• Korean Journal of Microbiology
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• v.29 no.1
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• pp.40-48
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• 1991

Two malonate-specific enzymes, malonyl-CoA synthetase and malonamidase, were found in free-living cultures of Rhizobium japonicum, Rhizobium meliloti, and Rhizobium trifolii, that infect plant roots where contain a high concentration of malonate. Malonyl-CoA synthetase catalyzes the formation of malonyl-CoA, AMP, and PPi directly from malonate, coenzyme A, and ATP in the presence of $Mg^{2+}$ Malonamidase is a novel enzyme that catalyzes hydrolysis and malonyl transfer of malonamate, and forms malonohydroxamate from malonate and hydroxylamine. Both enzymes are highly specific for malonate. These results show that Rhizobia have enzymes able to metabolize malonate and suggest that malonate may be used in symbiotic carbon and nitrogen metabolism.

• BMB Reports
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• v.32 no.3
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• pp.288-293
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• 1999

In order to understand the initial interaction of the substrates malonate, ATP, and CoA with malonyl-CoA synthetase, the catalytic product malonyl-CoA was characterized by NMR spectroscopy and molecular modeling. To assign proton and carbon chemical shifts, two-dimensional $^1H-^1H$ DQF-COSY and $^1H-^{13}C$ HMBC experiments were used. The structure of malonyl-CoA in the solution phase was determined based on distance constraints from NOESY and ROESY spectra. The structures were well-converged around the pantetheine region with the pairwise RMSD value of 0.08 nm. The solution structure exhibited a compact folded conformation with intramolecular hydrogen bonds among its carbonyl and hydroxyl groups. These findings will help us to understand the initial interaction of malonate and CoA with malonyl-CoA synthetase.

• Korean Journal of Microbiology
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• v.23 no.3
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• pp.230-234
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• 1985

A bacterium which can utilize malonate as a sole carbon source was isolated from soil. This strain was identified to be Acinetobacter calcoaceticus by morphological, cultural, phtsiological and biochemical examination. When this microorganism was grown on malonate as a aole carbon source, the enzymes, such as malonyl-CoA synthetase, isocitrate lyase and malate synthase were induced. These results suggest that in this microorganism, malonate is also assimilated through the proposed pathway in Pseudomonas fluorescens: $malonate{\rightarrow}malonyl-CoA{\rightarrow}acetyl-CoA{\rightarrow}glyoxylate\;cycle$.

• Bulletin of the Korean Chemical Society
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• v.15 no.5
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• pp.394-399
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• 1994

The catalytic mechanism of malonyl-CoA synthetase from Rhizobium trifolii was investigated by the steady state kinetics and intermediate identification. 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 $0.17{\pm}0.04 {\mu}M,\;0.24{\pm}0.18 {\mu}M\;and\;0.045{\pm}0.26 {\mu}$M for ATP, malonate and CoA, respectively. The TLC analysis of the $^{32}P-labelled$ products in reaction mixture containing $[{\gamma}-^{32}P]$ 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 $^{31}P-NMR$ spectra of AMP product isolated from the $^{18}O$ transfer experiment using $[^{18}O]$malonate. Two resonances were observed, corresponding to AMP labelled with zero and one atom of $^{18}O$, indicating that one atom of $^{18}O$ transferred from $[^{18}O]$malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the TLC analysis of reaction mixture containing $[{\alpha}-^{32}P]$ATP. These results strongly support the ordered Bi Uni Uni Bi Ping-Pong Ter Ter mechanism deduced from the initial velocity and product inhibition studies.

• Proceedings of the Korean Magnetic Resonance Society Conference
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• 2001.02a
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• pp.34-34
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• 2001

• Proceedings of the Korean Magnetic Resonance Society Conference
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• 1999.06a
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• pp.21-21
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• 1999

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.427-433
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• 2008

The cephabacins produced by Lysobacter lactamgenus are ${\beta}$-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, ${\beta}$-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of $His_6$-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-L-Ala-L-$^3H$-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with $^{14}C$-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.

• BMB Reports
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• v.35 no.5
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• pp.443-451
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• 2002

Malonate is a three-carbon dicarboxylic acid. It is well known as a competitive inhibitor of succinate dehydrogenase. It occurs naturally in biological systems, such as legumes and developing rat brains, which indicates that it may play an important role in symbiotic nitrogen metabolism and brain development. Recently, enzymes that are related to malonate metabolism were discovered and characterized. The genes that encode the enzymes were isolated, and the regulation of their expression was also studied. The mutant bacteria, in which the malonate-metabolizing gene was deleted, lost its primary function, symbiosis, between Rhizobium leguminosarium bv trifolii and clover. This suggests that malonate metabolism is essential in symbiotic nitrogen metabolism, at least in clover nodules. In addition to these, the genes matB and matC have been successfully used for generation of the industrial strain of Streptomyces for the production of antibiotics.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1143-1146
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• 2011

Metabolic engineering of plant-specific phenylpropanoid biosynthesis has attracted an increasing amount of attention recently, owing to the vast potential of flavonoids as nutraceuticals and pharmaceuticals. Recently, we have developed a recombinant Streptomyces venezuelae as a heterologous host for the production of flavonoids. In this study, we successfully improved flavonoid production by expressing two sets of genes predicted to be involved in malonate assimilation. The introduction of matB and matC encoding for malonyl-CoA synthetase and the putative dicarboxylate carrier protein, respectively, from Streptomyces coelicolor into the recombinant S. venezuelae strains expressing flavanone and flavone biosynthetic genes resulted in enhanced production of both flavonoids.