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Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction.

ABSTRACT: Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.

PROVIDER: S-EPMC1137833 | BioStudies | 1994-01-01

REPOSITORIES: biostudies

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Purification and properties of malonyl-CoA synthetase from Rhizobium japonicum.

Project description:A novel malonyl-CoA synthetase was found in Rhizobium japonicum bacteroid of the soybean nodule. The levels of the enzyme in the free-living cells grown on a variety of carbon sources including glucose were similar, indicating that this enzyme is not inducible. The malonyl-CoA synthetase from glucose-grown Rhizobium japonicum was purified to homogeneity. The Mr of the enzyme was determined to be 58,000 by gel filtration on a Sephacryl S-300 and by SDS/PAGE respectively, indicating a single polypeptide enzyme. N-Terminal amino acid of the enzyme was methionine but the enzyme preparation contained about 40% de-methionylated protein. The enzyme catalyses the formation of malonyl-CoA, AMP and PPi directly from malonate, CoA and ATP in the presence of Mg2+. High substrate specificity on malonate and ATP was revealed, but Mn2+ could be substituted for Mg2+ without any difference in activity. Optimum pH was 7.9. Kinetic constants, Km and Vmax, for malonate, CoA and ATP were 200 microM and 21.3 mumol/min per mg, 87 microM and 41.7 mumol/min per mg, and 33.3 microM and 29.4 mumol/min per mg respectively. Succinate inhibited the enzyme noncompetitively, whereas AMP and ADP inhibited competitively. Diethylpyrocarbonate and pyridoxal-5'-phosphate severely inhibited the enzyme, but iodoacetamide, p-chloromercuriphenylsulphonate, N-acetylimidazole and phenylglyoxal did not.

1991-01-01 | S-EPMC1149792 | BioStudies

Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii.

Project description:Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either. Kinetic analysis of the mutants revealed that Lys-170 and His-206 play a critical role for ATP binding and the formation of malonyl-AMP, whereas Arg-168 is critical for formation of malonyl-CoA and specificity for malonyl-AMP. Molecular modelling based on the crystal structures of luciferase and gramicidin S synthetase 1 provided MCS structure which could fully explain all these biochemical data even though the MCS model was generated by comparative modelling.

1999-01-01 | S-EPMC1220626 | BioStudies

The active site and substrates binding mode of malonyl-CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site-directed mutagenesis, and comparative modeling studies.

Project description:The active sites and substrate bindings of Rhizobium trifolii molonyl-CoA synthetase (MCS) catalyzing the malonyl-CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site-directed mutagenesis, and comparative modeling methods. The MCS-bound conformation of malonyl-CoA was determined from two-dimensional-transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural domains and consists of 16 alpha-helices, 24 beta-strands, and several long loops. The core active site was determined as a wide cleft close to the end of the small C-terminal domain. The catalytic substrate malonate is placed between ATP and His206 in the MCS enzyme, supporting His206 in its catalytic role as it generates reaction intermediate, malonyl-AMP. These findings are strongly supported by previous biochemical data, as well as by the site-directed mutagenesis data reported here. This structure reveals the biochemical role as well as the substrate specificity that conservative residues of adenylate-forming enzymes have.

2000-01-01 | S-EPMC2144687 | BioStudies

Studies on medium-chain fatty acyl-coenzyme a synthetase. Enzyme fraction II: mechanism of reaction and specific properties.

Project description:1. The mechanism of reaction of fatty acyl-CoA synthesis catalysed by fatty acyl-CoA synthetase from ox liver (fraction II; Bar-Tana, Rose & Shapiro, 1968) was investigated by a kinetic study of CoA disappearance dependent on butyrate plus ATP or butyryl-AMP (overall and partial reaction b respectively). 2. Contrary to findings with another enzyme (fraction I), a Bi Uni Uni Bi Ping Pong mechanism (Cleland, 1963a,b,c) corresponding to Berg's (1956) scheme of reaction was eliminated and an ordered Ter Ter mechanism with an A-C-B (standing for ATP, CoA and butyrate respectively) sequence of substrate entry for the overall reaction was established for fraction II. Partial reaction (b) was found to follow the ;Iso-Theorell-Chance' mechanism. 3. Also, in contrast with results obtained with fraction I, no allosteric properties could be demonstrated with fraction II.

1968-01-01 | S-EPMC1186786 | BioStudies

The Mammalian Malonyl-CoA Synthetase ACSF3 Is Required for Mitochondrial Protein Malonylation and Metabolic Efficiency.

