Zn‐finger domain of carboxyltransferase plays a role in nucleic acid binding and enzymatic activity

Abstract

Acetyl‐CoA carboxylase catalyzes the first, committed step in fatty acid synthesis, converting acetyl‐CoA to malonyl‐CoA. In contrast to the single polypeptide of eukaryotes, the bacterial form of the enzyme is a multi protein complex: a biotin carboxylase, a carrier protein, and a carboxyltransferase (CT). The crystal structures of the E. coli and S. aureus CT component reveal a small, independent, four cysteine Zn binding domain that lacks a complement in the eukaryotic homologue. Zn fingers are common features of proteins that bind nucleic acids. Through electrophoretic mobility shift assays, CT was found to bind DNA in a cooperative, non‐specific manner. Enzymatic analysis of single cysteine to alanine mutants reveals no change in Km for both substrates malonyl‐CoA and biocytin, and 0.7 to 12.7% of wildtype Vmax. No activity is observed for multi‐site mutants. DNA binding is similarly affected, with the single mutants having a 10‐fold increase in Kd; and multi‐site mutants losing all DNA binding ability. This shows that the Zn domain is required for catalysis as well as for DNA binding. What distinguishes CT from other DNA binding enzymes is that enzymatic function and DNA binding are inextricably linked through the Zn domain. The unique Zn domain, adjacent to the active site of CT, makes for a unique target for the development of novel antibiotics capable of highly specific binding and presumably marginal toxicity.