NTR: Transmembrane ascorbate ferrireductase activity

[lbreuza]

Hi,

wouldn't is make sense to create a term describing the molecular function of Cytochrome b561 protein family based on https://www.rhea-db.org/rhea/30403, EC 7.2.1.3 and described in this papers: PubMed=16911521, PubMed=24449903. Definition (tentative): Transmembrane ascorbate ferrireductase that that transfers electrons across a single membrane.

Reaction=Fe(3+)(out) + L-ascorbate(in) = Fe(2+)(out) + H(+) + monodehydro-L-ascorbate radical(in) [Rhea:30403]

It would be a child of GO:0016723. oxidoreductase activity, oxidizing metal ions, NAD or NADP as acceptor

It looks like thee creation of this term makes sense since the term GO:0000293 ferric-chelate reductase activity has been curated to some members of that protein family, but the mechanism described may not fit there. Maybe @kaxelsen would be interested in this query too.

Best, Lionel

[pgaudet]

This is similar to https://enzyme.expasy.org/EC/1.10.2.1 https://www.rhea-db.org/rhea/18677 (no annotation).

Some of those EC:7 terms are little confusing, mixing transporters and enzymatic activities. Is this reaction to transport iron or to reduce it ?

Thanks, Pascale

[lbreuza]

Hi Pascale, this is why I had also added @kaxelsen to this ticket. My understanding is that it oxidizes ascorbate on one side of the membrane while reducing iron on the other side of the membrane. Hence there is a flux of electron from in to out. This is why it is considered a transport reaction coupled to an oxido-reduction (EC.7...). The terms you mentioned are not describing reaction within a membrane and therefore there is not transport (only the oxido-reduction). Lionel

[pgaudet]

Do we know that EC/1.10.2.1 exists ? I just wonder if two similar terms were created. Maybe we can add the other xrefs to ~GO:0016723~  GO:0047063. In any case you ask for this term to be under oxidoreductase activity, not under transporter activity. Looks like the membrane is a mechanism rather than true transport ?

[kaxelsen]

I'm looking at it right now, and since EC 1.10.2.1 is based on only 1 article from 1969, I suspect that it might be the same as EC 7.2.1.3

[kaxelsen]

I have now found the article (there was no abstract in PubMed) and read the abstryt (in German) and this protein IS the same as EC 7.2.1.3 because the work with the microsomal (i.e. ER membrane) fraction. I will do the necessary for this entry to be deleted from the EC list.

[pgaudet]

Thanks @kaxelsen @lbreuza we can change the xrefs on GO:0047063 and update the term definition.

[lbreuza]

Did you have a look at the parent term of GO:0047063? It would not match with the function I am trying to capture.

[pgaudet]

@kaxelsen To add to the confusion, https://enzyme.expasy.org/EC/7.2.1.3 states "Formerly EC 1.16.5.1. ", which is an Oxidoreductases with a quinone or similar compound as acceptor

[pgaudet]

@amorgat @kaxelsen GO: was linking to https://www.rhea-db.org/rhea/18677 I think this also need to be merged into the new RHEA:30403

[pgaudet]

I propose this definition:

Oxidation of Fe(3+) to Fe(2+) outside of a membrane coupled to the reduction of L-ascorbate to monodehydro-L-ascorbate radical on the inner side of a membrane. Electrons get transferred across the membrane during the reaction."

@lbreuza please let me know

I will obsolete GO:0047063 since it's too different, and incorrect.

[pgaudet]

@kaxelsen We should review what's in the branch here:

https://enzyme.expasy.org/EC/1.10.3.-

Oxidoreductases Acting on diphenols and related substances as donors With oxygen as acceptor

Many of those are not acting on diphenols, as far as I can tell

[kaxelsen]

@kaxelsen To add to the confusion, https://enzyme.expasy.org/EC/7.2.1.3 states "Formerly EC 1.16.5.1. ", which is an Oxidoreductases with a quinone or similar compound as acceptor

Yes, and the reaction was ascorbate[side 1] + Fe(III)[side 2] = monodehydroascorbate[side 1] + Fe(II)[side 2]

