A PA01-ENCODED MOLYBDOPTERIN COFACTOR GENE (MOAA) OF ARTHROBACTER NICOTINOVORANS - CHARACTERIZATION AND SITE-DIRECTED MUTAGENESIS OF THE ENCODED PROTEIN

A gene homologous to moaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopte rin cofactor of Escherichia coli, was found to be located 2.7-kb upstr eam of the nicotine dehydrogenase (ndh) operon on the catabolic plasmi d pAO1 of Arthrobacter nicotinovorans. The MoaA protein, containing 35 4 amino acids, migrated on an SDS-polyacrylamide,eel with an apparent molecular weight of 40,000, in good agreement with the predicted molec ular weight of 38,880. The pAO1-encoded moaA gene from A. nicotinovora ns was expressed in E. coli as an active protein that functionally com plemented monA mutants. Its deduced amino acid sequence shows 43% iden tity 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 Meth anobacterium formicicum. N-terminal sequences, including the motif Cxx xCxYC, 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 fro m 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 monA mutation in E. coli. A second Cys-rich domain with the motif FCx xC(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. nicotino vorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.