly different pH-dependence than thiols, with selenols being active at much lower pH. Finally, selected typical enzymatic mechanisms which involve selenocysteine are critically discussed, also in view of the authors' own results.     

Recombinant human liver betaine-homocysteine S-methyltransferase: identification of three cysteine residues critical for zinc binding.

Betaine-homocysteine S-methyltransferase (BHMT; EC 2.1.1.5) catalyzes the transfer of an N-methyl group from betaine to homocysteine to produce dimethylglycine and methionine, respectively. The enzyme is found in the pathway of choline oxidation and is abundantly expressed in liver and kidney. We have recently shown that human BHMT is a zinc metalloenzyme [Millian, N. S., and Garrow, T. A. (1998) Arch. Biochem. Biophys. 356, 93-98]. To facilitate the rapid purification of human BHMT for further physical and mechanistic studies, including characterizing its metal binding properties, we have overexpressed the enzyme in E. coli as a fusion construct which facilitated its subsequent purification by a self-cleavable affinity tag system (IMPACT T7). Using this expression and purification system in conjunction with site-directed mutagenesis, we have identified Cys217, Cys299, and Cys300 as zinc ligands. Mutating any of these Cys residues to Ala results in the complete loss of activity and a significant reduction in the ability of the protein to bind zinc. Comparing the regions of BHMT amino acid sequence surrounding these Cys residues with similar amino acid sequences retrievable from protein databases, we have identified the following motif: G[ILV]NCX(20,100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I, which we propose to be a signature for a family of zinc-dependent methyltransferases that utilize thiols or selenols as methyl acceptors. Some of the members of this family include the vitamin B(12)-dependent methionine synthases, E. coli S-methylmethionine-S-homocysteine methyltransferase, and A. bisulcatus S-methylmethionine-selenocysteine methyltransferase.     

A selenomethionine-containing azurin from an auxotroph of Pseudomonas aeruginosa

The production and spectroscopic properties of an L-selenomethionine-containing homolog of Pseudomonas aeruginosa azurin are described. The amino acid substitution was carried out by developing an L-methionine-dependent bacterial strain from a fully functional ATCC culture. Uptake studies monitored using L-[75Se]methionine indicated that L-selenomethionine was incorporated into the protein synthetic pathway of Pseudomonas bacteria in a manner analogous to L-methionine. Several batches of bacteria were grown, and one sample of isolated and purified selenoazurin (azurin in which methionine was substituted by selenomethionine) was found (by neutron activation analysis) to contain 5.2 +/- 0.8 seleniums/copper. Correspondingly, a residual 0.35 methionines, relative to 6.0 in the native protein, were found by amino acid analysis in this azurin sample. The redox potential and extinction coefficient of this selenoazurin were found to be 333 +/- 1 mV (pH 7.0, I = 0.22) and 5855 +/- 160 M-1 cm-1 at 626 +/- 1 nm, respectively. Visible electronic, CD, and EPR spectra are reported and Gaussian curve fitting to the former spectrum allowed assignment of the selenomethionine Se----Cu(II) transition to a band found at 18034 cm-1, based upon an observed 450 cm-1 shift to the red from the analogous band position in the native protein. The data are consistent with a relatively more covalent copper site stabilizing the reduced, Cu(I), form in the selenoprotein. A role for the methionine as a modulator of the blue copper site redox potential by metal----ligand back bonding from Cu(I) is discussed in terms of a ligand sphere which limits the valence change at copper to much less than 1 during a redox cycle.