title: Metal-catalyzed oxidation and mutagenesis studies on the iron(II) binding site of 1-aminocyclopropane-1-carboxylate oxidase.
abstract: The final step in the biosynthesis of the plant signaling molecule ethylene is catalyzed by 1-aminocyclopropane-1-carboxylate (ACC) oxidase, a member of the non-heme iron(II) dependent family of oxygenases and oxidases, which has a requirement for ascorbate as a co-substrate and carbon dioxide as an activator. ACC oxidase (tomato) has a particularly short half-life under catalytic conditions undergoing metal-catalyzed oxidative (MCO) fragmentation. Sequence comparisons of ACC oxidases with isopenicillin N synthase (IPNS) and members of the 2-oxoglutarate Fe(II) dependent dioxygenases show an aspartate and two of six ACC oxidase conserved histidine residues are completely conserved throughout this subfamily of Fe(II) dependent oxygenases/oxidases. Previous mutagenesis, spectroscopic, and crystallographic studies on IPNS indicate that the two completely conserved histidine and aspartate residues act as Fe(II) ligands. To investigate the role of the conserved aspartate and histidine residues in ACC oxidase (tomato fruit), they were substituted via site-directed mutagenesis. Modified ACC oxidases produced were H39Q, H56Q, H94Q, H177Q, H177D, H177E, D179E, D179N, H177D&D179E, H211Q, H234Q, H234D, and H234E. Among those histidine mutants replaced by glutamine, H39Q, H56Q, H94Q, and H211Q were catalytically active, indicating these histidines are not essential for catalysis. Mutant enzymes H177D, H177Q, D179N, H177D&D179E, H234Q, H234D, and H234E were catalytically inactive consistent with the assignment of H177, D179, and H234 as iron ligands. Replacement of H177 with glutamate or D179 with glutamate resulted in modified ACC oxidases which still effected the conversion of ACC to ethylene, albeit at a very low level of activity, which was stimulated by bicarbonate. The H177D (inactive), H177E (low activity), D179E (low activity), and H234Q (inactive) modified ACC oxidases all underwent MCO fragmentation, indicating that they can bind iron, dioxygen, ACC, and ascorbate. The results suggest that MCO cleavage results from active site-mediated reactions and imply that, while H177, D179, and H234 are all involved in metal ligation during catalysis, ligation to H234 is not required for fragmentation. It is possible that MCO fragmentation results from reaction of incorrectly folded or "primed" ACC oxidase.
authors.affiliation: The Oxford Centre for Molecular Sciences and the Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, U.K.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.forename: J N
authors.initials: JN
authors.lastname: Barlow
authors.forename: J E
authors.initials: JE
authors.lastname: Baldwin
authors.forename: C J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism / Q000494:pharmacology; D000598:Amino Acids, Cyclic; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005030:Ethylenes / Q000096:biosynthesis; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019159:Nonheme Iron Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D017382:Reactive Oxygen Species / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
chemical_list: D000596:Amino Acids; D000598:Amino Acids, Cyclic; D005030:Ethylenes; D005296:Ferrous Compounds; D019159:Nonheme Iron Proteins; D017382:Reactive Oxygen Species; C023863:1-aminocyclopropane-1-carboxylic acid; C036216:ethylene; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase