Abstract The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molyb- dopterin guanine dinucleotide. The mob locus of Rhodo- bacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inac- tive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-re- quiring enzymes), but the activity of xanthine dehydroge- nase, a molybdopterin enzyme, is unaffected. The inabil- ity of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms follow- ing iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumu- late wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compro- mised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor in- sertion is not necessarily a pre-requisite for export. Keywords Rhodobacter sphaeroides · Dimethylsulfoxide reductase · Molybdenum cofactor · Molybdopterin guanine dinucleotide · Twin arginine translocation · Protein FA · MobA Introduction Mononuclear molybdenum enzymes are found in all three kingdoms of life. Molybdenum in these enzymes is al- ways found in association with a unique pterin, molyb- dopterin (Kisker et al. 1997). Molybdopterin, which is synthesised by an evolutionarily conserved multi-step pathway, is a tricyclic pyranopterin containing a dithio- lene group to which Mo is co-ordinated. Although molyb- dopterin constitutes the active form of the cofactor present in all eukaryotic and some prokaryotic molybdoenzymes, most bacterial enzymes require a modification of this ba- sic structure in order to be functionally active. This modi- fication involves attachment of a nucleotide moiety, usu- ally either GMP or CMP, onto the terminal phosphate group of the molybdopterin side chain via a phosphodi- ester link. Bacterial molybdoenzymes are almost always specific for one form of the cofactor. Thus enzymes of the dimethylsulfoxide (DMSO) reductase family only contain molybdopterin guanine dinucleotide, whilst the carbon monoxide and nicotine dehydrogenases bind molyb- dopterin cytosine dinucleotide (Hille 1996; Dobbek et al. 1999). The synthesis of molybdopterin guanine dinucleotide from molybdopterin and a guanine nucleotide is catalysed by protein(s) encoded at the mob locus. In Escherichia coli the mob locus encodes two genes (Plunkett III et al. 1993; Iobbi-Nivol et al. 1995). The first gene encodes MobA (also termed protein FA), which has previously been purified to homogeneity and the X-ray crystal struc- ture determined (Palmer et al. 1994; Lake et al. 2000; Stevenson et al. 2000). The MobA protein catalyses the time-dependent in vitro activation of molybdopterin gua- nine dinucleotide-requiring molybdoenzymes from crude cell extracts of mob mutants (Eaves et al. 1997). Very re- cently the purified recombinant DMSO reductase from Rhodobacter sphaeroides has been isolated from an E. coli mob mutant. The enzyme can be activated over a period of several hours in a process requiring only puri- fied MobA, Mg 2+ , GTP and a source of molybdopterin Grant Buchanan · Jochen Kuper · Ralf R. Mendel · Günter Schwarz · Tracy Palmer Characterisation of the mob locus of Rhodobacter sphaeroides WS8: mobA is the only gene required for molybdopterin guanine dinucleotide synthesis Received: 31 January 2001 / Revised: 12 April 2001 / Accepted: 24 April 2001 / Published online: 22 May 2001 ORIGINAL PAPER G. Buchanan · T. Palmer Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia Norwich NR4 7TJ, United Kingdom G. Buchanan · T. Palmer (✉) Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom e-mail: tracy.palmer@bbsrc.ac.uk, Tel.: +44-1603-450726, Fax: +44-1603-450778 J. Kuper · R.R. Mendel · G. Schwarz Botanical Institute, Technical University of Braunschweig, 38023 Braunschweig, Germany Arch Microbiol (2001) 176 : 62–68 DOI 10.1007/s002030100291 © Springer-Verlag 2001