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

Santini, Claire‐Lise; Iobbi‐Nivol, Chantal; Romane, C; Boxer, David H.; Giordano, Gérard

doi:10.1128/jb.174.24.7934-7940.1992

Cited by 19 publications

(28 citation statements)

References 29 publications

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“…However, its extent corresponds to only 5-10% of the expected level. In these reconstitution experiments, it has been suggested that the source of Mo-MPT is the purified NarGH dimer (13,15). These results contrast with those reported for DmsABC, in which no MPT or molybdenum was detected in the enzyme studied in a mobAB background (16).…”

contrasting

confidence: 56%

“…This result contrasts with reports that the NarGH dimer purified from a mobAB mutant contains Mo-MPT (13,15). To reconcile these results, we subjected membrane-bound NarGHI holoenzyme to acid denaturation followed by I 2 and KI oxidation to produce the fluorescent Form A derivative of MPT (12).…”

Section: Effect Of the Mobab Mutation On The Presence Of Mo-mgd Inmentioning

confidence: 64%

“…ϩ :nitrate oxidoreductase activity of the purified NarGH dimer from a mobAB mutant strain in which the only source of Mo-MPT has been suggested to be the purified enzyme (13,15). The lack of fluorescent derivatives obtained from the mobAB mutant membranes prompted us to reinvestigate the Mo-MPT content of NarGH purified from a mobAB mutant strain.…”

Section: Effect Of the Mobab Mutation On The Presence Of Mo-mgd Inmentioning

confidence: 99%

“…The gene product of mobA, MobA, has been shown to be responsible for nucleotide addition (13), whereas the gene product of mobB, MobB, enhances nucleotide addition but is not essential for it (14). The effect of mobAB mutants on E. coli NarGHI and DmsABC has been studied in detail (15,16). Purified soluble NarGH dimer prepared from a mobAB strain can be incubated in the presence of MobA and GTP, generating enzyme that is able to reduce nitrate with benzyl viologen (BV .…”

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confidence: 99%

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The Molybdenum Cofactor of Escherichia coli Nitrate Reductase A (NarGHI)

Rothery¹,

Magalon²,

Giordano³

et al. 1998

Journal of Biological Chemistry

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We have studied the effect of a mobAB mutation and tungstate on molybdo-molybdopterin-guanine dinucleotide (Mo-MGD) insertion into Escherichia coli nitrate reductase (NarGHI). Preparation of fluorescent oxidized derivatives of MGD (Form A and Form B) indicates that in a mobAB mutant there is essentially no detectable cofactor present in either the membrane-bound (NarGHI) or purified soluble (NarGH) forms of the enzyme. Electron paramagnetic resonance characterization of membrane-bound cofactor-deficient NarGHI suggests that it has altered electrochemistry with respect to the dithionite reducibility of the Escherichia coli, when grown anaerobically with nitrate as respiratory oxidant, develops a respiratory chain terminated by a membrane-bound quinol:nitrate oxidoreductase (NarGHI) 1 (1-3). This enzyme is a heterotrimeric complex iron-sulfur molybdoenzyme comprising a molybdenum cofactor-containing catalytic subunit (NarG; 139 kDa), an [Fe-S] cluster-containing electron transfer subunit (NarH; 58 kDa), and a heme-containing membrane anchor subunit (NarI; 26 kDa). NarGHI is an excellent example of a family of bacterial iron-sulfur molybdoenzymes, which includes E. coli Me 2 SO reductase (DmsABC (4)), formate dehydrogenase N (FdnGHI (5)), and Wolinella succinogenes polysulfide reductase (PsrABC (6)). Sequence comparisons between NarG and a large number of bacterial molybdoenzymes indicate that this subunit binds the molybdenum cofactor and is the site of nitrate reduction (3, 4, 7).

