ubiquinone biosynthesis process clean up

[ValWood]

I'm not sure that we need these terms, they are all describing the source of the intermediate 4-Hydroxybenzoate (4-HB) required by the second step performed by cox2 in higher eukaryotes and UbiA in bacteria. So the linear pathway, although it has differneces in eukaryotes and bacteria, the differences are not defined by the source of this intermedaite.

GO:0032180    ubiquinone biosynthetic process from tyrosine
via tyrosine to 4-hydroxybenzoate. PMID:11583838
onverts 4-hydroxybenzaldehyde into 4-hydroxybenzoate for ubiquinone anabolism
2 EXP annotation SGD HFD1 (this annotation is incorrect because although humans use tyrosine as a source of 4-HB, yeast doesnt

GO:0032193    ubiquinone biosynthetic process via 2-polyprenylphenol
The chemical reactions and pathways resulting in the formation of ubiquinone, via the intermediates 2-polyprenylphenol and 2-polyprenyl-6-hydroxyphenol. PMID:11583838
0 annotations
bacterial pathway (UbiI/UbiD)

GO:0032194    ubiquinone biosynthetic process via 3,4-dihydroxy-5-polyprenylbenzoate
eukaryotic/eukaryotic not human
3 EXP annotations

GO:0032150    ubiquinone biosynthetic process from chorismate
bacterial/eukaryotic, not human
5 EXP annotations

Most fungi, including Saccharomyces cerevisiae (yeast), use the chorismate pathway to generate 4-HB as a precursor for ubiquinone.
(GO:0032194 and GO:0032150) seem to be equivalent but describe the different parts of the upstream pathway

Plants synthesize ubiquinone and other aromatic compounds from chorismate-derived intermediates.
4-Hydroxybenzoate (4-HB) is a key intermediate in plant ubiquinone biosynthesis.

I propose merging all into parent
GO:0006744 ubiquinone biosynthetic process

[ValWood]

for reference, this is the pathway
http://noctua.geneontology.org/workbench/noctua-visual-pathway-editor/?model_id=gomodel%3A662af8fa00000408

http://noctua.geneontology.org/workbench/noctua-alliance-pathway-preview/?model_id=gomodel%3A662af8fa00000408

[pgaudet]

Hi @ValWood
I dont quite follow your proposal - you indicate that the pathways are different in different organisms, and you propose to merge them. It seems that if the pathways do not represent the same biological 'module', we should keep them? Also, potentially they link to different MetaCyc pathways? MetaCyc has at least one euk and one prok pathway

[ValWood]

From my understanding, most of these are describing the source of the HB (first step), this does not affect the subsequent pathway.

I'd never come across ubiquinol-7 before, but maybe we don't need a term for specific chain lengths?
Pombe and human make ubiquinol-10, and cerevisiae ubiquinol-6 but it's that identical pathway (with different numbers of elongation step)

CHAT-GPT
Ubiquinol-7 is a form of coenzyme Q7, a naturally occurring quinone. It is mainly synthesized by bacteria, particularly those in the genera Rhodobacter, Paracoccus, and some strains of Escherichia. These bacteria produce ubiquinol-7 as part of their electron transport chain for cellular respiration or photosynthesis, depending on the species.
In addition, certain archaeal species and some eukaryotes, like yeast, can produce variants of ubiquinol with similar chain lengths, including Q7, depending on their environmental adaptations and metabolic needs.

[ValWood]

UPDATED:

The early and late decarboxylation step in the EcoCyc terms could be an artefact.

The eukaryotic pathway was not fully fleshed out. We had a gap between coq3 and co3 5 recently which was presumed to be filled by coq4 but the precise activity was not known.
it turns out that this is 2 steps, one being 3-demethoxyubiquinone 3-hydroxylase (NADH) activity by coq6
and coq4 as a decarboxylase.

I didn't know about this until I saw
#29305
which pointed me to
https://pubmed.ncbi.nlm.nih.gov/38425362/ from earlier this year, which fleshed out the eukaryotic pathway more fully.....
"In animals, biosynthesis of coenzyme Q is currently attributed to ten different proteins, with COQ3, COQ4, COQ5, COQ6, COQ7 and COQ9 forming the iconic COQ metabolon. Yet several reaction steps conducted by the metabolon remain enigmatic"
so probably the eukaryotic and bacterial pathways are not dissimilar.

