Formation of catalytically active S‐acetyl(malonate decarboxylase) requires malonyl‐coenzyme A

Abstract

Functional malonate decarboxylase of Klebsiella pneumoniae is an acetyl-S-enzyme with an acetylated phosphoribosyl dephospho-CoA prosthetic group. The mdcH gene product acts as a malonyl-CoA:ACP transacylase and initiates the activation of (deacetyl)malonate decarboxylase by malonyl-transfer to the prosthetic group. The malonyl residue is subsequently decarboxylated to an acetyl residue by the decarboxylase itself. Purified malonate decarboxylase consists of the four subunits MdcA, D, E and C in an apparent 1 : 1 : 1 : 1 stoichiometry. In addition, the preparation contains substoichiometric amounts of MdcH comigrating on SDS/PAGE with MdcD. Malonate decarboxylase isolated from strains with a deletion of the mdcH gene was not activated with malonyl-CoA. Activity could be gained, however, in the additional presence of MdcH that has been synthesized in Escherichia coli and purified from inclusion bodies. Substrates for MdcH are malonyl-CoA or methylmalonyl-CoA but not acetyl-CoA. The enzyme has K m values of 16 mm for both substrates and V max for malonyl-CoA of 190 U´mg ±1 and for methylmalonyl-CoA of 37 U´mg ±1 . Transfer of the methylmalonyl-residue to the prosthetic group proceeds via the covalent methylmalonyl-MdcH intermediate. The transacylase is specifically inhibited by N-ethylmaleimide, and preincubation with malonyl-CoA or methylmalonyl-CoA protects the enzyme from this inhibition.Keywords: enzyme activation; fatty acid biosynthesis; Klebsiella pneumoniae; malonate decarboxylase; malonylCoA:acyl carrier protein transacylase.In spite of the widely used diagnostic criterion of bacterial growth on malonate [1], the enzymic and genetic basis for this behavior was resolved only recently (for a review, see [2]). The key element is a specific malonate decarboxylase that converts malonate directly into acetate and CO 2 . Malonate is chemically rather inert, especially at neutral pH, where both carboxylic residues are dissociated (pK a1 = 2.85, pK a2 = 5.69). The dicarboxylate is therefore activated for the decarboxylation reaction by transiently forming a thioester with the enzyme. The catalytically active enzyme carries an acetyl thioester residue that is exchanged in the first partial reaction by a malonyl thioester residue. This is subsequently decarboxylated with regeneration of the acetyl-S-enzyme (Fig. 1). This malonate decarboxylation mechanism was discovered for the enzyme from Malonomonas rubra, an anaerobic bacterium capable to grow entirely from the free energy of this decarboxylation reaction [3]. Later, aerobic bacteria known to grow on malonate, e.g. Klebsiella pneumoniae [4], Acinetobacter calcoaceticus [5], Pseudomonas putida [6] or Pseudomonas fluorescens [7] were found to dispose of malonate decarboxylases that activate the substrate by the same mechanism forming malonyl-S-enzyme derivates. These aerobic decarboxylases release CO 2 directly from the malonyl-S-enzyme intermediates, forfeiting the free energy of the decarboxylation reaction. In contrast, the M. rubra decarboxylase system catalyze...

The Malonate Decarboxylase Operon of Acinetobacter calcoaceticus KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY

“…Dimroth's group and our laboratory have extensively characterized malonate metabolism in Malonomonas rubra, Klebsiella pneumoniae, and Acinetobacter calcoaceticus (2,10,14,15,(18)(19)(20)30). The first enzyme in the malonate metabolism, malonate decarboxylase, which catalyzes the decarboxylation of malonate to acetate, has also been studied in various other bacteria (6,13,18,20,30).…”

Malonate Decarboxylase in Bacteria and Its Application for Determination of Intracellular Acyl-CoA Thioesters

“…However, alternate malonate decarboxylases with ACP as a component of the enzyme were discovered in Malonomonas rubra [5][6][7]20,[29][30][31]76) , Klebsiella pneumoniae 20,[32][33][34][35]78) , Acinetobacter calcoaceticus 8,24,43,49,50) , and P. fluorescens 8,24,44) , and molecular biology-based analyses concerning the enzyme reaction mechanism have been developed. In 1998, we reanalyzed the subunit composition of the Pseudomonas enzyme, and discovered subunits d and e in addition to subunits a, b, and g 14) .…”

“…In the former from a microaerotolerant anaerobic bacterium, Malonomonas rubra, biotin carrier protein is responsible for the decarboxylation of malonyl-S-ACP [5][6][7]20,[29][30][31]76) . The enzymes from aerobic and facultatively anaerobic bacteria, Pseudomonas putida 11,13,14,83) , P. fluorescens 8,24,44) , Acinetobacter calcoaceticus 8,24,43,49,50) , and Klebsiella pneumoniae 20,[32][33][34][35]78) belong to the latter category. However, the prosthetic group is common to both categories of malonate decarboxylase, i.e., every enzyme contains ACP with a covalently attached 2'-(5''-phosphoribosyl)-3'-dephospho-CoA 6,20,50,78) (Fig.…”

“…The former consist of four subunits a, b, g, and d, and do not possess a malonyl-CoA:ACP transacylase corresponding to the e subunit of the P. putida enzyme. In these bacteria, malonyl-CoA:ACP transacylase independently exists in the cytoplasm 20,50,78) or part of the proteins substoichiometrically associate with the four subunits 32) , and participates in the initial step of malonate decarboxylation (Fig. 1A).…”

“…MdcH consists of 306 amino acids (32,623 Da) in the e subunit, which catalyzes the transfer of the malonyl residue of malonyl-CoA to ACP, and is easily dissociated from the other subunits 14) . The amino acid sequence alignment of the gene poduct shows high similarities with various malonylCoA:ACP transacylases (EC 2.3.1.39), e.g., MdcHs from K. pneumoniae (57%) and A. calcoaceticus (23%) and FabDs from Bacillus subtilis (30%, U59433) 62) , E. coli (36%, Z11565 and M87040) 58,92) , and Haemophilus influenzae (36%, U32701) 22) .…”