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contrasting

confidence: 56%

Section: Effect Of the Mobab Mutation On The Presence Of Mo-mgd Inmentioning

confidence: 64%

Section: Effect Of the Mobab Mutation On The Presence Of Mo-mgd Inmentioning

confidence: 99%

mentioning

confidence: 99%

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The Molybdenum Cofactor of Escherichia coli Nitrate Reductase A (NarGHI)

Rothery¹,

Magalon²,

Giordano³

et al. 1998

Journal of Biological Chemistry

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“…In analogy to the known dinucleotide syntheses [54Ϫ56], the reaction is likely to be catalysed by a cytidylyltransferase which still remains to be demonstrated in H. pseudoflava. The reaction would also be analogous to the MobAcatalysed and MobB-catalysed formation of Mo-MGD from Mo-MPT and GTP in E. coli [57,58]. The Mo-MCD cofactor is subsequently integrated into the folding CO dehydrogenase polypeptides.…”

Section: Discussionmentioning

confidence: 99%

Effect of molybdate and tungstate on the biosynthesis of CO dehydrogenase and the molybdopterin cytosine‐dinucleotide‐type of molybdenum cofactor in Hydrogenophaga pseudoflava

Hänzelmann

Meyer

1998

European Journal of Biochemistry

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The molybdenum-containing iron-sulfur flavoprotein CO dehydrogenase is expressed in a catalytically fully competent form during heterotrophic growth of the aerobic bacterium Hydrogenophaga pseudoflava with pyruvate plus CO. We have adopted these conditions for studying the effect of molybdate (Mo) and tungstate (W) on the biosynthesis of CO dehydrogenase and its molybdopterin (MPT) cytosine-dinucleotide-(MCD)-type molybdenum cofactor. W was taken up by the Mo transport system and, therefore, interfered with Mo transport in an antagonistic way. Depletion of Mo from the growth medium as well as inclusion of excess W both resulted in the absence of intracellular Mo and led to the biosynthesis of CO dehydrogenase species of proper L 2 M 2 S 2 subunit structure that carried the two 2Fe:2S type-I and type-II centers and two FAD molecules. EPR, ultraviolet/visible and CD spectroscopies established the full functionality of the cofactors. Due to the absence of the Mo-MCD cofactor, the enzyme species were catalytically inactive. Unexpectedly, the following cytidine nucleotides were present in inactive CO dehydrogenase: CDP, dCDP, CMP, dCMP, CTP or dCTP. The sum of cytidine nucleotides was two/mol enzyme. The binding specificities of inactive CO dehydrogenase for cytidine nucleotides (oxy Ͼ deoxy; diphosphate Ͼ monophosphate Ͼ triphosphate), and the absence of MPT suggest that, in active CO dehydrogenase, the cytidine diphosphate moiety of Mo-MCD provides the strongest interactions with the protein and determines the specificity for the type of nucleotide. In H. pseudoflava, the biosynthesis of MPT (identified as form A) was independent of Mo. Mo was, however, strictly required for the conversion of MPT to MCD (identified as form-AϪCMP) as well as the insertion of Mo-MCD into CO dehydrogenase. These data support a model for the involvement of Mo in the biosynthesis of the Mo-MCD cofactor and of fully functional CO dehydrogenase in which the synthesis and insertion of Mo-MCD require Mo, and protein synthesis including integration of the FeS-centers and FAD are independent of Mo. Abbreviations. di(cam), di(carboxamidomethyl); MCD, molybdopterin cytosine dinucleotide; MPT, molybdopterin; INT, 1-phenyl-2-(4-iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium chloride; MPMS, 1-methoxyphenazine methosulfate.Enzyme. Carbon monoxide dehydrogenase (EC 1.2.99.2).nase [3,4], carboxylic acid reductase [5,6] and aldehyde dehydrogenase [7,8]. Most mesophilic, MoϪdependent organisms, when grown in the presence of tungstate, produce either inactive enzymes lacking any metal or W-substituted enzymes that have little or no catalytic activity [2]. This effect of tungstate has been termed in the literature as the antagonistic effect of tungstate over molybdate (e.g. [9]). Pyrococcus furiosus exclusively uses W to synthesize catalytically active forms of the three tungstoenzymes aldehyde ferredoxin oxidoreductase, formaldehyde ferredoxin oxidoreductase and glyceraldehyde-3-phosphate dehydrogenase, and active Mo-containing or V-containing isoenzymes ...

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The hydrogenases and formate dehydrogenases ofEscherichia coli

Sawers¹

1994

Antonie van Leeuwenhoek

223

205

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Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.

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

Cited by 19 publications

References 29 publications

The Molybdenum Cofactor of Escherichia coli Nitrate Reductase A (NarGHI)

The Molybdenum Cofactor of Escherichia coli Nitrate Reductase A (NarGHI)

Effect of molybdate and tungstate on the biosynthesis of CO dehydrogenase and the molybdopterin cytosine‐dinucleotide‐type of molybdenum cofactor in Hydrogenophaga pseudoflava

The hydrogenases and formate dehydrogenases ofEscherichia coli

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