Either way they seem to be linear with the same input (from different sources) and the same output (ubiquinone).

[ValWood]

The enzymes responsible for the C1 modifications are currently unknown (Fig. 1b). In bacteria, a dedicated enzyme pair, UbiX and UbiD, catalyse aromatic decarboxylation and utilize the highly reactive prenylated flavin mononucleotide cofactor, prFMN29,30. Likewise, a dedicated enzyme, UbiH performs the hydroxylation at C1 (ref. 31). So far, no biochemically equivalent proteins have been found in animals, suggesting an alternative mode of function. We reasoned that, in animals, the biosynthetic pipeline could follow two possibilities: a hydroxylation-through-decarboxylation reaction that directly transforms 3 into 4b, or a two-step process with one protein dedicated to the decarboxylation, 3 to 4a, and one to the hydroxylation, 4a to 4b (Fig. 1b and Supplementary Fig. 6).

Within the amidohydrolase superfamily there are Zn2+- and Mn2+-dependent ortho-(de)carboxylases that coordinate both the negatively charged carboxy and phenolate groups in a bidentate fashion for substrate decarboxylation32,33. Out of the currently known COQ proteins, COQ4 is the only system that possesses a canonical Zn-binding motif, HD−xx−H−(x)11−E (Fig. 3a), as first observed by Clarke and colleagues34. 3, however, does not possess a hydroxy group at either the C2 or C6 position, ortho to the carboxy substituent. We hypothesized that the electron delocalization properties of the C4-hydroxy group, para to the carboxy moiety, in combination with the Lewis acidic properties of Zn2+, may nonetheless be able to facilitate the decarboxylation reaction. In line with ortho-(de)carboxylases that do not require co-substrates, COQ4 was incubated overnight with 3 at 30 °C. Remarkably, we found that COQ4 exercised decarboxylase activity and production of 4a (Fig. 3b). No C1 hydroxylation, corresponding to 4b production, was observed suggesting that animals perform two separate and subsequent biosynthetic transformations for C1 modification.

[ValWood]

With COQ4 demonstrating decarboxylase activity, we turned to the C1-hydroxylation step (Fig. 1b). We speculated that the reaction could be fulfilled by COQ6, considering its ability to perform aromatic hydroxylation. FDXR, FDX2 and COQ6 were incubated overnight at 30 °C with 4a in the presence of an NADPH-regenerating system. We were able to detect product formation using GC/MS analysis, albeit at low levels, using both the truncated and full-length COQ6 proteins (Fig. 4a,b and Supplementary Fig. 7a). To substantiate the C1-hydroxylating role of COQ6, we exploited an in-house endpoint assay where the phenolic compounds, 4a and 4b, were conjugated to 4-aminoantipyrine using horseradish peroxidase35. The differing Λ of the adducts formed by 4a and 4b provided a qualitative assay to confirm product formation (Supplementary Fig. 7b). Indeed, COQ6, in the presence of FDXR and FDX2, produced the 4b adduct corroborating its dual functionality as both a C5 and C1 hydroxylase (Fig. 4c). COQ6 displayed typical Michaelis−Menten kinetics at saturating concentrations of NADPH over a range of 4a concentrations and exhibited molecular oxygen consumption in accordance with its role as a co-substrate and source for hydroxylation, with a KM of 20 μM and a kcat of 11 min−1 (Fig. 4d and Extended Data Fig. 2). COQ6 showed no activity in the presence of 3 and thus cannot function as a decarboxylase hydroxylase (see proposed reaction mechanism in Supplementary Fig. 6b). Finally, we found that coupling COQ6 with COQ4 resulted in the generation of both 4a and 4b starting from 3 (Fig. 4b).

[ValWood]

Illustrated here for the tyrosine part.
Its 'upstream' pombase/mitochondrion#13 (comment)

[amorgat]

Hi @ValWood
@kaxelsen and I are currently trying to summarize old and new literature on ubiquinone biosynthesis.
We have already created new reactions and EC numbers for new steps.
As you have seen it's a very complex subject ;-)
We will come back to you ASAP with a document to share.

