A novel
dehydratase that catalyzes the stoichiometric
dehydration of Z-
phenylacetaldoxime to
phenylacetonitrile has been purified 483-fold to homogeneity from a cell-free extract of Bacillus sp. strain OxB-1 isolated from soil. It has a M(r) of about 40 000 and is composed of a single
polypeptide chain with a loosely bound
protoheme IX. The
enzyme is inactive unless
FMN is added 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 maximum 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 Fe(2+), Sn(2+), SO(3)(2)(-), and NaN(3).
Metal-chelating
reagents, carbonyl
reagents, electron donors, and ferri- and
ferrocyanides strongly inhibit the
enzyme with K(i) values in the micromolar range. The
enzyme is active with arylalkylaldoximes and to a lesser extent with alkylaldoximes. 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 cloned into plasmid pUC18, and a 1053 base-pair open reading frame that codes for 351
amino acid residues was identified as the oxd gene. A
nitrilase, which participates in
aldoxime metabolism in the organism, was found to be coded by the region just upstream from the oxd gene. In addition an open reading frame (orf2), whose gene product is similar to bacterial regulatory (
DNA-binding) proteins, was found just upstream from the coding region of the
nitrilase. These findings provide genetic evidence for a novel gene cluster that is responsible for
aldoxime metabolism in this microorganism.