Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis

Abstract

Aldoxime dehydratase (OxdA), which is a unique heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. Unlike the utilization of H 2 O 2 or O 2 as a mediator of catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron. Here, we determined the crystal structure of OxdA. We then constructed OxdA mutants in which each of the polar amino acids lying within ∼6 Å of the iron atom of the heme was converted to alanine. Among the purified mutant OxdAs, S219A had completely lost and R178A exhibited a reduction in the activity. Together with this finding, the crystal structural analysis of OxdA and spectroscopic and electrostatic potential analyses of the wildtype and mutant OxdAs suggest that S219 plays a key role in the catalysis, forming a hydrogen bond with the substrate. Based on the spatial arrangement of the OxdA active site and the results of a series of mutagenesis experiments, we propose the detailed catalytic mechanism of general aldoxime dehydratases: (i) S219 stabilizes the hydroxy group of the substrate to increase its basicity; (ii) H320 acts as an acid-base catalyst; and (iii) R178 stabilizes the heme, and would donate a proton to and accept one from H320.hemoprotein | reaction mechanism | resonance Raman | distal histidine

Substrate, product, and cofactor: The extraordinarily flexible relationship between the CDE superfamily and heme

“…27,28 Like Clds and DyPs, they have a proximally histidine-ligated heme and a mostly-hydrophobic distal pocket. However, unlike Clds or DyPs, OxdAs are catalytically active in the ferrous form, and the substrate-bound ferric form of the protein is catalytically dead.…”

“…28 The aldoxime (R-CH=NOH) substrate directly binds the heme iron through its nitrogen atom, 27 where it forms hydrogen bonds to conserved serine and histidine residues through its –OH group. The histidine is adjacent to a conserved arginine which, like in the canonical peroxidases, is an arrangement that is proposed to stabilize the distal histidine in its neutral/deprotonated form.…”

A New Synthetic Route to N -Benzyl Carboxamides through the Reverse Reaction of N -Substituted Formamide Deformylase

“…The active site of N-substituted formamide deformylase has never been identified experimentally. Further studies on N-substituted formamide deformylase from the standpoint of its three-dimensional structure could provide information about the evolutionary relationship of this enzyme and other enzymes involved in the cleavage and synthesis of a C-N bond, such as nitrilase (56,57), nitrile hydratase (58), amidase (49,50), and aldoxime dehydratase (59).…”

Nitrile-synthesizing enzyme: Gene cloning, overexpression and application for the production of useful compounds

“…(Konishi et al, 2006;Nomura et al, 2013) of nitriles not only from a basic standpoint but also from an applied point of view (Herai et al, 2004;Kobayashi and Shimizu, 1998;Komeda et al, 1996b).It has been reported that microorganisms and plants can degrade cyanides to less toxic compounds through biochemical reactions. Some of these are degradative pathways involving cyanide hydratase, nitrile hydratase, cyanidase, nitrilase, thiocyanate hydrolase, cyanide dioxygenase and cyanase.…”

“…(Konishi et al, 2006;Nomura et al, 2013) of nitriles not only from a basic standpoint but also from an applied point of view (Herai et al, 2004;Kobayashi and Shimizu, 1998;Komeda et al, 1996b).…”