Abstract
Isoquinoline 1-oxidoreductase, which catalyzes the hydroxylation of isoquinoline to 1-oxo-1,2-dihydroisoquinoline with concomitant reduction of a suitable electron acceptor, was purified from the isoquinoline degrading bacterium Pseudomonas diminuta 7 to apparent homogeneity. The native enzyme was a heterodimer with a molecular mass of 95 kDa consisting of a 16- and a 80-kDa subunit. It contained 0.85 g atom molybdenum, 3.95 g atom iron, 3.9 g atom acid-labile sulfur, 2.1 mol of phosphate, and 1 mol of CMP/mol of enzyme. CMP and phosphate are suggested to originate from molybdopterin cytosine dinucleotide of the pterin molybdenum cofactor. It is assumed that the iron and the acid-labile sulfur 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 acceptors served as oxidizing substrates, whereas O2 and NAD were not used. Isoquinoline 1-oxidoreductase revealed a high specificity toward the reducing substrates isoquinoline, 5-hydroxyisoquinoline, quinazoline, and phthalazine. Isoquinoline 1-oxidoreductase was inactivated by methanol, arsenite, p-hydroxymercuribenzoate, 1,10-phenanthroline, and cyanide. 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-hydroxy-isoquinoline, which rapidly and very effectively inactivated the enzyme in the presence as well as in the absence of the electron acceptors iodonitrotetrazolium chloride, phenazine methosulfate, or ferricyanide.
Highlights
Among the enzymes catalyzing the hydroxylation of N-heterocyclic compoundsq, uinoline2-oxidoreductases(28, 291, quinoline 4-carboxylic acid2-oxidoreductase (311, quinaldine sulfur are arrangedin two (2Fe-2s)clusters
These results indicate that the firstenzyme of the isoquinoline catabolism, isoquinoline 1-oxidoreductase, is the only molybdenum-containing enzyme in the degradation pathway
We propose that theisoquinoline l-oxidoreductase from I! diminuta 7 exists as a heterodimer
Summary
0.09; FMN 0.19; NAD 0.94; NADP 0.47; methylene blue 0.01; 1.2-naph- with a x-ray fluorescence spectrometer To test putative inhibitorsfor their time- and concentration-depend- Tris-HC1 buffer, pH 8.5. Proteins used for calibration were as follows: ent influence on the enzymatic activity, they were incubated together y-globulin (169 kDa), phosphorylase b (rabbit muscle, 94 kDaa)l,cohol with the isoquinoline 1-oxidoreductase in 20mM Tris-HC1 buffer, 0.3% dehydrogenase (horse liver, 80 kDa), serum albumin (bovine, 67 kDa), Triton X-100, pH 8.5, at room temperature. Tested were asfollows:p-hydroxymercuribenzoate in concentrations of polyacrylamide gel electrophoresis. 2.5,3.5,5, and 25p~ with 1.0 unit/ml enzyme; sodium-meta-arsenite Theconversion of 5-hydroxyisoquinoline was as- calculation of the sedimentation coefficient and the estimation of the sayed in the presencoef 0.06 mM PMS or0.5, 1,2, and3.6 mM ferricya- molecularmass of isoquinoline1-oxidoreductasewascarriedout as nide instead of INT. Tration 1.11M) was addedt o the standard assay mixture usin1gunitlml enzyme.Quinacrine(0.86 mM finalconcentration),acriflavine(0.43 mM), EDTA (4.31 mM), 2,2'-bipyridyl(0.43 mM), 1,lO-phenanthroline
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