Saccharomyces cerevisiae CBR1 encodes a single-pass membrane flavoprotein NADH-cytochrome b5 reductase with cytosolic-facing catalytic activity at microsomal and mitochondrial outer membranes. Cbr1 reduces ferricytochrome b5 from NADH and is the major physiological NADH-dependent reductase for Dph3, supplying the reduced Dph3 electron/iron carrier that supports both Elp3-dependent mcm5s2U tRNA wobble uridine modification and the Dph1-Dph2-dependent first step of diphthamide biosynthesis.
Definition: Catalysis of the reaction: 2 Fe(3+)-[Dph3] + NADH = 2 Fe(2+)-[Dph3] + NAD(+) + H(+).
Justification: RHEA:71231 captures the NADH-dependent reduction of Dph3, and PMID:27694803 shows that S. cerevisiae Cbr1 performs this reaction in the physiological reduction system for the Dph1-Dph2 and Elp3 radical-SAM enzymes. GO:0090560 describes the downstream 2-(3-amino-3-carboxypropyl) histidine synthase reaction catalyzed by Dph1-Dph2 rather than the Cbr1 reductase step.
Parent term: oxidoreductase activity, acting on NAD(P)H
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0002098 tRNA wobble uridine modification | IMP PMID:27694803 Cbr1 is a Dph3 reductase required for the tRNA wobble uridin... | MODIFY | Summary: Cbr1 acts in Dph3/Elp3-dependent tRNA wobble uridine modification, but the descendant mcm5s2U-specific term better captures the affected wobble chemistry. Reason: Cbr1 is the major physiological NADH-dependent reductase of Dph3 in budding yeast, and cbr1 deletion impairs the Dph3-dependent mcm5s2U wobble uridine modification assayed by gamma-toxin cleavage. GO:0002098 is true as a broad parent, but this same paper supports the more specific GO:0002926 row already present on CBR1. Proposed replacements: tRNA wobble base 5-methoxycarbonylmethyl-2-thiouridinylation Supporting Evidence: PMID:27694803 formation of tRNA modifications was partially impaired |
| GO:0002926 tRNA wobble base 5-methoxycarbonylmethyl-2-thiouridinylation | IMP PMID:27694803 Cbr1 is a Dph3 reductase required for the tRNA wobble uridin... | ACCEPT | Summary: Cbr1 is directly involved in Elp3/Dph3-dependent mcm5s2U wobble-base modification through NADH-dependent reduction of Dph3. Reason: Lin et al. identified Cbr1 as the major physiological Dph3 reductase and showed that a cbr1 deletion strain has reduced mcm5s2U-dependent gamma-toxin cleavage of glu-tRNA. That places Cbr1 upstream of the radical-SAM Elp3 reaction as the reductase that helps supply reduced Dph3 for mcm5s2U formation. Supporting Evidence: PMID:27694803 CBR1 deletion alone conferred some resistance to Ξ³-toxin, suggesting that formation of tRNA modifications was partially impaired |
| GO:0003954 NADH dehydrogenase activity | IDA PMID:27694803 Cbr1 is a Dph3 reductase required for the tRNA wobble uridin... | MODIFY | Summary: PMID:27694803 assays NADH-dependent reduction of Dph3, so the broad NADH dehydrogenase parent should be replaced by a Dph3 reductase term. Reason: This paper identifies Cbr1 as an NADH-specific reductase for oxidized Dph3 and reconstitutes Dph1-Dph2 radical-SAM activity with the Cbr1/NADH/Dph3 reduction system. GO:0003954 is far too broad for that activity, and the Dph3 electron-acceptor specificity is not captured by GO:0090524, so this row should be moved to an NADH-dependent Dph3 reductase term when GO has one. Proposed replacements: NADH-dependent Dph3 reductase activity Supporting Evidence: PMID:27694803 Upon addition of NADH to initiate the reaction, Dph3 was rapidly reduced by Cbr1 |
| GO:0003954 NADH dehydrogenase activity | IEA GO_REF:0000117 | MODIFY | Summary: The ARBA model infers the correct NADH reductase fold but should use the more specific NADH-dependent cytochrome-b5 reductase term. Reason: Cbr1 is experimentally characterized as an FAD-containing NADH:ferricytochrome-b5 reductase with EC 1.6.2.2 activity. GO:0003954 omits the cytochrome-b5 acceptor and is an avoidably broad parent beside the GO:0090524 row. Proposed replacements: cytochrome-b5 reductase activity, acting on NADH Supporting Evidence: PMID:14930 The presence of NADH-cytochrome b5 reductase [EC 1.6.2.2] in microsomes from anaerobically grown yeast was confirmed by its isolation and purification. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IBA GO_REF:0000033 | MODIFY | Summary: PTN001064671 places cytochrome-b5 reductase activity on the CBR1-like Saccharomycetes branch; that parent activity should be narrowed to the NADH-specific child term. Reason: The PAINT IBA is correctly placed for S. cerevisiae CBR1, and the SGD CBR1 self-seed is valid PAINT evidence rather than circular support. The asserted GO:0004128 term is the NAD(P)H parent and should be narrowed to GO:0090524, which names the NADH-dependent cytochrome-b5 reductase reaction assayed for Cbr1. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN001064671 Β· PANTHER:PTN001064671 SUPPORTS TRANSFER This node covers the CBR1-like Saccharomycetes branch and carries the cytochrome-b5 reductase IBD seeded by S. cerevisiae CBR1. Proposed replacements: cytochrome-b5 reductase activity, acting on NADH Supporting Evidence: file:interpro/panther/PTHR19370/PTHR19370-paint.tsv PTHR19370 PTN001064671 GO:0004128 F IBD false |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IDA PMID:14930 Studies on the microsomal electron-transport system of anaer... | MODIFY | Summary: The old microsomal purification directly supports cytochrome-b5 reductase activity but the NADH-specific GO:0090524 row captures it more exactly. Reason: Kubota et al. purified the anaerobically induced yeast microsomal enzyme and showed that it used NADH to reduce cytochrome b5. The broader NAD(P)H term should therefore be narrowed to GO:0090524, which names the actual reductant assayed in the paper. Proposed replacements: cytochrome-b5 reductase activity, acting on NADH Supporting Evidence: PMID:14930 The reductase required NADH as a specific electron donor, and could reduce some redox dyes as well as cytochrom b5. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IDA PMID:27694803 Cbr1 is a Dph3 reductase required for the tRNA wobble uridin... | MODIFY | Summary: The 2016 paper supports NADH-dependent reduction of Dph3 rather than a cytochrome-b5 assay, so this molecular-function row needs the Dph3 reductase NTR. Reason: Lin et al. identified Cbr1 in the Dph3 interactome and directly demonstrated NADH-dependent reduction of purified Dph3 by Cbr1. The supported activity is therefore reduction of the Dph3 iron protein, not the ferricytochrome-b5 activity named by GO:0004128. Proposed replacements: NADH-dependent Dph3 reductase activity Supporting Evidence: PMID:27694803 Here, using a proteomic approach, we identified Saccharomyces cerevisiae cytochrome b5 reductase (Cbr1) as a NADH-dependent reductase for Dph3. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IDA PMID:34154323 Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating On... | MODIFY | Summary: PMID:34154323 uses the Cbr1/Dph3/NADH system to support Dph1-Dph2, so it points to Dph3 reductase activity rather than ferricytochrome-b5 reduction. Reason: The 2021 mechanism paper tests how reduced Dph3 maintains the Dph1-Dph2 Fe-S cluster during the radical-SAM step of diphthamide biosynthesis. Cbr1 is the NADH-dependent reductase in that system, whereas Dph1-Dph2 is the enzyme catalyzing ACP transfer to eEF2. Proposed replacements: NADH-dependent Dph3 reductase activity Supporting Evidence: PMID:34154323 the small iron-containing protein Dph3 donates one Fe atom to convert the [3Fe-4S] cluster in Dph1-Dph2 to a functional [4Fe-4S] cluster during the radical-SAM enzyme catalytic cycle. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IEA GO_REF:0000117 | MODIFY | Summary: The ARBA inference of cytochrome-b5 reductase activity is sound but should be narrowed from the NAD(P)H parent to the NADH-specific child. Reason: Cbr1 has direct biochemical evidence for NADH-dependent cytochrome-b5 reduction and lacks evidence for an NADPH-dependent cytochrome-b5 reaction, so GO:0090524 is the best replacement for this broader GO:0004128 prediction. Proposed replacements: cytochrome-b5 reductase activity, acting on NADH Supporting Evidence: PMID:14930 The reductase required NADH as a specific electron donor, and could reduce some redox dyes as well as cytochrom b5. |
| GO:0005634 nucleus | HDA PMID:26928762 One library to make them all: streamlining the creation of y... | MARK AS OVER ANNOTATED | Summary: The N-terminal SWAT-GFP nucleus call likely overreports soluble/nuclear localization by placing GFP directly upstream of Cbr1's only transmembrane anchor. Reason: The SWAT-GFP paper created an N-terminally tagged endomembrane-focused library and assigned subcellular localizations at high throughput. Cbr1 is a single-pass membrane protein whose only transmembrane helix is at residues 7-27, so an N-terminal GFP tag is placed directly upstream of the membrane anchor and could plausibly mask Cbr1's targeting determinant. This unreplicated nucleus call should therefore be treated as an over-annotation rather than as a core site for a membrane-anchored NADH-cytochrome-b5/Dph3 reductase. Supporting Evidence: PMID:26928762 To establish this strategy we constructed a library containing ~1,800 strains where all proteins with known or predicted localization to the yeast endomembrane system are tagged with an amino terminal file:genes/yeast/CBR1/CBR1-uniprot.txt TRANSMEM 7..27 |
