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An enormous amount of nitrile hydratase (NHase) is inducibly produced by Pseudomonas chlororaphis B23 after addition of methacrylamide as the sole nitrogen source to a medium. The expression pattern of the P. chlororaphis B23 NHase gene cluster in response to addition of methacrylamide to the medium was investigated. Recently, we reported that the NHase gene cluster comprises seven genes (oxdA, amiA, nhpA, nhpB, nhpC, nhpS, and acsA). Sequence analysis of the 1.5-kb region upstream of the oxdA gene revealed the presence of a 936-bp open reading frame (designated nhpR), which should encode a protein with a molecular mass of 35,098. The deduced amino acid sequence of the nhpR product showed similarity to the sequences of transcriptional regulators belonging to the XylS/AraC family. Although the transcription of the eight genes (nhpR, oxdA, amiA, nhpABC, nhpS, and acsA) in the NHase gene cluster was induced significantly in the P. chiororaphis B23 wild-type strain after addition of methacrylamide to the medium, transcription of these genes in the nhpR disruptant was not induced, demonstrating that nhpR codes for a positive transcriptional regulator in the NHase gene cluster. A reverse transcription-PCR experiment revealed that five genes (oxdA, amiA, nhpA, nhpB, and nhpC) are cotranscribed, as are two other genes (nhpS and acsA). The transcription start sites for nhpR, oxdA, nhpA, and nhpS were mapped by primer extension analysis, and putative -12 and -24 [[sigma].sup.54]-type promoter binding sites were identified. NhpR was found to be the first transcriptional regulator of NHase belonging to the XylS/AraC family.

The isolation and characterization of the NADH-cytochrome b5 reductases (CbR) gene from Mortierella alpina 1S-4 and the purification of the CbR expressed in Aspergillus oryzae are described. It was found that the purified CbR used NADH but not NADPH as an electron donor. When NADH was utilized as the electron donor, DCPIP was observed to be reduced by the purified CbR. Results provide support for the hypothesis that the cDNA cloned is definitely a CbR gene.

Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 [right arrow] Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 [right arrow] Asn substitution as well as Ser195 [right arrow] Aia substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-[NH.sub.2]) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

The derived amino acid sequence of peptides yielded by cleavage of Rhodococcus rhodochrous K22 was used to design an oligonucleotide probe for cloning of the nitrilase encoding gene. Isopropyl beta-D-thiogalactopyranoside-induced overproduction of active nitrilase was done in a pNK21 plasmid vector with a lac promoter. Nitrilase was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analysis of the gene sequence predicts a 383-residue protein with only one cysteine. Cys-170 mutants exhibited complete loss of nitrilase activity indicating that this residue is essential for the catalytic activity.