Crystal Structure of the β-Subunit of Acyl-CoA Carboxylase:  Structure-Based Engineering of Substrate Specificity^,

Abstract

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.

The enzymes of biotin dependent CO2 metabolism: What structures reveal about their reaction mechanisms

“…75 The biotin-binding site is lined with hydrophobic residues, which as pointed out by Diacovich et al, is very similar to the hydrophobic and van der Waals interactions observed between biotin and streptavidin. 74 The carbonyl oxygen of the ureido ring binds in an oxyanion hole, which is formed by the peptidic NH group of Ala 420 and the positive end of a helix dipole moment [ Fig.…”

“…72,74 The best characterized, with respect to three-dimensional architecture, is the b-subunit of propionyl-CoA carboxylase from S. coelicolor in which the binding pockets for both propionyl CoA and biotin have been identified. 75 In all of these enzymes, the acyl-CoA/CoA ligands bind in bent or U-shaped conformations with their carbonyl oxygens residing in oxyanion holes. For the 12S subunit of transcarboxylase, the carbonyl oxygen of methylmalonyl-CoA, and by inference its enolate ion, forms hydrogen bonds to the peptidic NH group of Ala 143 and a water molecule [ Fig.…”

“…The structure of the b-subunit of propionyl-CoA carboxylase liganded to both substrates allowed Diacovich et al, 75 to propose a catalytic mechanism for the enzyme that by inference is applicable to other carboxyltransferase subunits (Fig. 16).…”

OPINIONS Acetyl-coenzyme A carboxylase – an attractive enzyme for biotechnology

“…The interface of the CT domains within dimer is composed of highly conserved amino acids, which also create an active site, and are involved in interactions with thiol group of CoA. Based on molecular modeling and structure of related enzyme -propionylo-coenzyme A carboxylase, a biotin substrate is expected to approach the CT active site from C-subdomain of the other CT monomer (Diacovich et al, 2004).…”

An Asymmetric Model for Na+-translocating Glutaconyl-CoA Decarboxylases

“…5A). This bi-domain active site arrangement is common among members of the crotonase superfamily (47) and is characteristic for all structurally characterized biotin-dependent decarboxylases/carboxyltransferases (53).…”