Substrate analysis of homoserine acyltransferase from Bacillus cereus

Substrate specificity within the family of enzymes designated as homoserine transsuccinylases is variable, with some organisms utilizing succinyl-CoA and other organisms utilizing acetyl-CoA. In this study it is shown that the enzyme from Bacillus cereus uses acetyl-CoA as its acyl donor, but its catalytic rate is significantly lower than other HTS family members. BcHTS is inactivated by both iodoacetamide and diethyl pyrocarbonate and the enzyme can be partially protected from inactivation by the presence of succinyl-CoA. This leads to the conclusion that BcHTS can bind both acetyl-CoA and succinyl-CoA and suggests that it may represent an intermediate between the succinate-transferring HTS family members and the acetate-transferring HTS family members. The B. cereus enzyme was unable to rescue growth of an Escherichia coli strain lacking a functional transsuccinylase, however.     

Assessing the roles of essential functional groups in the mechanism of homoserine succinyltransferase

Homoserine acyltransferases catalyze the commitment step to methionine and other important biological precursors which make this class of enzymes essential for the survival of bacteria, plants and fungi. This class of enzymes is not found in humans, making them an attractive new target for antimicrobial design. Homoserine O-succinyltransferase (HST) is a representative from this class, with little known about the key amino acids involved in substrate specificity and catalysis. HST from Escherichia coli has been cloned, purified and kinetically characterized. Through site-directed mutagenesis and steady-state kinetic studies the residues that comprise a catalytic triad for HST, the catalytic cysteine nucleophile, an active site acid-base histidine, and the base orienting glutamate, have been identified and characterized. Several residues which confer substrate specificity for both homoserine and succinyl-CoA have also been identified and kinetically evaluated. Mutations of an active site glutamate to either aspartate or alanine drastically increase the K(m) for homoserine, assigning this glutamate to a binding role for the alpha-amino group of homoserine. An active site arginine orients the carboxyl moiety of homoserine, while the carboxyl moiety of succinyl-CoA is positioned for catalysis by a lysine residue. Removing functionality at either of these positions alters the enzyme's ability to effectively utilize homoserine or succinyl-CoA, respectively, reflected in an increased K(m) and decreased catalytic efficiency. The data presented here provides new details of the catalytic mechanism of succinyltransferases, resolves a controversy between alternative mechanistic hypotheses, and provides a starting point for the development of selective inhibitors of HST.     

Crystal structure of homoserine O-acetyltransferase from Leptospira interrogans

Homoserine O-acetyltransferase (HTA, EC 2.3.1.31) initiates methionine biosynthesis pathway by catalyzing the transfer of acetyl group from acetyl-CoA to homoserine. This study reports the crystal structure of HTA from Leptospira interrogans determined at 2.2 Å resolution using selenomethionyl single-wavelength anomalous diffraction method. HTA is modular and consists of two structurally distinct domains—a core α/β domain containing the catalytic site and a helical bundle called the lid domain. Overall, the structure fold belongs to α/β hydrolase superfamily with the characteristic ‘catalytic triad’ residues in the active site. Detailed structure analysis showed that the catalytic histidine and serine are both present in two conformations, which may be involved in the catalytic mechanism for acetyl transfer.     

Expression of the metA gene of Escherichia coli K-12 in recombinant plasmids

Abstract The expression of the metA gene coding for the first enzyme in the methionine biosynthethic pathway was studied in wild-type and in deregulated strains of Escherichia coli K-12 carrying the gene on multicopy plasmids.
We looked at (a) in vitro activity of the metA product—The enzyme homoserine transsuccinylase (HTS); (b) resistance of cells carrying metA plasmids to the analogue α-methylmethionine which specifically inhibits HTS, and (c) the metA polypeptide in mini cells.
The results indicate that the M _r value of the polypeptide synthesized by the metA gene is 40 000. The synthesis of HTS, even when the metA gene is cloned on a multicopy plasmid, is under the negative control of the regulatory metJ gene.     

The Mode of Action of a Carboxypeptidase from Malt

Certain methionine auxotrophs of Arthrobacter paraffineus and Bacillus species produce large amounts of O-acetylhomoserine (OAH). The methionine requirement of these auxotrophs could be satisfied by either cystathionine or homocysteine but not by homoserine. The cell-free extacts from the auxotrophs were found to be deficient in cystathionine ?-synthase activity. OAH and O-succinylhomoserine (OSH) could replace methionine in the auxotrophs which are deficient in homoserine-O-transacetylase. A methionine auxotroph of Corynebacterium glutamicum also produced OAH, and the blocked step in the auxotroph appeared to be between cystathionine and homocysteine.

