C14 -galactose metabolism in yeast - (Oct/23/2007 )

Hi,
I am planning an experiment where I will take radiolabelled galactose (C-14 labelled) and feed to yeast. Then I will look at the end products of the metabolism to see if my metabolite of interest has been formed, and if yes, how much. My metabolite of interest is a compound called artemisinic acid. The galactose would have to go through glycolyssi and then through the mevalonate pathway for artemisinic acid to be formed.

The problem is, I have found out that the commercially available C14-Gal is labelled at carbon-1. My question is, how to find out if the carbon-1 will be transferred through the metabolic pathway to artemisinic acid. What is the carbon-1 forms some other metabolite in the course of the pathway? How to find this out?

Thanks for any help.

I would have a look on the pathways to find out how Gal is metabolized. I found several websites that will lead you to your product step by step. You just need to follow C1. Good luck, it`s quite confusing. In my opinion it reaches the artemisinic acid. I know it makes it all the way down to pyruvate. From there on I got lost several times. But have a look youself.

http://www.biochem.arizona.edu/classes/bio...se4fig14-11.jpg
http://fig.cox.miami.edu/~cmallery/255/255...sis_pathway.jpg
http://en.wikipedia.org/wiki/HMG-CoA_reductase_pathway
http://www.nsf.gov/eng/cbet/nuggets/1491/i...91_keasling.jpg
http://www.freepatentsonline.com/7183089-0-large.jpg

Galactose is isomerized to glucose, and it comes down to 2 pyruvate after glycolysis. The C1 is in the carboxy terminal of one pyruvate. In yeast it can be fermented to alcohol, then the C1 becomes CO2, and is out of the cell.

The mevalonate pathway starts with a Acetyl-CoA + Aceto-acetyl-CoA reaction. So we want Acetlyl-CoA basically. That comes from the cytric acid cycle, in which the COOH of the pyruvate (half of which is C1) is also transformed to CO2. So I'd guess it is all gone. But I might be wrong, its just a guess.

Maybe the acetyl-CoA can come from a differrent pathway, and then its all wrong. But I dont know which one.

True is that the C1 of Gal is conserved as C1 (in methyl group) of 50 % of the pyruvate made. The other 50 % of pyruvate made during glycolysis consist of C4-6.
Pyruvate is either fermented or converted to acetyl-CoA. Acetyl-CoA is also made from acetate. From the pool of acetyl-CoA some goes into the TCA cycle and some into the mevalonate pathway and other pathways I guess.
Basically, some labeled C1 will end up as your acid, but how much of it is really dependent on the growth conditions. Fermentation should be avoided to generate more acetyl-CoA. Hope that is true.

Yeah, I wrote some stupid stuff, but it was late What I meant was that when Acetyl-CoA is generated from pyruvate, then the C1 becomes CO2 doesn't it?

Still think that C1 is in the methyl group (H3C1-CO-COOH) of pyruvate and therefore is also in the methyl group of acetyl-CoA (H3C1-CO-SCoA).

Nothing to be sorry about ) Took me ages to figure that out and I am still not 100 % sure...