Cloning and enhanced expression of the cytochrome P450nor gene (nicA; CYP55A5) encoding nitric oxide reductase from Aspergillus oryzae

Abstract

We cloned and characterized the gene and cDNA of Aspergillus oryzae cytochrome P450nor (Anor). The Anor gene (nicA; CYP55A5) has a different gene structure from other P450nor genes in that it has an extra intron. There were not only two kinds of mRNA but also two sets of TATA-box and CCAAT-box, and it appears that this gene has two expression patterns, like CYP55A1 of Fusarium oxysporum. A reporter analysis using the uidA gene indicated that gene expression of CYP55A5 was induced under anaerobic conditions, like CYP55A1. When the CYP55A5 gene was overexpressed in A. oryzae, a large amount of active Anor were accumulated as intracellular protein. Anor employed both NADH and NADPH as electron donors for reducing nitric oxide to nitrous oxide. Anor measured the amount of NO generated from 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)- 1-propanamine (NOC5) with a spectrophotometer. The sensitivity was 10 nmol/ml.