The “cytochrome b5 fold”: Structure of a novel protein superfamily

Guiard, Bernard; Lederer, Florence

doi:10.1016/0022-2836(79)90169-4

Cited by 78 publications

(37 citation statements)

References 48 publications

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“…This region shows 42% identity to the bovine liver cytochrome b5 catalytic domain. The histidine residues at positions 572 and 595 are conserved in all NR sequences as well as in all members of the cytochrome b5 superfamily (Guiard and Lederer 1979) in which they are considered to function as heme-ligands. Amino acids 634-902 show 41% identity to the catalytic domain of human NADH-cytochrome b5 reductase indicating that the FAD domain is situated at the C-terminal part of the protein.…”

Section: Comparison Of P Infestans Nr With That Of Other Organismsmentioning

confidence: 99%

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

et al. 1995

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The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene and its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a niadeletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia-mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli P-glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.C. M. J. Pietersel J. van't Klooster G. C. M. van den Berg-Velthuis F. Govers (2i)

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Section: Comparison Of P Infestans Nr With That Of Other Organismsmentioning

confidence: 99%

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

et al. 1995

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“…Cyt b, belongs to a family of hemoproteins characterized by the so-called 'cytochrome b, fold' [14], while reductase is a member of a recently discovered flavoenzyme family of dehydrogenases-electron transferases [15]. Proteins of both families have diverse intracellular localizations.…”

Section: Structure and Function Of The Ermentioning

confidence: 99%

NADH‐cytochrome b₅ reductase and cytochrome b₅ isoforms as models for the study of post‐translational targeting to the endoplasmic reticulum

et al. 1993

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Cytochrome b5 and NADH-cytochrome b, reductase are integral membrane proteins with cytosolic active domains and short membrane anchors, which are inserted post-translationally into their target membranes. Both are produced as different isoforms, with different localizations, in mammalian cells. In the rat, the reductase gene generates two transcripts by an alternative promoter mechanism: a ubiquitous mRNA coding for the myristylated membrane-bound form, and an erythroid mRNA which generates both the soluble form and a nonmyristylated membrane-binding form. The available evidence indicates that the ubiquitous myristylated form binds to the cytosolic face of both outer mitochondrial membranes and ER. In contrast, two genes code for two homologous forms of cytochrome b5, one of which is found on outer mitochondrial membranes, the other on the ER. The gene specifying the ER form probably also generates an erythroid-specific mRNA by alternative splicing, which codes for soluble cytochrome bS. Possible molecular mechanisms responsible for the observed locahzations of these different enzyme isoforms are discussed.

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“…This enzyme was chosen because flavocytochrome hl is a bifunctional protein which incorporates in one molecule the equivalent of cytochrome h5 and its reductase. The heme-binding domain of flavocytochrome h2 has been shown to be homologous to cytochrome h5 [9]. Nothing is known yet concerning a possible homology between the flavodehydrogenase part and the reductase but, in any event, in view of the exceptional similarity between the electron acceptors, one would expect the flavin-binding features responsible for the electron transferase activity to be identical in flavocytochrome bz and cytochrome b5 reductase.…”

Section: Enzvmementioning

confidence: 99%

Reconstitution of Liver NADH: Cytochrome b₅ Oxidoreductase and of Desulfovibrio vulgaris Flavodoxin with 1‐Carba‐1‐deazaflavin

Pompon

Guiard

Lederer

1982

European Journal of Biochemistry

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Flavin-free cytochrome bs reductase was reconstituted with 1-deazaflavin and 5-deazaflavin rnononucleotides and dinucleotides. The 5-deazaenzyme functioned in transhydrogenation reactions but lacked electron transferase activity. The 1-deazaenzyme was fully competent for both input and output reactions. The flavin reduction rate was lowered about sevenfold upon N-1 substitution of FAD, but hydrogen abstraction from NADH remained the limiting step. Autoxidation of the reduced enzyme was more rapid than with the normal cofactor. Oxidation was accompanied by appearance of a transient blue-type semiquinone and superoxide ion production.Flavin-free apoflavodoxin was reconstituted with 1 -deaza-I-carbaflavin mononucleotide (1 -deaza-FMN). Its behaviour toward dithionite and oxygen was qualitatively highly similar to that of native flavodoxin.These observations contrast with the fact that apoflavocytochrome bl could not be reconstituted with 1-deaza-FMN Eur. J. Biochem. 96, 571 -5791. These results, as well as other data from the literature, are discussed in the light of existing hypotheses, which try to correlate flavin protein interactions and flavoprotein function.Flavin analogs have become increasingly popular tools in flavoprotein studies. Among them, the deazaanalogs were proposed primarily as mechanistic tools. The chemical properties of the free cofactors have been reviewed [l -31. 5-Deazacofactors proved somewhat disappointing; they behaved in a similar fashion when bound to proteins catalyzing either one or two-electron redox reactions, insofar as they lack oneelectron chemistry and reactivity with oxygen. On the other hand, 1-deazaflavins still possess these two properties [l], and may provide more insight into protein influence on flavin reactivity. In particular a number of oxidases have been found to utilize I-deazaflavin for their normal catalytic reaction [4] We were the first to report the attempted reconstitution of a dehydrogenase/electron transferase with 1-deazaflavin [7,8]. We found that flavocytochrome bz did not detectably bind 1-deaza-FMN'. In order to check whether this lack of affinity was a general property of this flavoenzyme class, or was specific of the particular protein used, we decided to investigate the reconstitution of NADH : cytochrome b5 oxidoreductase Ahhrcviationc. EPR, electron paramagnetic resonance, 5-deaza-FMN, 5-deaza-Scarbaflavin mononucleotide ; 1 -deaza-FMN, 1 -deaza-I-carbaflavin mononucleotide. ___ - Enzvme.Cytochrome hs reductase (EC 1.6.2.2).Since our first report [7]. we have obtained a better estimate of the upper limit for the affinity of 1-deaza-FMN for flavin-free cytochromehz, by using the apoflavodoxin trap described by others for lowering the residual activity before reconstitution. No activity above a background of 6 x times the normal reconstituted activity was observed when 15 pM 1-deaza-FMN was incubated in the presence of flavin-free cytochrome hZ. It can be thus estimated that the affinity of 1-deaza-FMN is more than a 1000-fold weaker than that ofwith...

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The “cytochrome b5 fold”: Structure of a novel protein superfamily

Cited by 78 publications

References 48 publications

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

NADH‐cytochrome b₅ reductase and cytochrome b₅ isoforms as models for the study of post‐translational targeting to the endoplasmic reticulum

Reconstitution of Liver NADH: Cytochrome b₅ Oxidoreductase and of Desulfovibrio vulgaris Flavodoxin with 1‐Carba‐1‐deazaflavin

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The “cytochrome b5 fold”: Structure of a novel protein superfamily

Cited by 78 publications

References 48 publications

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

NADH‐cytochrome b5 reductase and cytochrome b5 isoforms as models for the study of post‐translational targeting to the endoplasmic reticulum

Reconstitution of Liver NADH: Cytochrome b5 Oxidoreductase and of Desulfovibrio vulgaris Flavodoxin with 1‐Carba‐1‐deazaflavin

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NADH‐cytochrome b₅ reductase and cytochrome b₅ isoforms as models for the study of post‐translational targeting to the endoplasmic reticulum

Reconstitution of Liver NADH: Cytochrome b₅ Oxidoreductase and of Desulfovibrio vulgaris Flavodoxin with 1‐Carba‐1‐deazaflavin