Residues involved in the catalytic mechanism and substrate specificity of the beta subunit in acyl-CoA carboxylases of S. coelicolor

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

Pinamar, Buenos Aires

Congreso; XLI Reunion Anual de la Sociedad Argentina de Bioquimica y Biologia Molecular (SAIB). X Congreso PABMB. Panamerican Association for Biochemistry and Molelucar Biology.; 2005

Sociedad Argentina de Bioquimica y Biologia Molecular y Panamerican Association for Biochemistry and Molelucar Biology.

Acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) catalyze the carboxylation of acetyl- and propionyl-CoA to generatemalonyl and ethylmalonyl-CoA, respectively. These molecules are essential building units for the fatty acid and polyketide biosynthesis. The polyketide compounds, which comprise many pharmaceutically relevant natural products, are synthesized by polyketide synthases in a combinatorial fashion. Understanding the substrate recognition of ACCases could lead to the generation of mutants with relaxed substrate specificity. These mutants can provide novel extender units to polyketide synthases and make “unnatural” natural polyketide products. Each ACCase complex is composed of three different subunits. Both complexes share the same biotinylated -subunit , AccA2. The and the subunits are specific for each of the complexes (AccB-AccE and PccBPccE to ACC and PCC respectively). The two complexes show very low activity in the absence of the corresponding subunits and the addition of PccE or AccE dramatically increased the specific activity of the enzymes. PccB and AccB are 360 kDa homo-hexamers, catalyzing the transcarboxylation between biotin and acyl-CoAs. Apo and substrate bound crystal structures of PccB hexamers were solved to 2.0- 2.8 Å. The hexamer assembly forms a ring-shaped complex. The hydrophobic, highly conserved biotin-binding pocket was identified for the first time. The di-domain, dimeric interaction is crucial for enzyme catalysis, stability and substrate specificity. Based on the amino acid sequence alignment, propionyl-CoA bound structure and structure comparison between AccB and PccB, the molecular basis of substrate specificity was investigated: an active site residue was identified as the possible recognition feature for substrate specificity of AccB (accepting C2-C4 substrates) and PccB (accepting only C3 and C4 substrates). The substitution of this residue in AccB by the corresponding one in PccB, and vice versa, rose a PccB mutant D422I capable of carboxylase Acetyl-CoA. The construction of futher mutants confirm the relevance of the hypothesized residues involved in the substrate specificity and catalytic mechanism.