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fatty acid alpha-hydroxylase activity (GO:0080132) #27541

Closed sjm41 closed 4 months ago

sjm41 commented 7 months ago

I think "GO:0080132 fatty acid alpha-hydroxylase activity" equates to EC:1.14.18.6 (RHEA:46520), but I would appreciate a second opinion. If this is true, then the tasks are:

Here's what I see:

id: GO:0080132 name: fatty acid alpha-hydroxylase activity namespace: molecular_function def: "Catalysis of the conversion of a fatty acid to an alpha-hydroxylated fatty acid. A hydroxyl group is added to the second carbon, counted from the carboxyl end, of a fatty acid chain." [PMID:19054355] synonym: "fatty acid 2-hydroxylase" EXACT [] is_a: GO:0016491 ! oxidoreductase activity created_by: dhl creation_date: 2009-05-06T04:43:36Z

The reference (PMID:19054355) used here only describes this activity tangentially, saying "Mitchell and Martin (1997) reported previously that in Arabidopsis, At2g34770 (AtFAH1) was homologous to ScFAH1, and that it had fatty acid 2-hydroxylation activity in yeast."

The primary Mitchell and Martin (1997) reference is PMID: 9353282, and it was used as the source for this EC and corresponding RHEA:

EC:1.14.18.6 (https://www.enzyme-database.org/query.php?ec=1.14.18.6): Name: 4-hydroxysphinganine ceramide fatty acyl 2-hydroxylase Comments: The enzyme, characterized from yeast and mammals, catalyzes the hydroxylation of carbon 2 of long- or very-long-chain fatty acids attached to (4R)-4-hydroxysphinganine during de novo ceramide synthesis. The enzymes from yeast and from mammals contain an N-terminal cytochrome b5 domain that acts as the direct electron donor to the desaturase active site.

RHEA:46520: an N-(1,2 saturated acyl)-(4R)-hydroxysphinganine + 2 Fe(II)-[cytochrome b5] + 2 H(+) + O2 = an N-(2R-hydroxyacyl)-4R-hydroxysphinganine + 2 Fe(III)-[cytochrome b5] + H2O

GO:0080132 has 13 EXP annotations, including: Yeast SCS7 (including from Mitchell and Martin (1997) = PMID: 9353282) Rat Fa2h Mouse Fa2h Human FA2H Fly Fa2h

RHEA:46520 has one annotation in UniProt, to Yeast SCS7. EC:1.14.18.6 has 4 annotations in UniProt, including yeast SCS7.

Other sources for EC:1.14.18.6 / RHEA:46520 include papers referenced on the human FA2H UniProt page: Alderson, N.L., Rembiesa, B.M., Walla, M.D., Bielawska, A., Bielawski, J. and Hama, H. The human FA2H gene encodes a fatty acid 2-hydroxylase. J. Biol. Chem. 279 (2004) 48562–48568. [DOI] [PMID: 15337768] Guo, L., Zhang, X., Zhou, D., Okunade, A.L. and Su, X. Stereospecificity of fatty acid 2-hydroxylase and differential functions of 2-hydroxy fatty acid enantiomers. J. Lipid Res. 53 (2012) 1327–1335. [DOI] [PMID: 22517924]

deustp01 commented 7 months ago

GO:0080132 has 13 EXP annotations

And one Reactome mapping, R-HSA-5693761 "FA2H hydroxylates 1,2-saturated fatty acids", which looks like it should have a cross-reference to RHEA:38855 "a 1,2-saturated fatty acid + 2 Fe(II)-[cytochrome b5] + 2 H(+) + O2 = a (R)-2-hydroxy fatty acid + 2 Fe(III)-[cytochrome b5] + H2O" but does not, for reasons I need to check.

sjm41 commented 7 months ago

Thanks @deustp01. In that case, it seems the "GO:0080132 fatty acid alpha-hydroxylase activity" term may have been used more as a 'grouping term' to annotate different, albeit related, activities? In that case, the proposal here would be to create some more specific child terms, including the two RHEAs referred to above, i.e:

