Study of Substrate Specificity by Structural and Mutagenesis Analysis of Carboxyltranferase Subunits of the Streptomyces coelicolor A3(2)

DIACOVICH L., GAGO G., TSAI, S-C., AND GRAMAJO HUGO.

Angra do Rei, Brasil

Congreso; 1st Latin American Protein Society Meeting; 2004

Streptomyces coelicolor as being used to produce over two thirds of naturally derived antibiotics in current use, as well as antitumor agents, herbicides and pharmacologically active metabolites. In all, Streptomyces make over 6,000 different chemicals. An important percentage of those compounds are polyketide and the biosynthesis is limited both for the regulation of biosynthetic pathways and for the disposability of precursor molecules, malonyl- and methylmalonyl-CoA. Those compounds are synthesized by the carboxylation of acetyl-CoA  and propionyl-CoA, catalyzed  by the acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) complexes. Each complex is compounds by three different subunits. Both complexes shared the same biotinylated subunit a, AccA2. The b and the e  subunits were specific from each of the complexes (AccB-AccE and PccB-PccE to ACC y PCC respectively). The two complexes showed very low activity in the absence of the corresponding e subunits, 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, resulted in the inter-conversion of their corresponding substrate specificity of the ACC and PCC complexes. This indicates that molecular recognition in this multi-subunit complex may be implemented by the active site residues of the b (carboxyltransferase) subunit. Our finding enables bioengineering of the acyl-CoA carboxylase substrate specificity to provide novel extender units for the combinatorial biosynthesis of polyketide. The hexameric structure also helps visualize different oligomeric architectures of ACC and PCC from different organisms, as well as the identifiction of future drug design targets.