Cysteine 42 is important for maintaining an integral active site for O-acetylserine sulfhydrylase resulting in the stabilization of the alpha-aminoacrylate intermediate.

O-Acetylserine sulfhydrylase-A (OASS-A) is a pyridoxal 5'-phosphate (PLP) dependent enzyme from Salmonella typhimurium that catalyzes the beta-replacement of acetate in O-acetyl-L-serine (OAS) by sulfide to give L-cysteine. The reaction occurs via a ping-pong kinetic mechanism in which alpha-aminoacrylate in Schiff base with the active site PLP is an intermediate [Cook, P. F., Hara, S., Nalabolu, S. R., and Schnackerz, K. D. (1992) Biochemistry 31, 2298-2303]. The sequence around the Schiff base lysine (K41) has been determined [Rege, V. D., Kredich, N. M., Tai, C.-H., Karsten, W. E., Schnackerz, K. D., & Cook, P. F. (1996) Biochemistry 35, 13485-13493], and the sole cysteine in the primary structure is immediately C-terminal to the lysine. In an effort to assess the role of C42, it has been changed to serine and alanine by site-directed mutagenesis. The mutant proteins are structurally nearly identical to the wild-type enzyme on the basis of UV-visible, fluorescence, far-UV and cofactor-induced CD, and 31P NMR studies, but subtle structural differences are noted. Kinetic properties of both mutant proteins differ significantly from those of the wild-type enzyme. The C42S mutant exhibits a > 50-fold increase in the OAS:acetate lyase activity and a 17-fold decrease in V for the cysteine synthesis compared to the wild-type enzyme, while decreases of > 200-fold in the OAS: acetate lyase activity and a 30-fold decrease in V for the cysteine synthesis are found for the C42A mutant enzyme. In both cases, however, the pH dependence of kinetic parameters for cysteine synthesis and OAS: acetate lyase activity yield, within error, identical pK values. In the three-dimensional structure of OASS-A, cysteine 42 is located behind the cofactor, pointing away from the active site, toward the interior of the protein. The dramatic change in the OAS:acetate lyase activity of OASS-A in the C42S and C42A mutant proteins likely results from a localized movement of the serine hydroxyl (compared to the cysteine thiol) toward additional hydrophilic, hydrogen-bonding groups in C42S, or away from hydrophilic groups for C42A, repositioning structure around and including K41. Subtle movement of the epsilon-amino group of K41 may change the geometry for nucleophilic displacement of the amino acid from PLP, leading to changes in overall activity and stability of the alpha-aminoacrylate intermediate. Data indicate that single amino acid substitutions that yield only subtle changes in structure can produce large differences in reaction rates and overall mechanism.

A change in the internal aldimine lysine (K42) in O-acetylserine sulfhydrylase to alanine indicates its importance in transimination and as a general base catalyst.

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes a beta-replacement reaction forming L-cysteine and acetate from O-acetyl-L-serine (OAS) and sulfide. The pyridoxal 5'-phosphate (PLP) is bound at the active site in Schiff base linkage with a lysine. In the present study, the Schiff base lysine was identified as lysine 42, and its role in the OASS reaction was determined by changing it to alanine using site-directed mutagenesis. K42A-OASS is isolated as an external aldimine with methionine or leucine and shows no reaction with the natural substrates. Apo-K42A-OASS can be reconstituted with PLP, suggesting that K42 is not necessary for cofactor binding and formation of the external Schiff base. The apo-K42A-OASS, reconstituted with PLP, shows slow formation of the external aldimine but does not form the alpha-aminoacrylate intermediate on addition of OAS, suggesting that K42 is involved in the abstraction of the alpha-proton in the beta-elimination reaction. The external aldimines formed upon addition of L-Ala or L-Ser are stable and represent a tautomer that absorbs maximally at 420 nm, while L-Cys gives a tautomeric form of the external aldimine that absorbs at 330 nm, and is also seen in the overall reaction after addition of primary amines to the assay system. The use of a small primary amine such as ethylamine or bromoethylamine in the assay system leads to the initial formation of an internal (gamma-thialysine) or external (ethylamine) aldimine followed by the slow formation of the alpha-aminoacrylate intermediate on addition of OAS. Activity could not be fully recovered, and only a single turnover is observed. Data suggest a significant rate enhancement resulting from the presence of K42 for transimination and general base catalysis.

