Acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) catalyze the carboxylation of acetyl- and propionyl-CoA to generate malonyl- and methylmalonyl-CoA, respectively. Understanding the substrate specificity of ACC and PCC will (1) help in the development of novel structure-based inhibitors that are potential therapeutics against obesity, cancer, and infectious disease and (2) facilitate bioengineering to provide novel extender units for polyketide biosynthesis. ACC and PCC in Streptomyces coelicolor are multisubunit complexes. The core catalytic beta-subunits, PccB and AccB, are 360 kDa homohexamers, catalyzing the transcarboxylation between biotin and acyl-CoAs. Apo and substrate-bound crystal structures of PccB hexamers were determined to 2.0-2.8 A. The hexamer assembly forms a ring-shaped complex. The hydrophobic, highly conserved biotin-binding pocket was identified for the first time. Biotin and propionyl-CoA bind perpendicular to each other in the active site, where two oxyanion holes were identified. N1 of biotin is proposed to be the active site base. Structure-based mutagenesis at a single residue of PccB and AccB allowed interconversion of the substrate specificity of ACC and PCC. The di-domain, dimeric interaction is crucial for enzyme catalysis, stability, and substrate specificity; these features are also highly conserved among biotin-dependent carboxyltransferases. Our findings enable bioengineering of the acyl-CoA carboxylase (ACCase) substrate specificity to provide novel extender units for the combinatorial biosynthesis of polyketides.

Mycolic acids and multimethyl-branched fatty acids are found uniquely in the cell envelope of pathogenic mycobacteria. These unusually long fatty acids are essential for the survival, virulence, and antibiotic resistance of Mycobacterium tuberculosis. Acyl-CoA carboxylases (ACCases) commit acyl-CoAs to the biosynthesis of these unique fatty acids. Unlike other organisms such as Escherichia coli or humans that have only one or two ACCases, M. tuberculosis contains six ACCase carboxyltransferase domains, AccD1-6, whose specific roles in the pathogen are not well defined. Previous studies indicate that AccD4, AccD5, and AccD6 are important for cell envelope lipid biosynthesis and that its disruption leads to pathogen death. We have determined the 2.9-Å crystal structure of AccD5, whose sequence, structure, and active site are highly conserved with respect to the carboxyltransferase domain of the Streptomyces coelicolor propionyl-CoA carboxylase. Contrary to the previous proposal that AccD4 -5 accept long-chain acyl-CoAs as their substrates, both crystal structure and kinetic assay indicate that AccD5 prefers propionyl-CoA as its substrate and produces methylmalonyl-CoA, the substrate for the biosyntheses of multimethyl-branched fatty acids such as mycocerosic, phthioceranic, hydroxyphthioceranic, mycosanoic, and mycolipenic acids. Extensive in silico screening of National Cancer Institute compounds and the University of California, Irvine, ChemDB database resulted in the identification of one inhibitor with a K i of 13.1 M. Our results pave the way toward understanding the biological roles of key ACCases that commit acyl-CoAs to the biosynthesis of cell envelope fatty acids, in addition to providing a target for structure-based development of antituberculosis therapeutics.cell wall lipid ͉ multimethyl-branched fatty acid ͉ mycolic acid ͉ tuberculosis ͉ mycocerosic acid

The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologues multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities towards these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456 and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity towards acetyl-, propionyl- and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456 and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.

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