A pAO1-encoded molybdopterin cofactor gene ( moaA ) of Arthrobacter nicotinovorans : characterization and site-directed mutagenesis of the encoded protein

Abstract

A gene homologous to moaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor of Escherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encoded moaA gene from A. nicotinovorans was expressed in E. coli as an active protein that functionally complemented moaA mutants. Its deduced amino acid sequence shows 43% identity to the E. coli MoaA, 44% to the NarAB gene product from Bacillus subtilis, and 42% to the gene product of two contiguous ORFs from Methanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in the fixZ gene product from Rhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement the moaA mutation in E. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy of moaA may not be unique in the A. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.

Mutational Analysis of the sbo-alb Locus of Bacillus subtilis : Identification of Genes Required for Subtilosin Production and Immunity

“…AlbC very likely functions in the export of subtilosin, which it performs, perhaps, in conjunction with other alb products. We propose that AlbA, very likely a member of the MoeA/NifB/PqqE family (16), and AlbF, a member of a family of zinc endoproteinases, perform critical modifications of the presubtilosin peptide. AlbB, -C, and -D are required for immunity to subtilosin.…”

A Megaplasmid-Borne Anaerobic Ribonucleotide Reductase in Alcaligenes eutrophus H16

“…Both motifs are also present in pyruvate formate lyase (Pfl) activases and in the PqqE, NifB, and MoaA proteins (57). No specific physiological function has been assigned to PqqE, NifB, and MoaA, which are all involved in cofactor synthesis (32). a The activity of ␤-galactosidase is expressed in arbitrary units.…”

Genetic features of circular bacteriocins produced by Gram-positive bacteria

“…The putative coding sequences of the alb operon ( Table 6) encode proteins that potentially function in the processing and export of peptides: the albA and albF products are critical for production while the albB, albC and albD products have a role in export and immunity. AlbA, involved in the posttranslational modification of presubtilosin , was identified as a member of the MoaA/NifB/PqqE family (Menendez et al, 1995) and possesses two Cys clusters, located at an Fe-S centre, which serve as the active sites in reactions involving hydration or dehydration of substrate compounds. The polypeptides encoded by albC, -E, and -F show primary structural similarity to known proteins: AlbC probably functions in the export as an ABC transport complex, AlbE is a processing protease and AlbF is a member of a family of zinc endoproteinases that may perform critical modifications of the presubtilosin peptide.…”