Cell-free extracts of A. paraffineus, C. glutamicum and Bacillus species catalyzed the formation of OAH from acetyl-CoA and homoserine, while a corresponding reaction with succinyl-CoA was not detected. Cystathionine γ-synthases in extracts of C. glutamicum and Bacillus species were specific for OAH, while the enzyme in extract of A. paraffineus was rather specific for OSH though it reacted with OAH to a certain extent.

These results indicate that the biosynthesis of l-methionine in these bacteria involves OAH.     

Purification and characterization of Thermotoga maritima homoserine transsuccinylase indicates it is a transacetylase

The methionine biosynthetic pathway found in bacteria is controlled at the first step, acylation of the γ-hydroxyl of homoserine. This reaction is catalyzed by one of two unique enzymes, homoserine transacetylase or homoserine transsuccinylase, which have no amino acid sequence similarity. We cloned, expressed, and purified homoserine transsuccinylase from the thermophilic bacterium Thermotoga maritima. Substrate specificity experiments demonstrated that acetyl-coenzyme A (CoA) is the preferred acyl donor and is used at least 30-fold more efficiently than succinyl-CoA. Steady-state kinetic experiments confirm that the enzyme utilizes a ping-pong kinetic mechanism in which the acetate group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine. The maximal velocity, V/K _acetyl-CoA and V/K _homoserine, all exhibited bell-shaped pH curves with apparent pKs of 6.0–6.9 and 8.2–8.8. The enzyme was inactivated by iodoacetamide in a pH-dependent manner, with an apparent pK of 6.3, suggesting the presence of an active-site cysteine residue which forms an acetyl-enzyme thioester intermediate during catalytic turnover, similar to observations with other transsuccinylases. In addition, the enzyme is highly stable at elevated temperatures, maintaining full activity at 70°C. Taken together, these data suggest that the T. maritima enzyme functions biochemically as a transacetylase, despite having the sequence of a transsuccinylase.     

Effects of deregulation of methionine biosynthesis on methionine excretion in Escherichia coli

Several regulators of methionine biosynthesis have been reported in Escherichia coli, which might represent barriers to the production of excess l-methionine (Met). In order to examine the effects of these factors on Met biosynthesis and metabolism, deletion mutations of the methionine repressor (metJ) and threonine biosynthetic (thrBC) genes were introduced into the W3110 wild-type strain of E. coli. Mutations of the metK gene encoding S-adenosylmethionine synthetase, which is involved in Met metabolism, were detected in 12 norleucine-resistant mutants. Three of the mutations in the metK structural gene were then introduced into metJ and thrBC double-mutant strains; one of the resultant strains was found to accumulate 0.13 g/liter Met. Mutations of the metA gene encoding homoserine succinyltransferase were detected in alpha-methylmethionine-resistant mutants, and these mutations were found to encode feedback-resistant enzymes in a 14C-labeled homoserine assay. Three metA mutations were introduced, using expression plasmids, into an E. coli strain that was shown to accumulate 0.24 g/liter Met. Combining mutations that affect the deregulation of Met biosynthesis and metabolism is therefore an effective approach for the production of Met-excreting strains.     

Corynebacterium glutamicum utilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis

A direct sulfhydrylation pathway for methionine biosynthesis in Corynebacterium glutamicum was found. The pathway was catalyzed by metY encoding O-acetylhomoserine sulfhydrylase. The gene metY, located immediately upstream of metA, was found to encode a protein of 437 amino acids with a deduced molecular mass of 46,751 Da. In accordance with DNA and protein sequence data, the introduction of metY into C. glutamicum resulted in the accumulation of a 47-kDa protein in the cells and a 30-fold increase in O-acetylhomoserine sulfhydrylase activity, showing the efficient expression of the cloned gene. Although disruption of the metB gene, which encodes cystathionine gamma-synthase catalyzing the transsulfuration pathway of methionine biosynthesis, or the metY gene was not enough to lead to methionine auxotrophy, an additional mutation in the metY or the metB gene resulted in methionine auxotrophy. The growth pattern of the metY mutant strain was identical to that of the metB mutant strain, suggesting that both methionine biosynthetic pathways function equally well. In addition, an Escherichia coli metB mutant could be complemented by transformation of the strain with a DNA fragment carrying corynebacterial metY and metA genes. These data clearly show that C. glutamicum utilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis. Although metY and metA are in close proximity to one another, separated by 143 bp on the chromosome, deletion analysis suggests that they are expressed independently. As with metA, methionine could also repress the expression of metY. The repression was also observed with metB, but the degree of repression was more severe with metY, which shows almost complete repression at 0.5 mM methionine in minimal medium. The data suggest a physiologically distinctive role of the direct sulfhydrylation pathway in C. glutamicum.     

