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

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Abstract

A gene homologous tomoaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor ofEscherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 ofArthrobacter 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-encodedmoaA gene fromA. nicotinovorans was expressed inE. coli as an active protein that functionally complementedmoaA mutants. Its reduced amino acid sequence shows 43% identity to theE. coli MoaA, 44% to the NarAB gene product fromBacillus subtilis, and 42% to the gene product of two contiguous ORFs fromMethanobacterium 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 thefixZ gene product fromRhizobium 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 themoaA mutation inE. 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 ofmoaA may not be unique in theA. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.

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Abbreviations

FDH :

Formate dehydrogenase

GST :

Glutathione S-transferase

IPTG :

Isopropyl-β-d-thiogalactopyranoside

MPT :

Molybdopterin cofactor

NDH :

Nicotine dehydrogenase

NR :

Nitrate reductase

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Authors and Affiliations

  1. Biochemisches Institut, Universität Freiburg, Hermann-Herder-Strasse 7, D-79104, Freiburg, Germany

    Cástor Menéndez, Hanspeter Henninger & Roderich Brandsch

  2. Institut für Biologie, Universität Freiburg, D-79104, Freiburg, Germany

    Gabor Igloi

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  1. Cástor Menéndez
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  2. Gabor Igloi
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  3. Hanspeter Henninger
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  4. Roderich Brandsch
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Correspondence to Roderich Brandsch.

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Menéndez, C., Igloi, G., Henninger, H. et al. A pAO1-encoded molybdopterin cofactor gene (moaA) ofArthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein. Arch. Microbiol. 164, 142–151 (1995). https://doi.org/10.1007/BF02525320

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  • DOI: https://doi.org/10.1007/BF02525320

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