Identification of a functionally impaired allele of human novel oxidoreductase 1 (NDOR1), NDOR1*1

Objectives: Human novel oxidoreductase 1 (NDOR1) is a diflavin reductase closely related to Cytochrome P450 reductase (POR) and nitric oxide synthase (NOS), which are involved in the metabolism of antitumour agents. A variant cDNA sequence of NDOR1, NDOR1 p.518-519ins9 or NDOR1_v1, has been deposited in GenBank (accession no. AK026089 and AY077845) that encodes an additional nine Amino acids, which led us to investigate NDOR1 polymorphism.

Methods and results: We analysed genomic DNA from 200 Caucasian and 49 Japanese individuals by PCR--restriction fragment length polymorphism analysis. The nine amino acid residue sequence was found to be present in all individuals, encoded by a 27 nt intronic nucleotide sequence at an intron/exon junction, suggesting that this variant did not represent a common genetic polymorphism, but may have arisen from an alternative mRNA splicing event. However, further analysis of NDOR1 revealed a polymorphic c.1564G>A transition (NDOR1*1), detected in 24/200 Caucasian and 1/49 Japanese individuals, producing a valine to isoleucine substitution at codon 522 in the NADPH binding region. Expression of the flavin adenine dinucleotide/reduced nicotinamide phosphate dehydrogenase (NADPH) domain in Escherichia coli showed a significant 74% reduction in potassium ferricyanide reductase activity, but no effect on NADPH binding. NDOR1_v1 showed a 10-fold decrease in affinity for NADPH, and a 90% reduction in ferricyanide reductase activity.

Conclusions: We have discovered a polymorphic variant of NDOR1, NDOR1*1, that produces a functionally impaired Enzyme. This will help define the structure and function of NDOR1 and its relationship to Cancer and Other Diseases.