The methionine biosynthetic pathway from homoserine in Pseudomonas putida involves the metW, metX, metZ, metH and metE gene products

Biosynthesis of methionine from homoserine in Pseudomonas putida takes place in three steps. The first step is the acylation of homoserine to yield an acyl-L-homoserine. This reaction is catalyzed by the products of the metXW genes and is equivalent to the first step in enterobacteria, gram-positive bacteria and fungi, except that in these microorganisms the reaction is catalyzed by a single polypeptide (the product of the metA gene in Escherichia coli and the met5 gene product in Neurospora crassa). In Pseudomonas putida, as in gram-positive bacteria and certain fungi, the second and third steps are a direct sulfhydrylation that converts the O-acyl-L-homoserine into homocysteine and further methylation to yield methionine. The latter reaction can be mediated by either of the two methionine synthetases present in the cells.     

Isolation of three cyclic-AMP-independent acetyl-CoA carboxylase kinases from lactating rat mammary gland and characterization of their effects on enzyme activity

Three cyclic AMP-independent acetyl-CoA carboxylase kinases (A, B1 and B2) have been isolated from lactating rat mammary gland, using phosphocellulose chromatography, high performance gel filtration, and affinity chromatography on casein-Sepharose and phosvitin-Sepharose. These protein kinases have been identified with previously described kinases by the following criteria. Kinase A phosphorylates the same sites on rabbit mammary acetyl-CoA carboxylase as acetyl-CoA carboxylase kinase 2, which was originally described as a contaminant of rabbit mammary acetyl-CoA carboxylase purified by the poly(ethylene glycol)procedure. Kinase A will henceforth be referred to as acetyl-CoA carboxylase kinase-2. Kinase B1 has been identified with casein kinase II by its heparin sensitivity, elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. Kinase B2 has been identified with casein kinase I by its elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. The three kinases phosphorylate distinct sites on acetyl-CoA carboxylase. Phosphorylation by either casein kinase I or II does not affect enzyme activity. However, acetyl-CoA carboxylase kinase 2 inactivates acetyl-CoA carboxylase reversibly, in an identical manner to cyclic-AMP-dependent protein kinase, and phosphorylates sites located on identical peptides. Acetyl-CoA carboxylase kinase-2 can, however, be distinguished from the free catalytic subunit of cyclic-AMP-dependent protein kinase by its molecular mass, its substrate specificity, its elution behaviour on phosphocellulose, and its complete lack of sensitivity to the protein inhibitor of cyclic-AMP-dependent protein kinase. We also present evidence that phosphorylation of acetyl-CoA carboxylase by cyclic-AMP-dependent protein kinase occurs directly and not via a bicyclic cascade system as proposed by other laboratories.     

The steady state concentrations of coenzyme A-SH and coenzyme A thioester, citrate, and isocitrate during tricarboxylate cycle oxidations in rabbit heart mitochondria.