| GO:0005739 mitochondrion | HDA PMID:14576278 The proteome of Saccharomyces cerevisiae mitochondria. | KEEP AS NON CORE | Summary: The mitochondrial proteome screen supports Cbr1 recovery from purified mitochondria as contextual localization evidence. Reason: This HDA row comes from a broad mass-spectrometry catalog of highly pure yeast mitochondria. It is consistent with the later mitochondrial outer-membrane assignment for Cbr1, but the broad mitochondrion term is a parent of that more exact compartment. Supporting Evidence: PMID:14576278 We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria. |
| GO:0005739 mitochondrion | HDA PMID:16823961 Toward the complete yeast mitochondrial proteome: multidimen... | KEEP AS NON CORE | Summary: The PROMITO mass-spectrometry dataset is acceptable support for mitochondrial recovery of Cbr1, but it is not as precise as the outer-membrane localization. Reason: Reinders et al. combined orthogonal proteomics methods to expand the yeast mitochondrial proteome. The resulting CBR1 HDA row is compatible with the protein's outer-membrane annotation but stays secondary because it reports the parent organelle. Supporting Evidence: PMID:16823961 A total of 851 different proteins (PROMITO dataset) were identified by use of multidimensional LC-MS/MS, 1D-SDS-PAGE combined with nano-LC-MS/MS and 2D-PAGE with subsequent MALDI-mass fingerprinting. |
| GO:0005739 mitochondrion | HDA PMID:24769239 Quantitative variations of the mitochondrial proteome and ph... | KEEP AS NON CORE | Summary: The quantitative isolated-mitochondria proteomics row reinforces mitochondrial recovery of Cbr1 but remains a broad localization parent. Reason: Renvoise et al. quantified proteins in isolated mitochondria from yeast grown on fermentative and respiratory carbon sources. This high-throughput evidence is compatible with Cbr1 at the mitochondrial outer membrane but does not improve on the more specific outer-membrane row. Supporting Evidence: PMID:24769239 we performed an overall quantitative proteomic and phosphoproteomic study of isolated mitochondria extracted from yeast grown on fermentative (glucose or galactose) and respiratory (lactate) media. |
| GO:0005741 mitochondrial outer membrane | HDA PMID:16407407 Proteomic analysis of the yeast mitochondrial outer membrane... | ACCEPT | Summary: Direct proteomic analysis of purified mitochondrial outer membranes supports retaining Cbr1 at the mitochondrial outer membrane. Reason: Zahedi et al. assayed highly purified yeast mitochondrial outer membranes and the resulting UniProt curation places Cbr1 in that compartment. This matches Cbr1's N-terminal membrane anchor and cytosolic catalytic domain. Supporting Evidence: PMID:16407407 We used a eukaryotic model organism, the yeast Saccharomyces cerevisiae, to determine the proteome of highly purified mitochondrial outer membranes. |
| GO:0005741 mitochondrial outer membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt's subcellular-location mapping to the mitochondrial outer membrane is consistent with the direct yeast outer-membrane proteomics evidence. Reason: The Swiss-Prot subcellular-location mapping reflects the curated mitochondrial outer-membrane localization of this single-pass membrane-anchored reductase and agrees with the Zahedi et al. HDA row. Supporting Evidence: PMID:16407407 We used a eukaryotic model organism, the yeast Saccharomyces cerevisiae, to determine the proteome of highly purified mitochondrial outer membranes. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PTN001064671 propagates a Candida-seeded plasma-membrane assertion across the CBR1-like Saccharomycetes branch; the assertion is plausible but secondary for budding yeast Cbr1. Reason: The PAINT row is a node assertion rather than a pairwise transfer, and the Candida seed is on the same CBR1-like branch as S. cerevisiae CBR1. Keep the plasma-membrane IBA as a valid inferred localization, while not treating it as evidence against the direct yeast mitochondrial outer-membrane rows. