Substrate tolerance of acyl‐CoA ligases

Abstract

Acyl‐CoA ligases catalyze the activation of a carboxylic acid to an acyl‐CoA ester in a two‐step reaction via an adenylate intermediate. CoA esters are precursors in various biochemical processes such as fatty acid synthesis and polyketide synthesis. These enzymes are also known to have a remarkably broad substrate tolerance.Five acyl‐CoA ligases were studied – cinnamate:CoA ligase and phenylacetate:CoA ligase from Streptomyces coelicolor, benzoate:CoA ligase from Rhodopseudomonas palustris, malonate:CoA ligase from Rhizobium trifolii, and 4‐coumarate:CoA ligase from Nicotina tabacum. All enzymes were expressed in Escherichia coli and purified to homogeneity in a single step using Ni2+‐chelating chromatography. Using the purified enzyme, various derivatives of cinnamic acid, 3‐phenylpropanoic acid, and benzoic acid were used to screen for a possible conversion of the various aromatic carboxylic acids to their corresponding acyl‐CoA esters. Aliphatic carboxylic acids were also screened as possible substrates. This study found that these enzymes were able to catalyze the CoA esterification of various aliphatic and aromatic carboxylic acids, highlighting the potential use of acyl‐CoA ligases as biosynthetic enzymes; the generated carboxyl‐CoA esters can be used as precursors in the biosynthesis of various secondary metabolites.This project was supported by grants from the Ministry of Education, Singapore.