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

Abstract

Pseudomonas putida is able to grow on malonate as a sole source of carbon and energy. Malonate decarboxylase is a key enzyme catabolizing malonate to acetate and CO2. The enzyme consists of the five different subunits, α (60 kDa), β (33 kDa), γ (28 kDa), δ (13 kDa), and e (30 kDa). The smallest subunit δ is an acyl-carrier protein (ACP) possessing 2'-(5-phosphoribosyl)-3'-dephospho-CoA as a prosthetic group. Acylation of ACP is the initial step triggering the decarboxylation of malonate in a cyclic manner, i.e., the δ subunit is activated to form acyl-S-ACP by the e subunit in the presence of acetyl-CoA or malonyl-CoA as an active acyl donor. The acetyl residue on the δ subunit is replaced with malonate by the α subunit, and the malonyl residue subsequently undergoes decarboxylation by the subunits β and γ, thereby regenerating acetyl-S-ACP. This unique cyclic reaction mechanism that amplifies acetate as its product in proportion to the amount of a given acetyl-CoA and/or malonyl-CoA is allows one to detect pmol levels of the CoA derivatives. Moreover, one can separately measure the three CoA molecular species, acetyl-CoA, malonyl-CoA, and nonesterified CoA (CoASH), by a combination of the elimination of acetyl-CoA with citrate synthetase (EC 4.1.3.7) or acetylation of CoASH with phosphate acetyltransferase (EC 2.3.1.8). The micromethod, named the acyl-CoA cycling method, is useful to define the rapid changes in vivo in the size and composition of the CoA pool. By this micromethod, it has been demonstrated that there is a remarkable difference in the size and composition of intracellular pools of CoASH and CoA thioesters between aerobic bacteria and facultatively anaerobic bacteria. It has also been revealed that the composition of CoA pools in facultatively anaerobic bacteria drastically changes within minuites in response to the quality and quantity of the carbon source in the medium, growth phase, pH, incubation temperature, osmotic stress, or antibiotics to inhibit energy yielding systems and fatty acid biosynthesis.