@amorgat @kaxelsen GO: was linking to https://www.rhea-db.org/rhea/18677 I think this also need to be merged into the new RHEA:30403

As we cannot merge reactions in Rhea, RHEA:18677 will be set to obsolete with a public note stating that it is now covered by RHEA:30403

[kaxelsen]

@kaxelsen We should review what's in the branch here:

https://enzyme.expasy.org/EC/1.10.3.-

Oxidoreductases Acting on diphenols and related substances as donors With oxygen as acceptor

Many of those are not acting on diphenols, as far as I can tell

I see what you mean, but they must go into the category "related substances" ;-)

[pgaudet]

ascorbate and iron are related to phenols ? OK I need to find my chemistry textbook....

[kaxelsen]

I agree, that ascorbate is really on the limit, but I think it is because it is an enol compound (or unsaturated alcohol that can loose one electron. I don't see any reactions with iron in EC 1.10.3.-

[lbreuza]

Hi, I believe the parent term should be : GO:0016491 because non of the child matches correctly the reaction described. Cheers, Lionel

[pgaudet]

I agree, we cannot be any more specific for now.

[pgaudet]

NTR +[Term] +id: GO:0140571 +name: transmembrane ascorbate ferrireductase activity +namespace: molecular_function +def: "Oxidation of Fe(3+) to Fe(2+) outside of a membrane coupled to the reduction of L-ascorbate to monodehydro-L-ascorbate radical on the inner side of a membrane. Electrons get transferred across the membrane during the reaction." [PMID:16911521, PMID:24449903, RHEA:30403] +synonym: "ascorbate-cytochrome b5 reductase activity" EXACT [] +synonym: "L-ascorbate-cytochrome-b5 reductase activity" EXACT [] +synonym: "L-ascorbate:ferricytochrome-b5 oxidoreductase activity" EXACT [] +xref: EC:7.2.1.3 +xref: MetaCyc:1.10.2.1-RXN +xref: RHEA:30403 +is_a: GO:0016491 ! oxidoreductase activity +created_by: pg +creation_date: 2020-12-18T16:25:06Z

[lbreuza]

Dear Pascale,

I am now curating CYB561 which has a very similar activity except that it reduces semidebydroascorbate into ascorbate within the lumen of secretory vesicles and uses cytoplasmic ascorbate as an electron donor to replenish the lumen of vesicles with ascorbate as shown by PubMed=1623014. Could you please consider creating a Molecular Function term to describe this activity. It is not identical to GO:0016656 monodehydroascorbate reductase (NADH) activity that uses NADH as an electron donor.

I suggest : NTR +[Term] +id: GO:xxxxxxx +name: transmembrane monodehydroascorbate reductase activity +namespace: molecular_function +def: "Oxidation of monodehydroascorbate outside of a membrane coupled to the reduction of L-ascorbate to monodehydro-L-ascorbate radical on the inner side of a membrane. Electrons get transferred across the membrane during the reaction."

L-ascorbate(in) + monodehydro-L-ascorbate radical(out)= H(+) + monodehydro-L-ascorbate radical(in) + L-ascorbate(out)

Btw I realized you did not provide the reaction in the transmembrane ascorbate ferrireductase activity term.

[PMID:1623014, RHEA:?] +xref: EC:7.2.1.- (no specific EC) +xref: RHEA:? (I have asked for the reaction) +is_a: GO:0016491 ! oxidoreductase activity

Thanks for your help. Best, Lionel

[pgaudet]

+[Term] +id: GO:0140575 +name: transmembrane monodehydroascorbate reductase activity +namespace: molecular_function +def: "Oxidation of monodehydroascorbate outside of a membrane coupled to the reduction of L-ascorbate to monodehydro-L-ascorbate radical on the inner side of a membrane. Electrons get transferred across the membrane during the reaction." [PMID:1623014] +xref: EC:7.2.1.- +is_a: GO:0016491 ! oxidoreductase activity +property_value: term_tracker_item https://github.com/geneontology/go-ontology/issues/20602 xsd:anyURI +created_by: pg +creation_date: 2021-01-07T16:20:04Z

