Purification and characterization of isoquinoline 1-oxidoreductase from Pseudomonas diminuta 7, a novel molybdenum-containing hydroxylase.
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.
Lehmann
M
M
Institut für Mikrobiologie, Universität Hohenheim, Stuttgart, Germany.
Tshisuaka
B
B
Fetzner
S
S
Röger
P
P
Lingens
F
F
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010710:Phosphates / Q000032:analysis; D011549:Pseudomonas / Q000201:enzymology* / Q000254:growth & development; D013053:Spectrophotometry; D013455:Sulfur / Q000032:analysis
D046911:Macromolecular Substances; D010710:Phosphates; D013455:Sulfur; D008982:Molybdenum; D007501:Iron; D006899:Mixed Function Oxygenases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C086842:isoquinoline 1-oxidoreductase