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN001064671 Β· PANTHER:PTN001064671 SUPPORTS TRANSFER This CBR1-like Saccharomycetes node carries the plasma-membrane IBD seeded by Candida CBR1; its placement supports the GOA IBA but does not negate S. cerevisiae mitochondrial outer-membrane evidence. Supporting Evidence: file:interpro/panther/PTHR19370/PTHR19370-paint.tsv PTHR19370 PTN001064671 GO:0005886 C IBD false |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro identifies Cbr1 as a flavoprotein oxidoreductase; the GO:0016491 parent is true but generic. Reason: Cbr1 contains the conserved FAD/NAD-binding domains of the flavoprotein pyridine nucleotide cytochrome reductase family and is an oxidoreductase. This InterPro2GO mapping is therefore biologically correct, but the exact functions are its NADH-dependent reductions of cytochrome b5 and Dph3. Supporting Evidence: PMID:14930 The reductase appeared to contain loosely-bound FAD as a prosthetic group. |
| GO:0017183 protein histidyl modification to diphthamide | IDA PMID:31463593 The asymmetric function of Dph1-Dph2 heterodimer in diphtham... | ACCEPT | Summary: Cbr1 contributes reducing equivalents to the Dph1-Dph2 radical-SAM system during diphthamide biosynthesis. Reason: Zhang et al. used the physiological Dph3/Cbr1/NADH reducing system to show that the Fe-S cluster in Dph2 facilitates electron transfer to the catalytic Dph1 cluster. Cbr1 does not perform ACP transfer itself, but it does the work of this process by reducing Dph3 for Dph1-Dph2. Supporting Evidence: PMID:31463593 the Dph1 cluster serves a catalytic role, while the Dph2 cluster facilitates the reduction of the Dph1 cluster by the physiological reducing system Dph3/Cbr1/NADH. |
| GO:0017183 protein histidyl modification to diphthamide | IDA PMID:34154323 Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating On... | ACCEPT | Summary: The aerobic Dph1-Dph2 repair mechanism reinforces Cbr1/Dph3-dependent participation in diphthamide biosynthesis. Reason: The 2021 study showed that Dph3 donates iron to restore a functional Dph1-Dph2 [4Fe-4S] cluster during the radical-SAM catalytic cycle, while Cbr1 uses NADH to reduce Dph3. That places Cbr1's Dph3 reductase activity within the first step of protein histidyl modification to diphthamide. Supporting Evidence: PMID:34154323 the small iron-containing protein Dph3 donates one Fe atom to convert the [3Fe-4S] cluster in Dph1-Dph2 to a functional [4Fe-4S] cluster during the radical-SAM enzyme catalytic cycle. |
| GO:0017183 protein histidyl modification to diphthamide | IEA GO_REF:0000041 | ACCEPT | Summary: The UniPathway mapping to diphthamide biosynthesis is consistent with Cbr1's experimentally established Dph3 reductase role. Reason: Cbr1 is not one of the enzymes that modify eEF2 directly, but its NADH-dependent reduction of Dph3 supplies the Dph1-Dph2 radical-SAM synthase system. The pathway-level IEA is therefore appropriate. Supporting Evidence: PMID:27694803 the Cbr1/NADH/Dph3 reduction system was able to reduce Dph1-Dph2 similarly to dithionite, a chemical reductant for Fe-S clusters |
| GO:0017183 protein histidyl modification to diphthamide | IMP PMID:27694803 Cbr1 is a Dph3 reductase required for the tRNA wobble uridin... | ACCEPT | Summary: Loss of CBR1 impairs Dph3-dependent diphthamide formation under increased eEF2 demand, supporting involvement in the pathway. Reason: A single cbr1 deletion did not abolish basal diphthamide formation, but cbr1 and cbr1 mcr1 deletion strains became resistant to diphtheria toxin when eEF2 was overexpressed. That phenotype is consistent with Cbr1 reducing Dph3 for the Dph1-Dph2 system and supports this process annotation. Supporting Evidence: PMID:27694803 purified eEF2 from cbr1Ξ strain over-expressing eEF2 showed a significant decrease in diphthamide modification level |
| GO:0090524 cytochrome-b5 reductase activity, acting on NADH | EXP PMID:14930 Studies on the microsomal electron-transport system of anaer... | ACCEPT | Summary: The microsomal yeast enzyme was purified and shown to reduce cytochrome b5 with NADH, directly supporting GO:0090524. Reason: Kubota et al. biochemically characterized the anaerobically induced yeast microsomal NADH-cytochrome b5 reductase and measured cytochrome-b5 reduction from NADH, so the NADH-specific child term is exactly supported. Supporting Evidence: PMID:14930 Michaelis constants of the reductase for NADH and calf liver cytochrome b5 were 6.3 and 1.5 micron M, respectively, and optimal pH for cytochrome b5 reduction was 5.6. |