[ValWood]

Excellent!
my Noctua model is here.
http://noctua.geneontology.org/workbench/noctua-visual-pathway-editor/?model_id=gomodel%3A662af8fa00000408
I had a gap between cox3 and cox5 where i am trying to put cox4 and cox6 but I don't know their activities.
My pathway (apart from the coq4 part which I just added), was reviewed by Makoto Kawamukai who has done all the work in fission yeast, so I know it was correct up to that point, if it helps.
v

[deustp01]

Ralf Stephen recently revised and updated the Reactome ubiquinol biosynthesis pathway - here's a link in case it's useful. And in case you disagree with our reactions, let us know - it would be great to arrive at a consensus view of the process! It is indeed complex and there are parts that remain poorly understood.

[ValWood]

More evidence, I'm trying to find out what is upstream of 4-hydroxybenzoate
in yeast, it is a precursor, and it can come from multiple sources, so it seems that these variations are not really "ubiquinone biosynthesis pathway"

CHat-GPT

In yeast, 4-hydroxybenzoate (4-HBA) is a precursor for the biosynthesis of ubiquinone (coenzyme Q). The enzyme upstream of 4-hydroxybenzoate in this pathway is aromatic amino acid aminotransferase (Aro8p or Aro9p) or other enzymes involved in the shikimate pathway, depending on the precursor source.

Here’s a simplified outline of the process:

Shikimate pathway: Produces aromatic compounds such as chorismate, which is a precursor for phenylalanine, tyrosine, and tryptophan.
Conversion of chorismate to 4-HBA: Chorismate can be converted to 4-HBA through intermediate steps, often involving 4-hydroxybenzoate synthase (though not all yeast species possess a direct equivalent of this enzyme, and the process may involve multi-step reactions).
Precursor pathways for CoQ biosynthesis: In yeast, phenylalanine or tyrosine may also act as upstream contributors to 4-HBA.
Would you like to explore more on the enzymatic details or related genes?

[ValWood]

I still haven't managed to figure out the upstream pathway,
but this is quite useful
https://www.sciencedirect.com/science/article/pii/S0005272816300846?via%3Dihub

The biochemical pathway responsible for CoQ biosynthesis is still incompletely characterized. Saccharomyces cerevisiae is able to use either para-aminobenzoic acid (pABA) or 4-hydroxybenzoate (4HB) as a precursor of CoQ [10], [11], while in mammals the precursor of the quinone ring is only 4HB, which is derived from tyrosine through an uncharacterized set of reactions.

[hattrill]

Sorry if I am repeating some of the points in this ticket, but there are inconsistencies in the way we are dealing with the prenyl chain length in the ubiquinone pathway MF steps.

For some steps we have chain agnostic terms that use 'polyprenyl', these are linked to chebi/rhea with suitably vague terms (which are fine, as we can specify the inputs and outputs using ChEBI IDs):
e.g. Coq3: 3,4-dihydroxy-5-polyprenylbenzoic acid O-methyltransferase activity GO:0010420 - IN: 3,4-dihydroxy-5-all-trans-polyprenylbenzoate + S-adenosyl-L-methionine; OUT: a 3-methoxy,4-hydroxy-5-all-trans-polyprenylbenzoate + S-adenosyl-L-homocysteine
and no child terms with specific chain lengths

but there are other steps that have specific chain length terms and no generic polyprenyl parent.

The issue here is that the chain length is specific for the organism and that in some organisms it is not exclusively one chain length. The length of the chain length is determined by the polyprenyl synthase enzyme (equivalant to sc COQ1):

"COQ1 ...defines the numbers (n) of isoprene units in the polyprenyl tail which is 6 for S. cerevisiae, 8 for E. coli, 9 in C. elegans, and 10 in humans." (PMID:25126050)

chatgpt "While the core biosynthetic machinery is conserved, the specificity for CoQ length is species-dependent and controlled early in the pathway by the polyprenyl synthase enzyme. Therefore, all types rely on the same enzymatic steps but are tailored to the biological context of the organism."

"The length of the isoprene chain differs between species; consequently, different organism may present a different major CoQ isoform. For example, Saccharomyces cerevisiae contains CoQ with six isoprene units (CoQ6), Escherichia coli CoQ8, rodents and Caenorhabditis elegans CoQ9, humans and S. pombe CoQ10. Nevertheless, some species have more than one CoQ form such as rodents that possess CoQ9 and CoQ10 and Drosophila melanogaster that contains CoQ8 (∼5 %), CoQ9 (∼82 %) and CoQ10 (∼13 %)" (PMID:39315151).