The mechanism of the auxotrophy for sulfur-containing amino acids imposed upon Escherichia coli by superoxide.

Defects in both of the genes coding for the cytosolic superoxide dismutases (SODs) of Escherichia coli impose an oxygen-dependent nutritional requirement for cysteine. This is now seen to be a bradytrophy, rather than an absolute auxotrophy, since lack of Cys merely imposed a growth lag and escape from this growth lag did not involve genetic reversion. This Cys bradytrophy was not seen in the SOD-competent parental strain, and it was relieved by a cell-permeant mimic of SOD activity; hence, it was due to O2-.. It was also relieved by an osmolyte, such as sucrose; hence, it appears due to leakage from the cell of some component needed for Cys biosynthesis. Medium conditioned by the aerobic growth of the SOD-defective strain relieved the growth lag. Bioassays with Cys mutants suggested that the conditioned medium contained SO3-3 or its equivalent, and sulfite per se was able to eliminate the growth lag. However, some component of the conditioned medium reacted with added sulfite and interfered with attempts to assay for it colorimetrically. These results suggest that the cell envelope of the SOD-defective strain was weakened, directly or indirectly, by O2 and then leaked sulfite. This prevents cysteine biosynthesis until sulfite accumulates in the medium.

In vitro characterization of constitutive CysB proteins from Salmonella typhimurium.

Expression of the cysteine regulon in Salmonella typhimurium and Escherichia coli is controlled by the LysR-type transcriptional activator CysB and by the inducer N-acetyl-L-serine. Sulphide and thiosulphate are anti-inducers. Two highly purified constitutive CysB proteins, CysB(T149M) and CysB(T149P), were found to bind to the cysJIH, cysK and cysP promoters, to activate transcription from the cysJIH and cysK promoters in the absence of N-acetyl-L-serine, and to be insensitive to the effects of anti-inducers. At 10 mM MgCl2, the in vitro transcription activity of CysB(T149M) was maximal without N-acetyl-L-serine, but that of CysB(T149P) was increased by inducer. At 2 mM MgCl2, both proteins were fully active without inducer. A third mutant protein, CysB(W166R), was totally inactive at 10 mM MgCl2, but gave constitutive expression of the cysK and cysJIH promoters at 2 mM MgCl2. Surprisingly, wild-type CysB was also constitutive for the cysK promoter at 2 mM mgCl2 but not at 10 mM MgCl2; it required inducer for cysJIH promoter activation at both concentrations. Mutagenic studies indicated that this difference between promoters is due to the distance between activation site half-sites, which are separated by 1 bp in the cysJIH promoter and by 2 bp in the cysK promoter. We speculate that inducer acts to decrease the distance between the binding domains of two CysB subunits that interact with an activation site. In vitro activities of wild-type and mutant CysB proteins correlated much better with in vivo behaviour at 2 mM than at 10 mM MgCl2, suggesting that the former is the more physiological concentration.

Chromosomal localization and catalytic properties of the recombinant alpha subunit of human lymphocyte methionine adenosyltransferase.

Human lymphocyte methionine adenosyltransferase (HuLy MAT) consists of heterologous subunits alpha and beta. The cDNA sequence of the alpha subunit of HuLy MAT from Jurkat leukemic T cells was identical to that of the human kidney alpha subunit and highly homologous to the sequence of the extrahepatic MAT from other sources. The 3'-untranslated sequence was found to be highly conserved, suggesting that it may be important in regulating the expression of MAT. The extrahepatic alpha subunit unit of MAT was found to be expressed also in human liver, and no differences were found in the sequence of the alpha subunit from normal and malignant T cells. The sequence of two unspliced introns found in the cDNA clones from the Jurkat library enabled us to isolate genomic clones harboring the human extrahepatic alpha subunit gene and to localize it to the centromere on chromosome arm 2p, an area that corresponds to band 2p11.2. Expression of the alpha subunit cDNA in Escherichia coli yielded two peptides with the immunoreactivity and mobilities of authentic alpha/alpha' subunits from HuLy. The Km of the recombinant alpha subunit was 80 microM, which is 20-fold higher than found for the (alpha alpha')x beta y holoenzyme purified from leukemic lymphocytes and 4-10-fold higher than found for the normal lymphocyte enzyme. The data suggest that the alpha/alpha' subunits mediate the enzyme catalytic activity and that the beta subunit may be a regulatory subunit of extrahepatic MAT.

Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium.

CysB is a transcriptional activator for the cysteine regulon and negatively autoregulates its own gene, cysB. Transcription activation also requires an inducer, N-acetyl-L-serine. CysB is known to bind to activation sites just upstream of the -35 regions of the positively regulated cysJIH, cysK, and cysP promoters and to a repressor site centered at about +1 in the cysB promoter. Additional accessory sites have been found in positively regulated promoters. The hydroxyl radical footprinting experiments reported here indicate that the activation sites CBS-J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are composed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp. N-Acetyl-L-serine stimulates binding to these sites as well as to the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites. A second topology is found in the accessory site CBS-K2 and the repressor site CBS-B, which contain divergently oriented 19-bp half-sites separated by one or two helical turns. N-Acetyl-L-serine inhibits binding to these two sites. A third topology is present in the cysK and cysP promoters, where an additional half-site is oriented toward the activation site and separated from it by one helical turn. Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending. The marked dissimilarities of these half-site arrangements and of their responses to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to them with different combinations of subunits.

Residue threonine-149 of the Salmonella typhimurium CysB transcription activator: mutations causing constitutive expression of positively regulated genes of the cysteine regulon.

In both Salmonella typhimurium and Escherichia coli, CysB is a LysR family transcriptional activator, which regulates genes of the cysteine regulon. Transcription activation of cys genes also requires an inducer, N-acetyl-L-serine, and cysB mutants that do not require inducer are termed constitutive, i.e. cysBc. After finding that two independently isolated cysBc mutants are substituted at amino acid residue threonine-149 (T149), we isolated the other 17 single-amino-acid substitutions by site-directed mutagenesis. Of the 19 mutant alleles, 11 supported normal growth on sulphate, and nine of these were cysBc. Four other mutants were 'leaky' cysB+, and four were cysB-. Insertions of up to 14 amino acids were also tolerated at T149, and two of three such mutants were cysBc. An allele containing a TAG translation terminator at codon 149 had no detectable function in a delta cysB strain, but gave a constitutive phenotype when introduced into either wild-type S. typhimurium or the E. coli strain NK1, which contains a cysB- mutation in a predicted helix-turn-helix region that interferes with specific binding of CysB to DNA and with autoregulation of cysB. The peptide encoded by the T149ter allele is proposed to interact with the wild-type CysB peptide or with the NK1 mutant peptide to form hetero-oligomers that do not require N-acetyl-L-serine for cys gene activation.

Stoichiometry of binding of CysB to the cysJIH, cysK, and cysP promoter regions of Salmonella typhimurium.

CysB is a member of the LysR family of transcriptional activators and regulates genes of the cysteine regulon in Salmonella typhimurium and Escherichia coli. CysB binds to specific sites just upstream of the -35 regions of the cysJIH, cysK, and cysP promoters, where, in the presence of N-acetyl-L-serine, it stimulates transcription initiation. The cysK and cysP promoters contain additional binding sites, and we have proposed that CysB bends these promoters by binding to adjacent sites. N-Acetyl-L-serine is thought to decrease the magnitude of such bending. Since stoichiometric data bearing on this model have been lacking, we analyzed complexes in gel mobility shift experiments with 35S-labeled CysB and 32P-labeled promoter fragments. CysB was found to bind as a tetramer, and N-acetyl-L-serine increased the electrophoretic mobilities of one-protein complexes of the multibinding site cysK and cysP promoters without changing their stoichiometry, indicating that a single CysB tetramer can bend these promoters and that N-acetyl-L-serine diminishes such bending. Bend angles for both promoters were calculated to be 100 and 50 degrees in the absence and presence of N-acetyl-L-serine. N-Acetyl-L-serine affected neither the stoichiometry nor the electrophoretic mobility of cysJIH promoter complexes, which are not known to contain bent DNA. DNA bending may be a mechanism for sequestering CysB at certain promoter sites by increasing their affinity for this protein in the absence of N-acetyl-L-serine.

Dissimilatory sulphite reductase from Archaeoglobus fulgidus: physico-chemical properties of the enzyme and cloning, sequencing and analysis of the reductase genes.

A dissimilatory sulphite reductase was isolated from the extremely thermophilic dissimilatory sulphate-reducing archaeon Archaeoglobus fulgidus. In common with other dissimilatory sulphite reductases thus far characterized, the enzyme has an alpha 2 beta 2-structure and contains sirohaem, non-haem iron atoms and acid labile sulphide. The oxidized enzyme exhibited absorption maxima at 281, 394, 545 and 593 nm with a weak band around 715 nm. We have cloned and sequenced the genes for the alpha and beta subunits of this enzyme, which we designate dsrA and dsrB, respectively. They are contiguous in the order dsrA dsrB and probably comprise an operon, since dsrA is preceded by sequences characteristic of promoters in methanogenic archaea, and dsrB is followed by a sequence resembling termination signals in extremely thermophilic sulphur-dependent archaea. dsrA and dsrB encode 47.4 kDa and 41.7 kDa peptides, which have 25.6% amino acid sequence identity, indicating that they may have arisen by duplication of an ancestral gene. Each deduced peptide contains cysteine clusters resembling those postulated to bind sirohaem-[Fe4S4] complexes in sulphite reductases and nitrite reductases from other species. The dsrB encoded peptide lacks a single cysteine residue in one of the two clusters, suggesting that only the alpha subunit binds a sirohaem-[Fe4S4] complex, and chemical analyses showed the presence of only two sirohaems per alpha 2 beta 2 enzyme molecule. Both deduced peptides also contain an arrangement of cysteine residues characteristic of [Fe4S4] ferredoxins, and chemical analyses were consistent with the presence of six [Fe4S4] clusters per alpha 2 beta 2 enzyme molecule, two of which would be expected to be associated with sirohaem while the other four could bind to the ferredoxin-like sites.

The molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli.

Most genes required for cysteine biosynthesis in Salmonella typhimurium and Escherichia coli are positively regulated by cysB, which encodes a transcriptional activator belonging to the LysR family of regulatory proteins. CysB protein binds just upstream of the -35 region of positively regulated promoters, where in the presence of inducer it facilitates formation of a transcription initiation complex. CysB protein also autoregulates its own synthesis by binding to the cysB promoter as a repressor. Cysteine down-regulates the pathway by inhibiting synthesis of O-acetylserine, a direct cysteine precursor and possibly an inducer of gene expression. O-Acetylserine spontaneously isomerizes to N-acetylserine, which is clearly an inducer. Sulphide and thiosulphate provide additional regulation by acting as anti-inducers. Inducer stimulates CysB protein binding to sites involved in positive regulation, and inhibits binding to the negatively autoregulated cysB promoter. For three sites with unknown function, binding is stimulated at one and inhibited at the other two.

The cysP promoter of Salmonella typhimurium: characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate.

The cysPTWA operons of Escherichia coli and Salmonella typhimurium encode components of periplasmic transport systems for sulfate and thiosulfate and are regulated as part of the cysteine regulons. In vitro transcription initiation from the cysP promoter was shown to require both CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, which act as inducers, and was inhibited by the anti-inducer sulfide. Thiosulfate was found to be even more potent than sulfide as an anti-inducer. DNase I protection experiments showed two discrete binding sites for CysB protein in the presence of N-acetyl-L-serine. CBS-P1 is located between positions -85 and -41 relative to the major transcription start site, and CBS-P2 is located between positions -19 and +25. Without N-acetyl-L-serine, the CysB protein protected the region between positions -63 and -11, which was designated CBS-P3. In gel mobility shift assays, the mobility of CysB protein-cysP promoter complexes was increased by O-acetyl-L-serine, N-Acetyl-L-serine had no effect in gel shift experiments, presumably because its anionic charge results in its rapid removal from the complex during electrophoresis. Comparison of DNA fragments differing with respect to binding site position indicated that complexes with CysB protein contain DNA that is bent somewhere between CBS-P1 and CBS-P2 and that O-acetyl-L-serine decreases DNA bending. Binding studies with fragments containing either CBS-P2 alone, CBS-P1 alone, or the entire cysP promoter region suggest a model in which the complex of bent DNA observed in the absence of O-acetyl-L-serine contains a single CysB protein molecule bound to CBS-P3. At relatively low CysB protein concentrations, O-acetyl-L-serine would cause a single CysB protein molecule to bind tightly to CBS-P1, rather than to CBS-P3, thereby decreasing DNA bending and increasing complex electrophoretic mobility. At higher CysB protein concentrations, O-acetyl-L-serine would cause a second molecule to bind at CBS-P2, giving a more slowly migrating complex.

