Structure based engineering of substrate specificity of the beta subunit of acyl-CoA carboxylase of Stretomyces coelicolor A3(2).

L. DIACOVICH, G. GAGO, A. ARABOLAZA, H. GRAMAJO.

Iguazu, Misiones

Congreso; XL Reunion Anual de la Sociedad Argentina de Bioquimica y Biologia Molecular (SAIB); 2004

Sociedad Argentina de Bioquimica y Biologia Molecular (SAIB)

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 help to develop novel structure-based inhibitors that are potential therapeutics against infectious disease. It would also facilitate bioengineering to provide novel extender units for polyketide biosynthesis. Streptomyces coelicolor ACC and PCC are multisubunit complexes. The core catalytic beta ƒnƒnsubunits, 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 solved 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.