| GO:0090524 cytochrome-b5 reductase activity, acting on NADH | IEA GO_REF:0000120 | ACCEPT | Summary: The Rhea/EC mapping captures the exact NADH:ferricytochrome-b5 reaction characterized for yeast Cbr1. Reason: The RHEA:46680 / EC 1.6.2.2 mapping names the same reaction established in the microsomal purification work: electron transfer from NADH to ferricytochrome b5. Supporting Evidence: PMID:14930 The presence of NADH-cytochrome b5 reductase [EC 1.6.2.2] in microsomes from anaerobically grown yeast was confirmed by its isolation and purification. |
| GO:0090560 2-(3-amino-3-carboxypropyl)histidine synthase activity | IDA PMID:24422557 Dph3 is an electron donor for Dph1-Dph2 in the first step of... | MODIFY | Summary: PMID:24422557 defines the Dph1-Dph2/Dph3 chemistry but predates discovery of Cbr1, and GO:0090560 names the downstream synthase rather than Cbr1's Dph3 reductase activity. Reason: Dong et al. showed that Dph3 is the electron carrier for Dph1-Dph2 and explicitly left the endogenous Dph3 reductase unknown. Lin et al. later identified that reductase as Cbr1. The annotation should therefore point Cbr1 to NADH-dependent Dph3 reductase activity, not the Dph1-Dph2 ACP-transfer activity described by GO:0090560; Cbr1's contribution to that complex-level activity is preserved in the core function as contributes_to rather than discarded. Proposed replacements: NADH-dependent Dph3 reductase activity Supporting Evidence: PMID:24422557 The identity of the endogenous reductase for Dph3 is unknown. PMID:27694803 Here, using a proteomic approach, we identified Saccharomyces cerevisiae cytochrome b5 reductase (Cbr1) as a NADH-dependent reductase for Dph3. |
| GO:0090560 2-(3-amino-3-carboxypropyl)histidine synthase activity | IDA PMID:34154323 Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating On... | MODIFY | Summary: The 2021 Dph1-Dph2/Dph3 study supports Cbr1 as a reducing-system component, but not as the ACP-transfer enzyme named by GO:0090560. Reason: GO:0090560 is the radical-SAM synthase activity of Dph1-Dph2. In this system Cbr1 uses NADH to reduce Dph3 upstream, and Dph3 then supplies iron and electrons needed by Dph1-Dph2. Cbr1 should be annotated to a Dph3 reductase term rather than as the enzyme directly attaching ACP to eEF2; Cbr1's membership in the broader reducing system is retained in the core function as a contributes_to relationship to GO:0090560. Proposed replacements: NADH-dependent Dph3 reductase activity Supporting Evidence: PMID:34154323 Under physiological conditions, the reduction of the [4Feβ4S] clusters in Dph1βDph2 requires Dph3 and Cbr1. |
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Download this section (compressed HTML)Q: Should GO add an NADH-dependent Dph3 reductase molecular-function term for RHEA:71231 so CBR1-like reductases can be annotated to their direct input into Dph3-dependent radical-SAM systems rather than to the Dph1-Dph2 synthase activity?
Q: Which membrane pool of S. cerevisiae Cbr1 supplies reduced Dph3 in vivo, and is Dph3 reduction spatially coupled to the ER, mitochondrial outer membrane, or both?
Q: Under which sterol-biosynthetic conditions does Cbr1 provide cytochrome-b5-dependent electrons to yeast cytochrome P450 enzymes in vivo, rather than acting primarily on Dph3?
Experiment: Retarget Cbr1 selectively to the ER or mitochondrial outer membrane in a cbr1 deletion background and measure mcm5s2U formation, diphthamide formation under eEF2 overexpression, and Dph3 redox state to determine which membrane pool supplies physiological Dph3 reductase activity.
Experiment: Measure sterol intermediates and cytochrome P450-dependent sterol flux in cbr1, mcr1, and cbr1 mcr1 strains under respiratory, anaerobic, and NADPH-cytochrome P450 reductase-limiting conditions to test the in vivo contribution of Cbr1 to the fungal cytochrome-b5 electron-transfer system.
Cytochrome b reductase and NADH-dependent reductase for Dph3p; required for diphthamide synthesis and tRNA wobble uridine modification; also detected in mitochondria; mutation in conserved NADH binding domain of the human ortholog results in type I methemoglobinemia
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