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

AmfS, a class III lantipeptide serves as a morphogen in Streptomyces griseus. Here, we constructed a high production system of AmfS in S. griseus. We isolated S. griseus Grd1 strain defective in glucose repression of aerial mycelium formation and found it suitable for the overproduction of AmfS. Two expression vectors carrying the strong and constitutive ermE2 promoter were constructed using a multicopy number plasmid, pIJ702. The use of the Grd1 strain combined with the expression vectors enabled high production of AmfS by S. griseus into its culture broth. The expression system was also effective for the generation of abundant AmfS derived from Streptomyces avermitilis. In addition, site-directed mutagenesis revealed the amino acid residues essential for the morphogen activity of AmfS. These results indicate that the constructed system enables efficient production of class III lantipeptides by Streptomyces.

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We have studied microbial metabolism of nitrile [R‐CN] at the protein and gene levels. We discovered a nitrile‐synthesizing enzyme linked with nitrile hydratase (which is an industrial enzyme used for the production of amide from nitrile) in the nitrile metabolism. The enzyme, aldoxime dehydratase contains a heme as the prosthetic group, and catalyzes the dehydration of aldoxime [R‐CH=N‐OH] into nitrile [R‐CN]. Unlike the utilization of H2O2 or O2 as a mediator of the catalysis by other heme‐containing enzymes (e.g., P450), this enzyme is notable for the direct binding of a substrate to the heme ion, experimental evidence of which was obtained by means of resonance Raman analysis with an isotope technique. This enzyme is also unique in the aspect of dehydration of the substrate even in the aqueous solution. We previously reported that ferrous enzyme, containing a five‐coordinated high‐spin heme and a histidine residue as its proximal ligand, is the reactive form of the enzyme, and that another histidine in the distal heme pocket would play a crucial role in donating a proton to a substrate during aldoxime dehydration. Considering the crystal structure of the enzyme and the amino acid sequence alignment among this enzyme family, we here constructed several mutant enzymes, where each of amino acid residues is replaced by alanine by site‐directed mutagenesis. They were expressed in E. coli and purified and the properties of the resultant purified mutant enzymes were characterized. The combination of mutagenesis, estimation of the heme contents and specific activities of the mutant enzymes and spectroscopic analyses led the identification of crucial amino acid residues for the catalysis.