The steady state mitochondrial content of coenzyme A-SH (CoA), acetyl-CoA, succinyl-CoA, and long chain acyl-CoA has been determined during the oxidation of palmitoylcarnitine by rabbit heart mitochondria. Variation of the substrate concentration during ADP-stimulated (state 3) respiration varies the mitochondrial content of long chain acyl-CoA and the rate of O2 uptake, and permits the conclusion that the Km of beta oxidation for intramitochondrial long chain acyl-CoA is approximately 1 nmol/mg of mitochondrial protein. At near saturating concentrations of palmitoylcarnitine, plus L-malate, the addition of ADP causes a decrease in acetyl-CoA, an increase in CoA and succinyl-CoA, and no clear change in long chain acyl-CoA content. These changes reverse upon the depletion of ADP (state 3 leads to 4 transition). Similar changes in CoA, acetyl-CoA, and succinyl-CoA are seen during state 4 leads to 3 leads to 4 transitions with pyruvate plus L-malate and octanoate plus L-malate as substrates. These results suggest a limitation of flux by citrate synthase during the controlled oxidation of these three substrates. The ratio acetyl-CoA/succinyl-CoA was determined not only during state 3 and state 4 oxidation of palmitoylcarnitine plus L-malate and pyruvate plus L-malate, but also during intermediate respiratory states (state 3 1/2) generated by adding glucose and varying amounts of hexokinase. These intermediate states are characterized by a high succinyl-CoA content, relative to either state 3 or state 4, and a suboptimal flux through citrate synthase, estimated either by pyruvate disappearance or by O2 uptake.     

New Methionine Structural Gene in Salmonella typhimurium

Eight metH mutants in Salmonella typhimurium with closely linked sites of mutation which could grow only on methionine were isolated from a metE mutant deficient in N(5)-methyltetrahydropteroyltriglutamate-homocysteine transmethylase; their deficiency in cobalamin-dependent N(5)-methyltetrahydrofolate-homocysteine transmethylase was supported by the results of enzyme studies of one of them. Cotransduction of metH and metA (homoserine O-transsuccinylase) mutants was obtained, thus revealing linkage between a second pair of the six known methionine structural genes. One metH mutant clearly differed from the rest in that it reverted at a higher frequency, was temperature sensitive, complemented all other metH mutants, and was located farthest from the metA gene.     

Control of methionine biosynthesis in Escherichia coli by proteolysis

Most bacterial proteins are stable, with half-lives considerably longer than the generation time. In Escherichia coli, the few exceptions are unstable regulatory proteins. The results presented here indicate that the first enzyme in methionine biosynthesis - homoserine trans-succinylase (HTS) - is unstable and subject to energy-dependent proteolysis. The enzyme is stable in triple mutants defective in Lon-, HslVU- and ClpP-dependent proteases. The instability of the protein is determined by the amino-terminal part of the protein, and its removal or substitution by the N-terminal part of beta-galactosidase confers stability. The effect of the amino-terminal segment is not caused by the N-end rule, as substitution of the first amino acid does not affect the stability of the protein. HTS is the first biosynthetic E. coli enzyme shown to have a short half-life and may represent a group of biosynthetic enzymes whose expression is controlled by proteolysis. Alternatively, the proteolytic processing of HTS may be unique to this enzyme and could reflect its central role in regulating bacterial growth, especially at elevated temperatures.     

The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The steady-state concentrations of coenzyme A, acetyl-coenzyme A and succinyl-coenzyme A in flight muscle and isolated mitochondria

(1) A ;cycling' method involving citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) was modified by the inclusion of succinyl-CoA synthetase (EC 6.2.1.5) and hexokinase (EC 2.7.1.1) to permit the determination of very small amounts of succinyl-CoA in addition to CoA and acetyl-CoA. (2) Application of this technique to blowfly (Phormia regina) flight-muscle extracts reveals no change in acetyl-CoA concentration, a slight fall in CoA concentration and a rise in succinyl-CoA concentration during flight. (3) Extraction of isolated mitochondria during controlled (state 4) pyruvate oxidation reveals essentially only acetyl-CoA. Activation of respiration by ADP (state 3) or uncoupling agents leads to a fall in acetyl-CoA and a rise in CoA and succinyl-CoA content. (4) The presence of glycerol phosphate in addition to pyruvate results in a lower acetyl-CoA content in state 4. (5) It is contended that these results are consistent with a primary control of one of the reactions of the tricarboxylate cycle, rather than of pyruvate dehydrogenase, during the state 4 oxidation of pyruvate by isolated mitochondria, and that the modulation of citrate synthase activity by the ratio of acetyl-CoA/succinyl-CoA is unimportant under these conditions.     

