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Nitrile‐synthetic enzyme involved in the formation of a carbon‐nitrogen triple bond
Author(s) -
KOBAYASHI MICHIHIKO,
NOMURA JUNPEI,
HASHIMOTO YOSHITERU
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.788.2
Subject(s) - chemistry , nitrile , nitrile hydratase , stereochemistry , enzyme , histidine , active site , heme , lyase , biochemistry , organic chemistry
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 H 2 O 2 or O 2 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.