RHEA:46520: an N-(1,2 saturated acyl)-(4R)-hydroxysphinganine + 2 Fe(II)-[cytochrome b5] + 2 H(+) + O2 = an N-(2R-hydroxyacyl)-4R-hydroxysphinganine + 2 Fe(III)-[cytochrome b5] + H2O

and

RHEA:38855: a 1,2-saturated fatty acid + 2 Fe(II)-[cytochrome b5] + 2 H(+) + O2 = a (R)-2-hydroxy fatty acid + 2 Fe(III)-[cytochrome b5] + H2O

deustp01 commented 7 months ago

it seems the "GO:0080132 fatty acid alpha-hydroxylase activity" term may have been used more as a 'grouping term'

It definitely has, by us as shown here. But also as intended by RHEA, whose entry lists two more specific forms of the reaction and two more general forms. This leads to an important tangent issue, the role of grouping terms in annotation of broad-specificity enzymes. My recollection of old biochemistry literature is that no description of the catalytic properties of a newly characterized enzyme or transporter is that most such papers featured tables to show the range of inputs that could be acted on at physiologically interesting rates. A grouping term is really useful for capturing that range in a compact annotation. @ukemi ?

pgaudet commented 7 months ago

It looks like the most general reaction in RHEA is https://www.rhea-db.org/rhea/39983 ?

sjm41 commented 7 months ago

It looks like the most general reaction in RHEA is https://www.rhea-db.org/rhea/39983 ?

Yes, RHEA has two very similar 'general' terms for hydroxylation of 'free' fatty acids, arranged like this:

RHEA:39983 (a 1,2-saturated fatty acid + 2 Fe(II)-[cytochrome b5] + 2 H(+) + O2 = a 2-hydroxy fatty acid + 2 Fe(III)-[cytochrome b5] + H2O)
   |_RHEA:38855 (a 1,2-saturated fatty acid + 2 Fe(II)-[cytochrome b5] + 2 H(+) + O2 = a (R)-2-hydroxy fatty acid + 2 Fe(III)-[cytochrome b5] + H2O)

Note that the other RHEA mentioned at the top of this ticket (RHEA:46520 = EC:1.14.18.6) is for hydroxylation of fatty acids "attached to (4R)-4-hydroxysphinganine during de novo ceramide synthesis".

From its definitions and usage, it seems that the current fatty acid alpha-hydroxylase activity (GO:0080132) term encompasses hydroxylation of both 'free' and 'attached' fatty acids. That's fine, and just means we could make some new GO terms for more specific children terms corresponding to:

In this view, the existing term GO:0102771 (EC:1.14.18.7 = RHEA:46512) would also become a child of GO:0080132 - that's for hydroxylation of fatty acids attached to sphinganine: "an N-(1,2-saturated acyl)sphinganine + 2 Fe(II)-[cytochrome b5] + 2 H+ + O2 = an N-[(2'R)-hydroxyacyl]sphinganine + 2 Fe(III)-[cytochrome b5] + H2O."

sjm41 commented 7 months ago

Just found this useful 2023 review about the fatty acid 2-hydroxylase enzyme and 2-hydroxylated fatty acids (2hFA) containing sphingolipids (2hFA-SL) : https://pubmed.ncbi.nlm.nih.gov/36902339/

A couple of quotes suggest that 2-hydroxylation of 'free' fatty acids may not be physiologically relevant: "it was concluded that acyl-CoAs are most likely not substrates for FA2H/Scs7p, whereas a ceramide fits well into the catalytic center of the enzyme. On the other hand, the only established in vitro enzymatic FA2H assay used free fatty acids as an efficient substrate [53]. It is an open question whether or to what extent free fatty acids may serve as substrates in vivo."

Whether free 2hFA have a physiological function is currently not known. However, exogenously added 2hFA can dramatically affect cell physiology

@deustp01 Does Reactome have evidence that free fatty acids are 2-hydroxylated in humans?

deustp01 commented 7 months ago

Does Reactome have evidence that free fatty acids are 2-hydroxylated in humans?

