AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in Mycobacterium tuberculosis

Abstract

The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II complex to produce meromycolic acids. The M. tuberculosis genome contains three biotin carboxylase subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with accD6 located in a genetic locus that contains members of the FAS II complex. We found by quantitative real-time PCR analysis that the transcripts of accA3, accD4, accD5, and accD6 are expressed at high levels during the exponential growth phases of M. tuberculosis in vitro. Microarray analysis of M. tuberculosis transcripts indicated that the transcripts for accA3, accD4, accD5, accD6, and accE were repressed during later growth stages. AccD4 and AccD5 have been previously studied, but there are no reports on the function of AccD6. We expressed AccA3 (␣ 3 ) and AccD6 (␤ 6 ) in E. coli and purified them by affinity chromatography. We report here that reconstitution of the ␣ 3 -␤ 6 complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1 nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the ␣ 3 -␤ 6 complex was inhibited by the subunit. The ␣ 3 -␤ 6 carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the ␤ 6 subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS II complex.Tuberculosis causes 2 million deaths each year, according to the World Health Organization. Mycobacterium tuberculosis, the pathogen that causes the disease, infects 8 million people each year and is one of the world's deadliest pathogens (9). The ongoing AIDS pandemic has developed a deadly synergy with tuberculosis, which is the leading cause of death among AIDS patients (2). Multidrug-resistant M. tuberculosis strains have been emerging rapidly (9), and the need for identifying novel drug targets in this pathogen has become urgent. The cell wall of M. tuberculosis is lipid enriched and acts as an impermeable barrier to many common broad-spectrum antibiotics (14).The first committed step of fatty-acid biosynthesis, which is the biotin-dependent carboxylation of acyl-coenzyme A (CoA) to produce malonyl-CoA and methylmalonyl-CoA, is catalyzed by the acyl-CoA carboxylase. The reaction consists of two catalytic steps, which involve the biotin carboxylase and the carboxyltransferase (8). In M. tuberculosis, the biotin carboxylation step is catalyzed by the ␣ subunit; there are three open reading frames (ORFs) that can encode the ␣ subunit (accA1 to -A3) in the genome. Carboxyl transfer is catalyzed by the ␤ subunit, and there are six ␤ subunits (accD1 to -D6) in the genome of the pathogen (6).Previously, the catalytic activities of the ␣ 3 , ␤ 4 , and ␤ 5 subunits were studied (10,11,22,24). However, the level...

AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium tuberculosis, Is Dispensable in a Nonpathogenic Strain

“…Daniel et al confirmed that M. tuberculosis accD6 (accD6 Mtb ) is highly expressed during intensive mycolate biosynthesis and showed that the AccD6 and AccA3 proteins could together reconstitute an enzyme that is able to carboxylate acetyl-CoA in vitro (see Fig. S1 in the supplemental material) (18). However, due to the essential nature of accD6, the possible involvement of AccD6 in mycolic acid biosynthesis has never been addressed in vivo.…”

“…The expression profiles of all nine genes predicted to encode the carboxylase subunits of M. tuberculosis have been evaluated previously (18). Quantitative real-time PCR (qRT-PCR) analysis revealed that only the genes encoding the AccA3, AccD4, AccD5, and AccD6 carboxylase subunits were expressed at high levels during M. tuberculosis exponential growth.…”

“…To date, the roles of accD4 and accD5 in mycolic acid biosynthesis have been studied (see Fig. S1 in the supplemental material) (21,28,38,51,58), and the expression profiles of all accD family members in M. tuberculosis are known (18).…”

“…S1 in the supplemental material): the mammalian-type multienzyme fatty acid synthase I (FAS-I) and fatty acid synthase II (FAS-II), which is a set of autonomic enzymes similar to those found in other bacteria (8,59,68,82). FAS-I catalyzes the de novo synthesis of short fatty acyl primers (typically C [16][17][18][19][20][21][22][23][24][25][26], and the FAS-II system subsequently elongates these short chains into long-chain fatty acids (C [48][49][50][51][52][53][54][55][56] ). Mycolic acid biosynthesis is also the target of powerful antitubercular drugs, such as isoniazid (INH), ethionamide, and thiolactomycin (3,27,31,44,53,70,72), and an emerging target for future inhibitors.…”

Crystal structure of the essential biotin‐dependent carboxylase AccA3 from Mycobacterium tuberculosis

“…AccA3–AccD5 have been shown to act mainly on propionyl‐CoA, producing methylmalonyl‐CoA, used for synthesis of methyl‐branched lipids, key components of the mycobacterial cell wall. AccA3–AccD6 acts mainly on acetyl‐CoA, producing malonyl‐CoA for fatty acid biosynthesis 11, 15, 21, 22.…”