Isolation of protein FA, a product of the mob locus required for molybdenum cofactor biosynthesis in Escherichia coli

Abstract

The mob mutants in Escherichia coli are pleiotropically defective in all molybdoenzyme activities. They synthesise molybdopterin, the unique core of the molybdenum cofactor, but are unable to attach the GMP moiety to molybdopterin to form molybdopterin guanine dinucleotide, the functional molybdenum cofactor in Escherichia coli. A partially purified preparation termed protein FA (protein factor d'association), is able to restore molybdoenzyme activities to broken cell preparations of mob mutants. A fragment of DNA capable of complementing mob mutants has been isolated from an E. coli genomic library. Strains carrying this DNA in a multicopy plasmid, express 30-fold more protein FA activity than the wild-type bacterium. Protein FA has been purified to homogeneity by a combination of ion-exchange, affinity and gel-filtration chromatography. Protein FA consists of a single polypeptide of molecular mass 22 kDa and is monomeric in solution. N-terminal amino acid sequencing confirmed that protein FA is a product of the first gene at the mob locus. The purified protein FA was required in stoichiometric rather than catalytic amounts in the process that leads to the activation of the precursor of the molybdoenzyme nitrate reductase, which is consistent with the requirement of a further component in the activation.Molybdoenzymes, with the exception of molybdodinitrogenase, contain a molybdenum cofactor which is composed in its simplest form of a unique pterin derivative, molybdopterin, to which the molybdenum is bound [l]. The chlorateresistant mutants of Escherichia coli (recently renamed mo [2]) are pleiotropically defective in all molybdoenzyme activities and are defective in the biosynthesis of active molybdenum cofactor. These mutants carry mutations which map to five distinct regions of the E. coli chromosome: moa, mob, mod, moe and mog. Each of the loci has been cloned and almost all have been sequenced [4-81. The overall functions encoded by most of the loci have been deduced. The moa and moe loci encode proteins which assemble the molybdopterin portion of the cofactor [9, 101. The mod locus encodes the molybdate uptake system of the cell [9, 111. The function of mog is not known [12]. For several years the role of the mob locus in cofactor biosynthesis was unclear since these mutants possessed molybdopterin [13, 141. However, recently it has been demonstrated that in E. coli the active form of the cofactor is molybdopterin guanine dinucleotide (MGD), a covalent complex of molybdopterin with guanosine monophosphate. Mutants defective at mob are unable to attach GMP to molybdopterin, suggesting that the products of the mob locus are responsible for this late step in molybdenum cofactor biosynthesis [15].

TorD, A Cytoplasmic Chaperone That Interacts with the Unfolded Trimethylamine N-Oxide Reductase Enzyme (TorA) in Escherichia coli

“…1 mg of pure TMAO reductase was obtained with a specific activity of 250 mol of TMAO reduced/min/mg of protein. The protein FA, product of the mobA gene, was purified as described by Palmer et al (12).…”

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

“…Probing the Effect of MGD Absence on the InteractionsFinally, two-hybrid assays were conducted into a mob strain (BTH101mob::Tn10) genetically able to synthesize active Moco (MPT-Mo) for MPT-containing enzymes but defective in the GMP attachment step (29,30). In such a strain, presence of either T18-MobA or T25-MobA fusion proteins leads to a full restoration of the phenotype revealed by nitrate reductase activity measurement (data not shown).…”

MocA Is a Specific Cytidylyltransferase Involved in Molybdopterin Cytosine Dinucleotide Biosynthesis in Escherichia coli

“…The biosynthesis of Moco has been divided into four major steps in Escherichia coli: (i) formation of precursor Z (3,4), (ii) formation of MPT from precursor Z (5, 6), (iii) insertion of molybdenum to form Moco via an adenylylated MPT intermediate (7)(8)(9), and (iv) additional modification by covalent addition of GMP to the C4Ј phosphate of MPT via a pyrophosphate bond, forming the molybdopterin guanine dinucleotide (MGD) cofactor (10,11). In E. coli, GMP attachment to Moco is catalyzed by the MobA and MobB proteins (12). Although MobA was shown to be essential for this reaction and acts as a GTP:molybdopterin guanylyltransferase (11), the role of MobB still remains uncertain.…”