Functions, diversity, and physiological functions of novel heme-containing enzyme "aldoxime dehydratase"

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We examined the following experiments in order to clarify 1) reaction mechanism of aldoxime dehydratase (Oxd), 2) diversity of aldoxime dehydratase, and 3) diversity and physiological function of "aldoxime-nitrile pathway".1. We constructed an overexpression system of nitrilase (Nit)-linked oxd gene from Bacillus sp. OxB-1 in a recombinant E. coli and obtained large quantities of Oxd protein (OxdB) in highly pure and homogenous form.2. We analyzed OxdB protein spectrophotometrically to identify its heme environment and functional residue during enzyme catalysis.3. We screened oxd genes genetically from stock cultures and cloned whole lengths oxd genes.4. We overexpressed new oxds in E.coli and examined their characters. Based on the findings, physiological functions of Oxds are studied.5. We genetically screened nitrile-degrading enzymes in strains having "aldoxime-nitrile pathway" and cloned their genes.6. We compared the characters and primary structures of the nitrile-degrading enzymes and studied the relationship between Oxds enzymologically and genetically. Physiology and evolutions of the pathway are also discussed.