PURIFICATION AND CHARACTERIZATION OF ISOQUINOLINE 1-OXIDOREDUCTASE FROM PSEUDOMONAS-DIMINUTA-7, A NOVEL MOLYBDENUM-CONTAINING HYDROXYLASE

Isoquinoline 1-oxidoreductase, which catalyzes the hydroxylation of is oquinoline to 1-oxo-1,2-dihydroisoquinoline with concomitant reduction of a suitable electron acceptor, was purified from the isoquinoline d egrading bacterium Pseudomonas diminuta 7 to apparent homogeneity. The native enzyme was a heterodimer with a molecular mass of 95 kDa consi sting of a 16- and a 80-kDa subunit. It contained 0.85 g atom molybden um, 3.95 g atom iron, 3.9 g atom acid-labile sulfur, 2.1 mol of phosph ate, and 1 mol of CMP/mol of enzyme. CMP and phosphate are suggested t o originate from molybdopterin cytosine dinucleotide of the pterin mol ybdenum cofactor. It is assumed that the iron and the acid-labile sulf ur are arranged in two (2Fe-2S) clusters. The isoelectric point of the isoquinoline 1-oxidoreductase was within the range of pH 6.2 to 6.8. Cytochrome c, ferricyanide, and several non-physiological electron acc eptors served as oxidizing substrates, whereas O2 and NAD were not use d. Isoquinoline 1-oxidoreductase revealed a high specificity toward th e reducing substrates isoquinoline, 5-hydroxyisoquinoline, quinazoline , and phthalazine. Isoquinoline 1-oxidoreductase was inactivated by me thanol, arsenite, p-hydroxymercuribenzoate, 1,10-phenanthroline, and c yanide. Additionally, the enzyme was inactivated upon incubation with its substrates isoquinoline, which slowly inhibited the enzyme in the absence of an electron acceptor, and 5-hydroxyisoquinoline, which rapi dly and very effectively inactivated the enzyme in the presence as wel l as in the absence of the electron acceptors iodonitrotetrazolium chl oride, phenazine methosulfate, or ferricyanide.