MOLECULAR-CLONING OF THE ISOQUINOLINE 1-OXIDOREDUCTASE GENES FROM PSEUDOMONAS-DIMINUTA-7, STRUCTURAL-ANALYSIS OF IORA AND IORB, AND SEQUENCE COMPARISONS WITH OTHER MOLYBDENUM-CONTAINING HYDROXYLASES

The iorA and iorB genes from the isoquinoline-degrading bacterium Pseu domonas diminuta 7, encoding the heterodimeric molybdo-iron-sulfur-pro tein isoquinoline 1-oxidoreductase, were cloned and sequenced. The ded uced amino acid sequences IorA and IorE showed homologies (i) to the s mall (gamma) and large (alpha) subunits of complex molybdenum-containi ng hydroxylases (alpha beta gamma/alpha(2) beta(2) gamma(2)) possessin g a pterin molybdenum cofactor with a monooxo-monosulfido-type molybde num center, (ii) to the N- and C-terminal regions of aldehyde oxidored uctase from Desulfovibrio gigas, and (iii) to the N- and C-terminal do mains of eucaryotic xanthine dehydrogenases, respectively. The closest similarity to IorE was shown by aldehyde dehydrogenase (Adh) hom the acetic acid bacterium Acetobacter polyoxogenes. Five conserved domains of IorB were identified by multiple sequence alignments. Whereas IorB and Adh showed an identical sequential arrangement of these conserved domains, in all other molybdenum-containing hydroxylases the relative position of ''domain A'' differed. IorA contained eight conserved cys teine residues. The amino acid pattern harboring the four cysteine res idues proposed to ligate the Fe/S I cluster was homologous to the cons ensus binding site of bacterial and chloroplast-type [2Fe-2S] ferredox ins, whereas the pattern including the four cysteines assumed to ligat e the Fe/S II center showed no similarities to any described [2Fe-2S] binding motif. The N-terminal region of IorB comprised a putative sign al peptide similar to typical leader peptides, indicating that isoquin oline 1-oxidoreductase is associated with the cell membrane.