Biochemical and Structural Characterization of an Essential Acyl Coenzyme A Carboxylase from Mycobacterium tuberculosis

Abstract

Pathogenic mycobacteria contain a variety of unique fatty acids that have methyl branches at an evennumbered position at the carboxyl end and a long n-aliphatic chain. One such group of acids, called mycocerosic acids, is found uniquely in the cell wall of pathogenic mycobacteria, and their biosynthesis is essential for growth and pathogenesis. Therefore, the biosynthetic pathway of the unique precursor of such lipids, methylmalonyl coenzyme A (CoA), represents an attractive target for developing new antituberculous drugs. Heterologous protein expression and purification of the individual subunits allowed the successful reconstitution of an essential acyl-CoA carboxylase from Mycobacterium tuberculosis, whose main role appears to be the synthesis of methylmalonyl-CoA. The enzyme complex was reconstituted from the ␣ biotinylated subunit AccA3, the carboxyltransferase ␤ subunit AccD5, and the subunit AccE5 (Rv3281). The kinetic properties of this enzyme showed a clear substrate preference for propionyl-CoA compared with acetyl-CoA (specificity constant fivefold higher), indicating that the main physiological role of this enzyme complex is to generate methylmalonyl-CoA for the biosynthesis of branched-chain fatty acids. The ␣ and ␤ subunits are capable of forming a stable ␣6-␤6 subcomplex but with very low specific activity. The addition of the subunit, which binds tightly to the ␣-␤ subcomplex, is essential for gaining maximal enzyme activity.

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

“…The reconstituted ␣ 3 -␤ 6 acyl-CoA carboxylase, reported here, preferred acetyl-CoA over propionyl-CoA, in contrast to the ␣ 3 -␤ 5 complex, which preferred propionyl-CoA over acetyl-CoA (10,22). This observation leads us to suggest that M. tuberculosis may utilize the ␤ 6 subunit with ␣ 3 to provide malonyl-CoA to FAS I and to the FAS II complex for de novo fatty-acid biosynthesis and mycolic acid biosynthesis, respectively, and the ␤ 5 subunit with ␣ 3 for providing methylmalonylCoA for branched-fatty-acid biosynthesis.…”

“…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 levels of expression of the various subunits have not been examined.…”

“…Previously, the catalytic activities of the ␣ 3 , ␤ 4 , and ␤ 5 subunits were studied (10,11,22,24). However, the levels of expression of the various subunits have not been examined.…”

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

“…Mycobacterium tuberculosis AccA3 thus provides the carboxylated biotin to all of the three essential AccD proteins 14, 15, 18, 19, 20. Moreover, the same interaction pattern has been found in MTb 11, 23.…”

“…MTb AccA3 has been shown to form functional Acetyl‐CoA Carboxylase complexes with AccD4, AccD5, and AccD6 14, 15, 18, 19, 20. The MTb AccA3‐AccD4 Acetyl‐CoA Carboxylase carboxylates long‐chain acyl‐CoA substrates.…”