The two steps I am having an issue with are the coq6 monooxygenase and the polyprenyl synthase step.

1. 4-hydroxy-3-all-trans-hexaprenylbenzoate oxygenase activity GO:0106364
   xrefs: RHEA:20361
   MetaCyc RXN3O-58
   is correct for Q6 biosynthesis

i.e. For s.cerevisiae, metacyc Q6:
ubiquinol-6 biosynthesis from 4-aminobenzoate (yeast)
ubiquinol-6 biosynthesis (late decarboxylation)
has this:
1.14.15.M7: 4-hydroxy-3-hexaprenylbenzoate + 2 reduced ferredoxin [iron-sulfur] cluster + dioxygen + 2 H+ → 3-all trans-hexaprenyl-4,5-dihydroxybenzoate + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

but this seems to have propagated to other species via InterPro (and other mechanisms), where Q6 is not the major species of ubiquinone biosythesis.

In metacyc the reactions for some other coenzyme Q lengths are:

For human, metacyc Q10:
ubiquinol-10 biosynthesis (late decarboxylation)
1.14.13.M81 -- 4-hydroxy-3-polyprenylbenzoate 5-hydroxylase
4-hydroxy-3-decaprenylbenzoate + NADPH + dioxygen + H+ → 3,4-dihydroxy-5-all-trans-decaprenylbenzoate + NADP+ + H2O

For Arabidopsis, metacyc Q9
ubiquinol-9 biosynthesis (late decarboxylation)
4-hydroxy-3-nonaprenylbenzoate + NADPH + dioxygen + H+ → 3,4-dihydroxy-5-all-trans-nonaprenylbenzoate + NADP+ + H2O

So, I think we could do with a parent term:
4-hydroxy-3-all-trans-polyprenylbenzoate oxygenase activity

We could also have the specific terms for decaprenyl, etc as well - I am ok with just a generic parent for my needs, but you might want a deca for s.pombe @ValWood?

2. polyprenyl diphosphate synthase step (equivalent to sc COQ1 reaction):
   This the step that determines the polyprenyl chain length.
   As D.mel has Q8, Q9 and Q10, I presume this reflects the output of the polyprenyl diphosphate synthase (Pdss2 + qless), but no direct assay that looks at chain length has been done.
   For Pdss2 + qless, we could use - GO:0120531 prenyl diphosphate synthase activity - this is an umbrella term for a lot of activities and has no direct annotations. But could specify inputs and outputs using chebi IDs.

There are child terms that could be used for specific polyprenyl chain lengths:
For Q10 biosynthesis pathway: GO:0097269 'all-trans-decaprenyl-diphosphate synthase activity'
For Q6 in sc, according to metacyc:
2.5.1.83: (2E,6E)-farnesyl diphosphate + 3 3-methylbut-3-en-1-yl diphosphate → all-trans-hexaprenyl diphosphate + 3 diphosphate
which maps to this term in GO, GO:0036423 hexaprenyl-diphosphate synthase ((2E,6E)-farnesyl-diphosphate specific) activity
https://enzyme.expasy.org/EC/2.5.1.83

However, the sc COQ1 has been annotated with heptaprenyl diphosphate synthase activity, so needs to be updated.

Ammending comment as GO:0120531 prenyl diphosphate synthase activity has been worked on recently in #20661

[ValWood]

I'm happy to go with the consensus here. detailed activities with different inputs and outputs might be better than multiple processes....

[sjm41]

Also noting that we currently have this:

GO:0006744 ubiquinone biosynthetic process
    |_GO:1901006 ubiquinone-6 biosynthetic process (4 EXP annotations by SGD)

But there are not BP terms for ubiquinone-10, ubiquinone-8 etc.

And that term is also part of the following hierarchy (which we don't have for other chain lengths):

GO:1901004 ubiquinone-6 metabolic process (0 direct annotations)
    |_GO:1901006 ubiquinone-6 biosynthetic process (4 EXP annotations by SGD)
    |_GO:1901005 ubiquinone-6 catabolic process (0 annotations)

So would be good to either have BP terms for all chain lengths or none. (My preference is for latter!)

[hattrill]

My pref too: I think we should have just a chain agnostic ubiquinone biosynthetic process - as the difference just lies in the properties of one enzyme.

[sjm41]

Obsoletion request: #29419