Crystal structures and mutational analyses of acyl-CoA carboxylase beta subunit of Streptomyces coelicolor

Abstract

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 homologue 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 toward 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 toward 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.

Fig. 1

Determination of the oligomeric state of PccB mutants. Each D422 mutant was analyzed by size exclusion chromatography (Superdex 200). The protein profiles (A215 in milli-absorbance units [mAU]) and molecular masses of protein standards used for calibration are detailed in the chromatograms.

Fig. 2

(A) The overall structures of all PccB mutants are highly similar as a hexamer, with each monomer in different color. (B) The PccB active site lies in between two monomers (in color and gray, respectively). (C) Zoom to the acyl-CoA binding pocket, showing the position of residue D422 at the “end” of the active site and the conformation of loops that contain N80 and R456 and R457, which define part of the acyl-CoA pocket entrance.

Fig. 3

(A) Interactions of the CoA phosphate groups with residues N80, R456 and R457 via hydrogen bonds in wild type PccB. (B) Enlarged view, sructural overlap between the wild-type (green) and D422N mutant (blue), showing the conformational changes near the 55 – 70 and 450 – 460 loops. (C) The Fo-Fc simulated annealing electron density map near residue 422 for D422L, contoured at 2.5 sigma. (D) Structural overlap of the biotin binding pockets of all the PccB mutant shows that there is no significant conformational change in the biotin binding pocket. In the enlarged panels (B), residue 422 is out of sight, but is located above the viewpoint.

Fig. 4

A comparison of acyl-CoA binding pocket between wild-type and PccB mutants. The molecular surface is colored from red to blue according to electronegative to electropositive surface potential. The labeled length measures the distance from the entrance (the N80 side chain) to the bottom (residue 422 side chain) of the acyl-CoA binding pocket. As a molecular rule, propionyl-CoA and biotin were docked into the apo mutant structures based on the co-crystal structure of the wild type substrate-biotin complex.

Fig. 5

Size exclusion chromatography of N80A, R456A and R456A-R457A mutants. The protein profiles (A215 in milli-absorbance units [mAU]) and molecular masses of protein standards used for calibration are detailed in the chromatogram.

Scheme 1

Stepwise reactions of ACCase.