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.
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Kadyrzhanova, D., McCully, T.J., Warner, T., Vlachonasios, K., Wang, Z., Dilley, D.R. (1999). Analysis of ACC Oxidase Activity by Site-Directed Mutagenesis of Conserved Amino Acid Residues. In: Kanellis, A.K., Chang, C., Klee, H., Bleecker, A.B., Pech, J.C., Grierson, D. (eds) Biology and Biotechnology of the Plant Hormone Ethylene II. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-4453-7_2
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DOI: https://doi.org/10.1007/978-94-011-4453-7_2
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