Regulation of S-adenosylmethionine synthetase activity in cultured human lymphocytes.

S-Adenosylmethionine (AdoMet), inorganic pyrophosphate (PPi) and inorganic phosphate (Pi) are potent product inhibitors of AdoMet synthetase and have been postulated to play a role in increasing AdoMet levels and turnover in peripheral blood mononuclear cells (PBM) after stimulation with phytohemagglutinin (PHA). Measurements of these metabolites in PHA-stimulated PBM showed the expected 2- to 3-fold increases in AdoMet after 8 h, and smaller increases in PPi and Pi. Since the kinetic model requires substantial decreases in PPi and Pi in response to PHA, product inhibition cannot explain the observed changes in AdoMet metabolism in this system. A 2.5-fold increase in AdoMet synthetase catalytic activity was found in crude extracts of PBM within 8 h of PHA-stimulation and probably accounts for increased cellular levels and utilization of AdoMet. Immunochemical analyses with a monoclonal antibody specific for the alpha/alpha' subunits of human lymphocyte AdoMet synthetase showed that these increases in catalytic activity were not associated with increases in immunoreactive protein. The ratio of catalytic activity to immunoreactivity in stimulated cells was 4-fold higher than in unstimulated controls and almost identical to that found in extracts from the human B-lymphocyte line WI-L2. Unstimulated PBM appear to contain substantial amounts of AdoMet synthetase alpha/alpha' subunit with reduced or absent catalytic activity, which can be activated by PHA-stimulation.

Negative autoregulation of cysB in Salmonella typhimurium: in vitro interactions of CysB protein with the cysB promoter.

CysB protein positively regulates genes of the Salmonella typhimurium cysteine regulon and negatively autoregulates cysB. The cysB promoter was characterized by primer extension of cellular RNA, which gave products identifying a major in vivo transcription start site located 95 bp upstream of the cysB start codon and two minor sites located 9 and 10 bp downstream of the major site. Gel shift binding studies and DNase I footprinting experiments showed that CysB protein binds to the cysB promoter from position -10 to +36 relative to the major transcription start site. We have designated this binding site CBS-B. CysB protein inhibited transcription initiation at the cysB promoter in an in vitro runoff assay, indicating that cysB is negatively autoregulated by the binding of CysB protein to the cysB promoter, where it acts as a repressor. N-Acetyl-L-serine, an inducer of the cysteine regulon, inhibited the binding of CysB protein to the cysB promoter and partially reversed the ability of CysB protein to inhibit transcription initiation. These effects are in contrast to those observed in studies of positively regulated cys promoters, in which N-acetyl-L-serine stimulates binding and causes CysB protein to activate transcription initiation.

High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor.

The flavoprotein and hemoprotein components of Escherichia coli B NADPH-sulfite reductase are encoded by cysJ and cysI, respectively. Plasmids containing these two genes overexpressed flavoprotein catalytic activity and apohemoprotein by 13- to 35-fold, but NADPH-sulfite reductase holoenzyme activity was increased only 3-fold. Maximum overexpression of holoenzyme activity was achieved by the inclusion in such plasmids of Salmonella typhimurium cysG, which encodes a uroporphyrinogen III methyltransferase required for the synthesis of siroheme, a cofactor for the hemoprotein. Thus, cofactor deficiency, in this case siroheme, can limit overexpression of a cloned enzyme. Catalytically active holoenzyme accounted for 10% of total soluble protein in a host containing cloned cysJ, cysI, and cysG. A 5.3-kb DNA fragment containing S. typhimurium cysG was sequenced, and the open reading frame corresponding to cysG was identified by subcloning and by identifying plasmid-encoded peptides in maxicells. Comparison with the sequence reported for the E. coli cysG region (J. A. Cole, unpublished data; GenBank sequence ECONIRBC) indicates a gene order of nirB-nirC-cysG in the cloned S. typhimurium fragment. In addition, two open reading frames of unknown identity were found immediately downstream of cysG. One of these contains 11 direct repeats of 33 nucleotides each, which correspond to the consensus amino acid sequence Asp-Asp-Val-Thr-Pro-Pro-Asp-Asp-Ser-Gly-Asp.

