Novel heme-containing lyase, phenylacetaldoxime dehydratase from Bacillus sp, strain OxB-1: Purification, characterization, and molecular cloning of the gene

A novel dehydratase that catalyzes the stoichiometric dehydration of Z-phen ylacetaldoxime to phenylacetonitrile has been purified 483-fold to homogene ity from a cell-free extract of Bacillus sp. strain OxB-1 isolated from soi l. It has a M-r of about 40000 and is composed of a single polypeptide chai n with a loosely bound protoheme IX. The enzyme is inactive unless FMN is a dded to the assay, but low activity is also observed when sulfite replaces FMN. The activity in the presence of FMN is enhanced 5-fold under anaerobic conditions compared to the activity measured in air. The enzyme has maximu m activity at pH 7.0 and 30 degrees C, and it is stable at up to 45 degrees C at around neutral pH. The aerobically measured activity in the presence of FMN is also enhanced by Fe2+, Sn2+, SO32-, and NaN3. Metal-chelating rea gents, carbonyl reagents, electron donors, and ferri- and ferrocyanides str ongly inhibit the enzyme with K-i values in the micromolar range. The enzym e is active with arylalkylaldoximes and to a lesser extent with alkylaldoxi mes, The enzyme prefers the Z-form of phenylacetaldoxime over its E-isomer. On the basis of its substrate specificity, the enzyme has been tentatively named phenylacetaldoxime dehydratase. The gene coding for the enzyme was c loned into plasmid pUC18, and a 1053 base-pair open reading frame that code s for 351 amino acid residues was identified as the oxd gene. A nitrilase w hich participates in aldoxime metabolism in the organism, was found to be c eded by the region just upstream from the oxd gene. In addition an open rea ding frame (orf2), whose gene product is similar to bacterial regulatory (D NA-binding) proteins, was found just upstream from the coding region of the nitrilase. These findings provide genetic evidence for a novel gene cluste r that is responsible for aldoxime metabolism in this microorganism.