AccD6, a Member of the Fas II Locus, is a Functional Carboxyltransferase Subunit of the Acyl-CoA Carboxylase in Mycobacterium tuberculosis

Burnett College of Biomedical Sciences, University of Central Florida, BMS 136, 4000 Central Florida Blvd., Orlando, FL 32816-2364

^* To whom correspondence should be addressed. Email: pk{at}mail.ucf.edu.

	Abstract

The Mycobacterium tuberculosis acyl-CoA carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I end-products by the FAS II complex to produce meromycolic acids. M. tuberculosis genome contains three biotin carboxylase subunits (AccA1-3) and 6 carboxyltransferase subunits (AccD1-6) of which AccD6 is 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 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.