In vitro interactions of CysB protein with the cysK and cysJIH promoter regions of Salmonella typhimurium.

The cysteine regulons of Salmonella typhimurium and Escherichia coli are positively regulated by CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, both of which act as inducers. Gel mobility shift assays and DNase I footprinting experiments showed that CysB protein binds to the S. typhimurium cysK promoter at two sites, one, designated CBS-K1, at positions -78 to -39 relative to the major transcription start site, and the other, designated CBS-K2, at positions -115 to -79. The S. typhimurium cysJIH promoter was found to contain a single binding site, designated CBS-JH, at positions -76 to -35. Acetyl-L-serine stimulated binding to CBS-K1 and CBS-J and inhibited binding to CBS-K2. In the absence of acetyl-L-serine, CysB protein bound to both CBS-K1 and CBS-K2 and gave a complex that migrated more slowly during gel electrophoresis than did that formed in the presence of acetyl-L-serine, in which case CysB protein bound only to CBS-K1. Complexes formed with DNA containing the two binding sites either at the middle or at one end of the fragment migrated differently, suggesting that DNA was bent in the slow complex formed in the absence of acetyl-L-serine and that DNA in the fast complex was less bent or not bent at all. An analysis of upstream deletions of the cysK promoter showed that only CBS-K1 is required for in vivo promoter activity. CBS-J is analogous in position to CBS-K1 and is probably also required for activity of the cysJIH promoter. CBS-K2 has no known function but may help sequester CysB protein at the cysK promoter.

Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase.

Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.

Regulation of human lymphocyte S-adenosylmethionine synthetase by product inhibition.

A steady-state kinetic analysis of human S-adenosylmethionine synthetase indicates that the reaction is Bi Ter with ordered addition of ATP and L-methionine and release of S-adenosylmethionine as the first product. Pyrophosphate and phosphate are then released randomly. I-Parabolic inhibition by phosphate with respect to ATP indicates that this product must bind to more than one site. A model in which phosphate binds to the pyrophosphate site gives a rate equation that is consistent with the kinetic data. Values have been determined for those constants in the equation that are large enough to evaluate, and the in vitro kinetic behavior of S-adenosylmethionine synthetase can be predicted at substrate and product concentrations that are expected intracellularly. Inhibition by combinations of products, especially pyrophosphate and phosphate, is synergistic. Of particular interest is the ability of pyrophosphate and phosphate to increase the sensitivity of the enzyme to inhibition by S-adenosylmethionine. This phenomenon may play a role in regulating steady-state cellular concentrations of S-adenosylmethionine.

In vitro interactions of CysB protein with the cysJIH promoter of Salmonella typhimurium: inhibitory effects of sulfide.

The cysteine regulon of Salmonella typhimurium is positively regulated by the CysB protein and an inducer, which can be either O-acetyl-L-serine or N-acetyl-L-serine. In vivo experiments confirmed that sulfide and L-cysteine (supplied as L-cystine) interfere with induction by exogenously supplied O-acetyl-L-serine and also showed the same effects when N-acetyl-L-serine was used as an inducer. In a gel shift assay, purified CysB protein bound specifically to a 278-base-pair DNA fragment containing the S. typhimurium cysJIH promoter region. Binding occurred in the absence of inducer but did not stimulate in vitro transcription initiation, indicating that binding alone is insufficient to cause formation of a transcription initiation complex. Addition of N-acetyl-L-serine or O-acetyl-L-serine was required for transcription initiation and also stimulated binding three- to eightfold. Sulfide inhibited both transcription initiation and binding by interfering with the stimulatory effects of inducer in a competitive manner. These findings indicate that sulfide is an anti-inducer and may explain why full expression of the cysteine regulon requires sulfur limitation. L-Cysteine did not affect in vitro transcription initiation or binding of CysB protein to the cysJIH promoter region. The in vivo effects of L-cysteine may be secondary to its degradation to sulfide by the inducible enzyme cysteine desulfhydrase.

Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase.

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.