When I feed a particular substance to pea plant, I see conversion to unknown compound.
I don't know, what is the product, I don't know (obviously), what is the reaction and I do not know required conditions (like cofactor(s) etc.).
How would you proceed to identify the responsible enzyme/gene?
I intend to identify the product, as that is the easiest thing to do right now, I guess. But I'm afraid that won't help me much, as I won't be able to specify the conditions.
Because any in vitro measurement/detection is ruled out because of the lack of knowledge about the reaction conditions, it seems to me like the only option is to go in planta. What I could come up with is either random mutagenesis or finding two genotypes with different activities and then trying to cross them.
However, for that I would need to have either reliable sensor to monitor amount of my compound in planta, or feed it to the plant and then isolate and measure on HPLC/MS (not very high-throughput).
I was also thinking about utilizing cDNA library, but so far, I did not come up with simple way to do so.
It would be a whole project, but you could try to fractionate a plant extract containing the activity, using the formation of the unknown substance from the substrate as the assay. Since you don't know what cofactors are involved, you could provide a protein-free extract of the plant (passed through an ultrafiltration membrane) as the source of any unknown low-molecular-weight cofactors. Protein fractions from various purification steps would be tested, and the best ones subjected to additional fractionation, until you had an enriched fraction that could be sent for protein identification by a mass spec lab to narrow down the list of possible enzymes.
Of course, this "grind-and-find" approach might fail if you can't supply the necessary conditions, such as an anerobic environment, or if a necessary enzyme is membrane-bound, or if there are multiple enzymes required for the conversion.
Hello Adam Adam B Shapiro,
thanks for your suggestion. This is actually relatable as I've been doing similar experiments for ages :)
I tried something similar, I suppose. Actually, I asked about it previously xD
https://www.researchgate.net/post/I_observe_a_metabolic_conversion_in_planta_how_do_I_purify_identify_the_responsible_enzyme
so I won't repeat it, but in short, I homogenized tissue sample, added phosphate buffer with Triton, DTT and my substrate DHZ and incubated for up to 24 hrs. I saw no conversion to my unknown compound.
I saw some other peak with similar retention time though. DHZ can be quickly converted to various conjugates (riboside, glucosides), so maybe it could be analogous compound originating from one of the conjugates. However, it did not increase in time.
Thank you, I appreciate your time.
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