Project description:Malonyl-coenzyme A (malonyl-CoA) is a central metabolite in mammalian fatty acid biochemistry generated and utilized in the cytoplasm; however, little is known about noncanonical organelle-specific malonyl-CoA metabolism. Intramitochondrial malonyl-CoA is generated by a malonyl-CoA synthetase, ACSF3, which produces malonyl-CoA from malonate, an endogenous competitive inhibitor of succinate dehydrogenase. To determine the metabolic requirement for mitochondrial malonyl-CoA, ACSF3 knockout (KO) cells were generated by CRISPR/Cas-mediated genome editing. ACSF3 KO cells exhibited elevated malonate and impaired mitochondrial metabolism. Unbiased and targeted metabolomics analysis of KO and control cells in the presence or absence of exogenous malonate revealed metabolic changes dependent on either malonate or malonyl-CoA. While ACSF3 was required for the metabolism and therefore detoxification of malonate, ACSF3-derived malonyl-CoA was specifically required for lysine malonylation of mitochondrial proteins. Together, these data describe an essential role for ACSF3 in dictating the metabolic fate of mitochondrial malonate and malonyl-CoA in mammalian metabolism.

| S-EPMC5482780 | BioStudies

Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of incubation with Ca2+, Mg2+ and ATP on enzyme activity in tissue extracts.

Project description:1. The effect of preincubation of extracts of lactating rat mammary gland with ATP, Mg2+ and micromolar concentrations of Ca2+ on the activity of acetyl-CoA carboxylase was studied. 2. Both Mg2+ and Ca2+ activated the enzyme. Activation with Mg2+ (5 mM) was larger than that with Ca2+ (calculated free Ca2+ concentration = 20-50 microM), but the activity decreased after reaching a peak. The activation obtained with Ca2+ was stable for up to 180 min. 3. Incubation with Ca2+ and Mg2+ together resulted in an activation that was slightly higher than that with Mg2+ only and was stable (compare the results for Ca2+ alone). 4. Preincubation in the absence of Mg2+, but not in the absence of Ca2+, resulted in the impairment of subsequent activation with either Mg2+ (when preincubation was with Ca2+ alone) or Mg2+ plus Ca2+. 5. KF (50 mM) prevented the activation of acetyl-CoA carboxylase by Ca2+ and Mg2+. 6. MgATP2- reversed (Mg2+ + Ca2+)-mediated activation and decreased the activity of acetyl-CoA carboxylase to about 10% of initial activity. Inhibition by ATP was unaffected by addition of cyclic AMP or cyclic AMP-dependent protein kinase inhibitor. 7. 32P was incorporated into acetyl-CoA carboxylase when incubations were carried out in the presence of [gamma-32P]ATP. Subsequent removal of ATP from the incubation medium resulted in rapid loss of 32P from acetyl-CoA carboxylase. 8. It is suggested that extracts of rat mammary gland contain endogenous protein kinase and phosphatase activities that modulate acetyl-CoA carboxylase activity through reversible phosphorylation and dephosphorylation. The phosphatase activity is sensitive to both Mg2+ and micromolar concentrations of Ca2+, whereas the kinase does not appear to be cyclic AMP-dependent.

1981-01-01 | S-EPMC1163586 | BioStudies

Structure-guided expansion of the substrate range of methylmalonyl coenzyme A synthetase (MatB) of Rhodopseudomonas palustris.

Project description:Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are two of the most commonly used extender units for polyketide biosynthesis and are utilized to synthesize a vast array of pharmaceutically relevant products with antibacterial, antiparasitic, anticholesterol, anticancer, antifungal, and immunosuppressive properties. Heterologous hosts used for polyketide production such as Escherichia coli often do not produce significant amounts of methylmalonyl-CoA, however, requiring the introduction of other pathways for the generation of this important building block. Recently, the bacterial malonyl-CoA synthetase class of enzymes has been utilized to generate malonyl-CoA and methylmalonyl-CoA directly from malonate and methylmalonate. We demonstrate that in the purple photosynthetic bacterium Rhodopseudomonas palustris, MatB (RpMatB) acts as a methylmalonyl-CoA synthetase and is required for growth on methylmalonate. We report the apo (1.7-Å resolution) and ATP-bound (2.0-Å resolution) structure and kinetic analysis of RpMatB, which shows similar activities for both malonate and methylmalonate, making it an ideal enzyme for heterologous polyketide biosynthesis. Additionally, rational, structure-based mutagenesis of the active site of RpMatB led to substantially higher activity with ethylmalonate and butylmalonate, demonstrating that this enzyme is a prime target for expanded substrate specificity.

| S-EPMC3426712 | BioStudies

Malonyl-coenzyme A reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus spp.

Project description:Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO2 fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO2 into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H+ --> malonate-semialdehyde + CoA + NADP+. In its native state it is associated with small RNA. Its activity was stimulated by Mg2+ and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (Km = 25 microM) and malonyl-CoA (Km = 40 microM). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.

| S-EPMC1698253 | BioStudies

Malonate catabolism does not drive N2 fixation in legume nodules.

Project description:Malonyl-coenzyme A (CoA) decarboxylase, malonyl-CoA synthetase, and malonate transporter mutants of Rhizobium leguminosarum bv. viciae and trifolii fixed N2 at wild-type rates on pea and clover, respectively. Thus, malonate does not drive N2 fixation in legume nodules.

| S-EPMC3697510 | BioStudies

Mammalian ACSF3 protein is a malonyl-CoA synthetase that supplies the chain extender units for mitochondrial fatty acid synthesis.

Project description:The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate.

| S-EPMC3190830 | BioStudies

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