[pgaudet]

Also created the corresponding process:

+[Term] +id: GO:0140576 +name: cellular ascorbate homeostasis +namespace: biological_process +def: "Any process involved in the maintenance of an internal steady state of ascorbate at the level of a cell." [PMID:1623014, PMID:32547589] +is_a: GO:0019725 ! cellular homeostasis +property_value: term_tracker_item https://github.com/geneontology/go-ontology/issues/20602 xsd:anyURI +created_by: pg +creation_date: 2021-01-07T16:50:44Z

[pgaudet]

@lbreuza I created the 2 new terms. Please let me know when the Rhea ID is available.

Thanks, Pascale

[kaxelsen]

It is RHEA:66524

[sjm41]

Hi @lbreuza Just wondering if you figured out how best to annotate the human CYB561 family members? (I'm looking at the fly orthologs, and struggling to work things out...)

Seems to be 5 relevant human genes/proteins from this page: https://www.genenames.org/data/genegroup/#!/group/497

Do you know if they all do both the reactions mentioned above, or do some do only one?

I see these manual annotations on protein2GO right now: CYB561 (P49447) - transmembrane monodehydroascorbate reductase activity CYBRD1 (Q53TN4) - transmembrane monodehydroascorbate reductase activity & transmembrane ascorbate ferrireductase activity CYB561A3 (Q8NBI2) - transmembrane ascorbate ferrireductase activity CYB561D1 (Q8N8Q1) - (neither annotation) CYB561D2 (O14569) - transmembrane monodehydroascorbate reductase activity & transmembrane ascorbate ferrireductase activity

This 2013 review may be helpful: https://pubmed.ncbi.nlm.nih.gov/23249217/

Another Q: Should 'electron transfer activity' be a parent of both terms?

[lbreuza]

Hi Steven, I have recently updated the human proteins containing a cytochrome b561 domain (the 5 human proteins you mention in your ticket). If you have access to the most recent GO annotations, then you can see my annotations in GO for these proteins. The best characterized protein in human is Q53TN4 : Plasma membrane reductase that uses cytoplasmic ascorbate as an electron donor to reduce extracellular Fe3+ into Fe2+ (PubMed:30272000). Probably functions in dietary iron absorption at the brush border of duodenal enterocytes by producing Fe2+, the divalent form of iron that can be transported into enterocytes (PubMed:30272000). It is also able to reduce extracellular monodehydro-L-ascorbate and may be involved in extracellular ascorbate regeneration by erythrocytes in blood (PubMed:17068337). May also act as a ferrireductase in airway epithelial cells (Probable). May also function as a cupric transmembrane reductase (By similarity).

The electron donor is ascorbate but acceptors may include iron and monodehydro-L-ascorbate. This is why I curated CYBRD1 with 2 GO terms. Other proteins containing this domain have either only one acceptor demonstrated or none yet which explains why they have only reaction annotated or none like CYB561D1.

These proteins catalyse an oxido-reduction reaction which results due to the fact that they are transmembrane proteins to the transfer of an electron to one side of a membrane to the other side.

Concerning the 'electron transfer activity' it sounds a reasonable annotation. I could add it. @pgaudet, do you have an opinion on that. I felt like it was redundant but maybe one could add it.

Lionel

[sjm41]

Thanks Lionel, that's helpful.

Since the 2 terms are related, I was thinking that a grouping term in the GO would be useful, so folks can easily find proteins annotated with either term. Something like "oxidoreductase activity, acting on ascorbate as donor" (similar to the existing grouping term "oxidoreductase activity, acting on diphenols and related substances as donors (GO:0016679))". What do you think?

Regarding the 'electron transfer activity' term, I was suggesting adding it as a parent to the GO term(s), rather than adding it as an extra annotation.

[lbreuza]

Hi Steven, what you suggest make sense and I basically agree. This is something we discussed with @pgaudet while quickly revisiting this part of the ontology and for some reasons, I cannot recall, we decided to keep it simple. Maybe Pascale wants to reconsider or comment on these suggestions. Best, Lionel