Overexpression and Kinetic Characterization of the Carboxyltransferase Component of Acetyl-CoA Carboxylase

Abstract

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report the overexpression of the genes for the carboxyltransferase component is described. The steady-state kinetics of the recombinant carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The initial velocity patterns indicated that the kinetic mechanism is equilibrium-ordered with malonyl-CoA binding before biocytin and the binding of malonyl-CoA to carboxyltransferase at equilibrium. The biotin analogs, desthiobiotin and 2-imidazolidone, inhibited carboxyltransferase. Both analogs exhibited parabolic noncompetitive inhibition, which means that two molecules of inhibitor bind to the enzyme. The pH dependence for both the maximum velocity (V) and the (V/K) biocytin parameters decreased at low pH. A single ionizing group on the enzyme with a pK of 6.2 or lower in the (V/K) biocytin profile and 7.5 in the V profile must be unprotonated for catalysis. Carboxyltransferase was inactivated by N-ethylmaleimide, whereas malonyl-CoA protected against inactivation. This suggests that a thiol in or near the active site is needed for catalysis. The rate of inactivation of carboxyltransferase by N-ethylmaleimide decreased with decreasing pH and indicated that the pK of the sulfhydryl group had a pK value of 7.3. It is proposed that the thiolate ion of a cysteine acts as a catalytic base to remove the N1 proton of biocytin.The first committed step in fatty acid biosynthesis is catalyzed by acetyl-CoA carboxylase (1). The enzyme is found in all animals, plants, and bacteria and catalyzes the biotin-dependent carboxylation of acetyl-CoA to form malonyl-CoA in the two steps of Reaction 1.In Escherichia coli the enzyme is composed of three components: biotin carboxylase, carboxyltransferase, and biotin carboxyl carrier protein, which contains the biotin covalently attached to the ⑀-nitrogen of lysine (2). Biotin carboxylase catalyzes the first half-reaction in which biotin is carboxylated to form carboxybiotin. Carboxyltransferase catalyzes the second half-reaction where the carboxyl group is transferred from biotin to acetyl-CoA to form malonyl-CoA. Both biotin carboxylase and the carrier protein are homodimers, whereas the carboxyltransferase component is an ␣ 2 ␤ 2 tetramer. Each of the three components can be isolated separately, and the biotin carboxylase and carboxyltransferase components retain catalytic activity in the absence of the other two components. Moreover, biotin carboxylase and carboxyltransferase will utilize free biotin as a substrate, which makes them ideal model systems for studying mechanistic aspects of biotin-dependent enzymes.Most mechanistic studies of acetyl-CoA carboxylase have focused on the biotin carboxylase component because the gene for the enzyme has bee...

Andrimid producers encode an acetyl-CoA carboxyltransferase subunit resistant to the action of the antibiotic

“…2B). Both AccA/AccD and AccA/AdmT complexes yielded Ͼ20 mg of soluble proteins per liter of E. coli culture and diminished the potential endogenous E. coli AccA binding to Ͻ0.1% (11,16).…”

“…Biocytin (in excess) was used as a BCCP surrogate (11,16). The production of acetyl-CoA was coupled to the citrate synthase/malate dehydrogenase reaction cycle, which requires NAD ϩ reduction and was monitored at 340 nm (11,16).…”

“…Biocytin (in excess) was used as a BCCP surrogate (11,16). The production of acetyl-CoA was coupled to the citrate synthase/malate dehydrogenase reaction cycle, which requires NAD ϩ reduction and was monitored at 340 nm (11,16). Kinetic analyses of AccA/AdmT revealed an apparent K m of 149 Ϯ 26 M for malonyl-CoA and 5.1 Ϯ 0.5 mM for biocytin, indicating that the purified AccA/AdmT forms an active CT with catalytic efficiency comparable to that of the E. coli AccA/AccD pair (6,11).…”

“…The His-tagged AccA/AccD, AccA/AdmT, and their mutant protein complexes were overproduced in E. coli BL21 DE3 in a manner analogous to that described previously (11). The proteins were purified to apparent homogeneity by Ni-NTA agarose column and buffer-exchanged to 25 mM Hepes (pH 7.5) and 500 mM NaCl.…”

“…For the in vitro assay of AccD and AdmT activities, they were cloned as one operon with the E. coli accA gene, respectively. A chimeric accA/accD gene containing an XhoI restriction site and a ribosomal binding site in between was first constructed as described previously (11). In brief, accA and accD genes were amplified from plasmids pLS151 (31) and pSJ9 by using primers 4 -7 (Table S1) and overlap-extended by using primers 4 and 7 to give the target chimeric accA/accD gene.…”