Analysis of ACC Oxidase Activity by Site-Directed Mutagenesis of Conserved Amino Acid Residues

Abstract

Site-directed mutagenesis of ACC oxidase (ACO) was used to determine the nature and role of conserved amino acid residues in the mechanism by which CO₂ activates the enzyme. Mutants of ACO were expressed in E. coli as His-Tag fusion proteins. A consensus sequence search of 38 known or putative ACO revealed 8 completely conserved lysine residues; K⁷², K¹⁴⁴, K¹⁵⁸, K¹⁷², K¹⁹⁹, K²³⁰ K²⁹² and K²⁹⁶. All of the lysine mutant forms were typically activated by CO₂ indicating that none of them is essential for CO₂ activation by a carbamylation mechanism. H¹⁷⁷, H²³⁴ and D¹⁷⁹ are essential ligands for Fe. The H¹⁷⁷, H²³⁴ and D¹⁷⁹ ligands for Fe in ACO have equivalent residues in isopenicillin N synthase (IPNS) as H²¹⁴, D²¹⁶ and H²⁷⁰. ACO, a non-heme Fe²⁺/ascorbate requiring enzyme, belongs to the IPNS protein structure family. The Cterminal sequence from K²⁹² through E³⁰¹ is important for enzyme activity and CO₂ activation; Arg²⁹⁹ may be involved in the mechanism of CO₂ activation. We prepared R244K and S246A mutants to determine if these Arg and Ser residues may serve as ligands for the carboxyl group of ACC. The R244K and S246A mutants were 5.4% and 35% as active, respectively, as the native enzyme but were typically activated by CO₂; the Km values for ACC for the R244K and S246A mutants were increased 2- to 3-fold compared to the native enxyme. This supports a putative role of Arg²⁴⁴ and Ser²⁴⁶ as ligands for the ACC carboxyl group.