Escherichia coli Molybdoenzymes Can Be Activated by Protein FA from Several Gram-negative Bacteria

Santini, Claire‐Lise; Karibian, Doris; Vasishta, Anil; Boxer, David H.; Giordano, Gérard

doi:10.1099/00221287-135-12-3467

Cited by 5 publications

(3 citation statements)

References 23 publications

(24 reference statements)

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“…We show that protein FA purified as described here exists as a monomer in solution. This is in agreement with an estimated molecular mass for active protein FA of 18 kDa, obtained following gel filtration of partially purified extracts of wildtype cells [30].…”

Section: Discussionsupporting

confidence: 90%

“…More likely another component required for the nitrate reductase activation assay is present in limiting quantities in the mob crude extract. Protein FA activity is susceptible to exposure to proteinases [30]. In order to limit any loss of protein FA activity during the activation by proteases present in the crude extract, a cocktail of protease inhibitors was added to the suspension of mob cells before disruption.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Palmer¹,

Vasishta²,

Whitty³

et al. 1994

European Journal of Biochemistry

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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].

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

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

Palmer¹,

Vasishta²,

Whitty³

et al. 1994

European Journal of Biochemistry

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“…It is constitutively expressed in E. coli. Protein FA has been purified from the soluble fraction of a chUA mutant and its apparent relative molecular mass was about 18,000 (26,27). Further characterization of the protein FA-dependent activation of the inactive nitrate reductase present in the soluble fraction of a chlB mutant showed that a heat-stable low-* Corresponding author.…”

mentioning

confidence: 99%

Molybdoenzyme biosynthesis in Escherichia coli: in vitro activation of purified nitrate reductase from a chlB mutant

et al. 1992

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All molybdoenzyme activities are absent in chlB mutants because of their inability to synthesize molybdopterin guanine dinucleotide, which together with molybdate constitutes the molybdenum cofactor in Escherichia coli. The chlB mutants are able to synthesize molybdopterin. We have previously shown that the inactive nitrate reductase present in a chlB mutant can be activated in a process requiring protein FA and a heat-stable low-molecular-weight substance. We show here that purified nitrate reductase from the soluble fraction of a chlB mutant can be partially activated in a process that requires protein FA, GTP, and an additional protein termed factor X. It appears that the molybdopterin present in the nitrate reductase of a chlB mutant is converted to molybdopterin guanine dinucleotide during activation. The activation is absolutely dependent upon both protein FA and factor X. Factor X activity is present in chU4, chlB, chWE, and chiG mutants.

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Purification and characterization of the molybdoenzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini

Nagel

Andreesen

1990

Arch. Microbiol.

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Escherichia coli Molybdoenzymes Can Be Activated by Protein FA from Several Gram-negative Bacteria

Cited by 5 publications

References 23 publications

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

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

Molybdoenzyme biosynthesis in Escherichia coli: in vitro activation of purified nitrate reductase from a chlB mutant

Purification and characterization of the molybdoenzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini

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