No, we don't. And in fact the reaction I cited above is the first step in the Reactome pathway "Sphingolipid de novo biosynthesis" (R-HSA-1660661), and while the output hydroxylated fatty acids are not consumed in any of the other reactions annotated in that pathway, that looks like a gap in our annotation, not evidence that 2-hydroxylated free fatty acids have a role elsewhere in metabolism.

sjm41 commented 7 months ago

OK, well in that case I think the proposal now is either:

  1. Keep GO:0080132 as a grouping term and make a new child term for EC:1.14.18.6, like this:
oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, another compound as one donor, and incorporation of one atom of oxygen (GO:0016716) = EC:1.14.18.- 
    |_fatty acid alpha-hydroxylase activity (GO:0080132)
        |_4-hydroxysphinganine ceramide fatty acyl 2-hydroxylase (NTR) = RHEA:46520/EC:1.14.18.6
        |_dihydroceramide fatty acyl 2-hydroxylase activity (GO:0102771) = RHEA:46512/EC:1.14.18.7

or:

  1. Abandon the grouping term and instead equate GO:0080132 to RHEA:46520/EC:1.14.18.6, like this: 
    oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, another compound as one donor, and incorporation of one atom of oxygen (GO:0016716) = EC:1.14.18.- 
|_4-hydroxysphinganine ceramide fatty acyl 2-hydroxylase (GO:0080132) = RHEA:46520/EC:1.14.18.6
|_dihydroceramide fatty acyl 2-hydroxylase activity (GO:0102771) = RHEA:46512/EC:1.14.18.7

I think I prefer version 1.

(In either scenario, we move GO:0080132 to be a child of GO:0016716 = EC:1.14.18.- )

pgaudet commented 7 months ago

Hi @sjm41

Looking at these 2 ECs: -EC:1.14.18.6 states that this enzyme is characterized from yeast and mammals. Looking at the annotations, we only have yeast SCS7 associated (https://www.uniprot.org/uniprotkb/Q03529/entry); and that is associated with both EC:1.14.18.6 and EC:1.14.18.7

Maybe a third possibility is to obsolete GO:0102771 dihydroceramide fatty acyl 2-hydroxylase activity and put both EC as NARRROW matches on fatty acid alpha-hydroxylase activity (GO:0080132) (no annotations; it's has a single IEA coming from the REAH mapping)

Otherwise, we will need to create all the children RHEA? https://www.rhea-db.org/rhea/39983

What do you think ?

sjm41 commented 7 months ago

The citations in ExplorEnz for EC:1.14.18.6mention FAH2 as the human/mammalian enzyme with this activity; however in UniProt FA2H is associated with a number of RHEAs, but neither RHEA:46520 nor RHEA:46512 (and no EC).

Right, the current FA2H annotations at UniProt are not consistent with the comments at ExplorEnz - I think this UniProt entry may be misannotated and should be reviewed.

Otherwise, we will need to create all the children RHEA? https://www.rhea-db.org/rhea/39983

The two RHEAs mentioned in my most recent proposal (RHEA:46520 and RHEA:46512) are not included in the RHEA:39983 branch, so I don't think we necessarily need to consider that branch here. The RHEA:39983 branch seems to just be for 2-hydroxylation of 'free' fatty acids, which has no associated ECs and may not be physiologically relevant (according to the review mentioned above). And as per other similar situations, I don't think there's any need to add the children for the specific chain-length children (ie. for eicosanoate, docosanoate, octadecanoate, tetracosanoate, hexadecanoate)

The major FA2H reaction (=EC:1.14.18.6) seems very important, so I think we should have a GO term with an exact xref match to it. I'm less bothered about GO:0102771//EC:1.14.18.7, but that seems important in plants.

sjm41 commented 4 months ago

I've carefully reviewed all the comments above and delved deeper into the annotations and literature for the GO/EC/RHEA terms involved here. As a reminder, this concerns annotating the described functions of the conserved FA2H/SCS7 protein, which is acknowledged to catalyse the 2-hydroxylation of free fatty acids and of fatty acyl chains within sphingolipids/ceramides (see PMID:36902339 for recent review).