Biosynthesis of Protein Amino Acids in Plant Tissue Culture IV Isotope Competition Experiments using Glucose-U-C and Potential Intermediates

The isotope competition method with glucose-U-¹⁴C as a carbon source has been used to determine whether or not selected compounds contribute carbon to the biosynthesis of protein amino acids in cells derived from Paul's Scarlet Rose. Of 48 compounds tested, 15 contributed carbon to protein amino acids. The results show for the first time that homoserine is an intermediate in threonine biosynthesis; that homoserine, cystathionine and homocysteine are intermediates in methionine biosynthesis and that histidinol is an intermediate in histidine biosynthesis in plant cells.     

The mechanism of antifungal action of (S)-2-amino-4-oxo-5-hydroxypentanoic acid, RI-331: the inhibition of homoserine dehydrogenase in Saccharomyces cerevisiae

We have explored the mechanism by which an antifungal antibiotic, (S)-2-amino-4-oxo-5-hydroxypentanoic acid, RI-331, preferentially inhibits protein biosynthesis in Saccharomyces cerevisiae, by inhibiting the biosynthesis of the aspartate family of amino acids, methionine, isoleucine and threonine. This inhibition was effected by inhibiting the biosynthesis of their common intermediate precursor homoserine. The target enzyme of RI-331 was homoserine dehydrogenase (EC.1.1.1.3) which is involved in converting aspartate semialdehyde to homoserine in the pathway from aspartate to homoserine. The enzyme is lacking in animals. So the antibiotic is selectively toxic to prototrophic fungi.     

Effect of technical threonine sources on homoserine biosynthesis by mutant Brevibacteruim flavum 2T

The effect of threonine technical sources on the homoserine biosynthesis by the threonine auxotroph Brevibacterium flavum 2T when cultivated on sucrose and acetic acid containing media was investigated. Various threonine sources (corn extract and fodder yeast, microbial biomass and soybean meal hydrolyzates) prepared by means of different hydrolyzing agents (acids, enzymes, autolysis) were used. The most effective substrate was protein--vitamin concentrate hydrolyzate, particularly combined with corn extract in the ratio 1: 0,25-0.5 (with respect to the dry weight of the initial material). The homoserine yield was 16.2 g/l on the sucrose containing medium and 18.4 g/l on the acetic acid containing medium which was in agreement with controls. The medium containing pure threonine was used as a control. With other threonine sources (corn extract, protein-vitamin concentrate autolyzate and enzymolyzate, fodder yeast and soybean meal hydrolyzates), the homoserine production was significantly lower, i.e. 40-70% of the control. The content of amino acids (threonine, isoleucine, methionine) in the initial material and their suitability for the homoserine biosynthesis were found to be correlated. The substrates with a high content of threonine (over 3.5%) and a low content of methionine (below 0.5%) proved most effective. The use of the material in which the ratio threonine: methionine was less than 6.0 caused the homoserine biosynthesis to be partially replaced with that of lysine.     

5'-adenosinephosphosulfate lies at a metabolic branch point in mycobacteria

Bacterial sulfate assimilation pathways provide for activation of inorganic sulfur for the biosynthesis of cysteine and methionine, through either adenosine 5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as intermediates. PAPS is also the substrate for sulfotransferases that produce sulfolipids, putative virulence factors, in Mycobacterium tuberculosis such as SL-1. In this report, genetic complementation using Escherichia coli mutant strains deficient in APS kinase and PAPS reductase was used to define the M. tuberculosis and Mycobacterium smegmatis CysH enzymes as APS reductases. Consequently, the sulfate assimilation pathway of M. tuberculosis proceeds from sulfate through APS, which is acted on by APS reductase in the first committed step toward cysteine and methionine. Thus, M. tuberculosis most likely produces PAPS for the sole use of this organism's sulfotransferases. Deletion of CysH from M. smegmatis afforded a cysteine and methionine auxotroph consistent with a metabolic branch point centered on APS. In addition, we have redefined the substrate specificity of the B. subtilis CysH, formerly designated a PAPS reductase, as an APS reductase, based on its ability to complement a mutant E. coli strain deficient in APS kinase. Together, these studies show that two conserved sequence motifs, CCXXRKXXPL and SXGCXXCT, found in the C termini of all APS reductases, but not in PAPS reductases, may be used to predict the substrate specificity of these enzymes. A functional domain of the M. tuberculosis CysC protein was cloned and expressed in E. coli, confirming the ability of this organism to make PAPS. The expression of recombinant M. tuberculosis APS kinase provides a means for the discovery of inhibitors of this enzyme and thus of the biosynthesis of SL-1.