I think the following is the safest and most accurate course of action:

GO:0080132 fatty acid alpha-hydroxylase activity

NTR for a child term specifically acting on free fatty acids, corresponding to RHEA:38855

name: free fatty acid 2-hydroxylase activity definition: Catalysis of the reaction: a 1,2-saturated fatty acid + 2 Fe(II)-[cytochrome b5] + 2 H+ + O2 = a (R)-2-hydroxy fatty acid + 2 Fe(III)-[cytochrome b5] + H2O. Note that the substrate is a free fatty acid, not a fatty acyl chain within a sphingolipids/ceramide. [RHEA:38855] xref: RHEA:38855 exactMatch is_a: GO:0080132

RHEA:38855 is currently annotated to three proteins FA2H_HUMAN, FA2H_MOUSE, FA2H_RAT, and RHEA:38855 matches the Reactome mapping R-HSA-5693761 "FA2H hydroxylates 1,2-saturated fatty acids", which says "Fatty acid 2-hydroxylase (FA2H), an ER membrane-associated enzyme, catalyzes the cytochrome b5-dependent hydroxylation of free fatty acids on the saturated C2 position. 2-Hydroxylation of free fatty acids occurs before de novo ceramide synthesis." - Reactome may want to re-annotate that reaction to this more specific term.

GO:0102771 dihydroceramide fatty acyl 2-hydroxylase activity

NTR for for a child term specifically acting on free fatty acids, corresponding to RHEA::46520/EC:1.14.18.6:

name: 4-hydroxysphinganine ceramide fatty acyl 2-hydroxylase activity definition: Catalysis of the reaction: an N-(1,2 saturated acyl)-(4R)-hydroxysphinganine + 2 Fe(II)-[cytochrome b5] + 2 H(+) + O2 = an N-(2R-hydroxyacyl)-4R-hydroxysphinganine + 2 Fe(III)-[cytochrome b5] + H2O xref: EC:1.14.18.6 broadMatch xref: RHEA:46520 exactMatch is_a: GO:0080132

We end up with a small hierarchy like this:

fatty acid 2-hydroxylase activity (GO:0080132)
    |_free fatty acid 2-hydroxylase activity (NTR = RHEA:38855)
    |_dihydroceramide fatty acyl 2-hydroxylase activity (GO:0102771 = EC:1.14.18.7 = RHEA:46512)
    |_4-hydroxysphinganine ceramide fatty acyl 2-hydroxylase activity (NTR = EC 1.14.18.6 = RHEA:46520/RHEA:64532)
sjm41 commented 4 months ago

New terms:

[Term] id: GO:0120520 name: free fatty acid 2-hydroxylase activity namespace: molecular_function def: "Catalysis of the reaction: a 1,2-saturated fatty acid + 2 Fe(II)-[cytochrome b5] + 2 H+ + O2 = a (R)-2-hydroxy fatty acid + 2 Fe(III)-[cytochrome b5] + H2O. Note that the substrate is a free fatty acid, not a fatty acyl chain within a sphingolipid." [GOC:sjm, RHEA:38855] xref: RHEA:38855 {source="skos:exactMatch"} is_a: GO:0080132 ! fatty acid 2-hydroxylase activity property_value: term_tracker_item "https://github.com/geneontology/go-ontology/issues/27541" xsd:anyURI created_by: sjm creation_date: 2024-07-12T08:40:48Z

[Term] id: GO:0120521 name: 4-hydroxysphinganine ceramide fatty acyl 2-hydroxylase activity namespace: molecular_function def: "Catalysis of the reaction: an N-(1,2 saturated acyl)-(4R)-hydroxysphinganine + 2 Fe(II)-[cytochrome b5] + 2 H+ + O2 = an N-(2R-hydroxyacyl)-4R-hydroxysphinganine + 2 Fe(III)-[cytochrome b5] + H2O." [RHEA:46520] xref: EC:1.14.18.6 {source="skos:broadMatch"} xref: MetaCyc:RXN3O-4042 {source="skos:exactMatch"} xref: RHEA:46520 {source="skos:exactMatch"} is_a: GO:0080132 ! fatty acid 2-hydroxylase activity property_value: term_tracker_item "https://github.com/geneontology/go-ontology/issues/27541" xsd:anyURI created_by: sjm creation_date: 2024-07-12T08:47:01Z

sjm41 commented 4 months ago

Adding EC synonym to GO:0102771 1.14.18.7: plant sphingolipid fatty acid 2-hydroxylase