Thursday, January 30, 2014

205 - Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool

Saccharomyces cerevisiae is a well-known species of yeast, commonly used for making bread, beer, wine, and other such fermented products. It can convert sugar effectively into ethanol (useful in alcohol production) and carbon dioxide (useful for creating pockets of air that make bread dough rise).

This yeast's products are inextricably linked to its anaerobic fermentative metabolism—what happens when it consumes food without consuming oxygen. (Note: S. cerevisiae does have the ability to use oxygen, but it chooses not to when its food is abundant, or when oxygen is not present, obviously.)

And an essential part of this metabolism is nucleotide molecules that carry protons and electrons from one cellular chemical reaction to another, oxidizing and reducing various compounds in the cell. These are called NAD+/NADH and NADP+/NADPH (the + refers to the oxidized form with one fewer electron and proton, and the H refers to the reduced form), and are slightly different, both in function and chemical structure.

NAD is used more in breaking down food and shuttling the electrons from this process to the final reactions of the metabolic process. For example, electrons from glucose transfer to NAD+, forming NADH. Then in an aerobic condition, the electrons would be pushed across the membrane in the Electron Transport Chain to generate energy for the cell, ending up on oxygen to form water; in anaerobic metabolism, they end up being dumped to generate ethanol and CO2 and regenerate NAD+ to repeat the cycle.

NADP is used more for synthesizing important compounds for cell growth, such as proteins and nucleotides.

There are some organisms that have enzymes that can transfer electrons between these two systems, usually from NADH to NADPH. These enzymes are called transhydrogenases, of which there are two kinds: those bound to the membrane, which are pretty common in mammals and bacteria; and those that are floating in the cytoplasm of the cell, found especially in bacteria such as E. coli, Pseudomonas, and Azotobacter vinelandii.

The authors suspected that if this kind of enzyme were present in S. cerevisiae, it might increase the amount of ethanol the yeast produces from the same amount of sugar, and since ethanol is a valuable product for biofuels (and for drinking), they thought it was worth a try.

But first they had to find the DNA sequence of the gene in A. vinelandii, since at this point the genome hadn't been published. To do this, they purified the transhydrogenase protein from the bacteria (detecting it by its chemical activity rather than its sequence) and determined the sequence of amino acids at one end of it, by chopping off one amino acid at a time, identifying it, then the next, up to the first four. Then, since a number of different DNA sequences can code for the same sequence of amino acids, they made primers that could bind to a number of similar but not identical sequences (called "degenerate primers") and used these for PCR to amplify the gene out of the bacteria. They named it sthA (for Soluble Transhydrogenase A).

Compared to the amino acid sequences from the other bacteria in which the enzyme is found, A. vinelandii's SthA protein is pretty similar. And the more closely related the species, the more similar the protein, as might be expected.

So they took the transhydrogenase gene from A. vinelandii, sthA, put it on a plasmid (a small circular piece of DNA) and put it into the yeast along with a gene that provides resistance to an anti-bacterial and anti-yeast compound, so they could be reasonably sure that any cells that could grow in this compound would also contain the gene of interest. The result was a strain of yeast called TN21.

When they tested extracts of this strain for STH activity, they found a decent amount, and none in strains TN1 and TN26 which lacked the gene. So the new protein seemed to be working in the yeast, and at higher levels than it is naturally in the bacteria. Producing the transhydrogenase also slowed down the growth rate of strain TN21, down to less than half of the parent strain, but did it work as they wanted?

They tested levels of an enzyme in a metabolic pathway called the Pentose Phosphate Pathway, which is a way cells break down sugar that is good for generating NADPH. Their hypothesis was that the SthA activity might reduce the activity of this pathway, since the cell would already have plenty of NADPH converted directly from NADH. So they tested the activity of one of the enzymes in this pathway, but found that it was just as active in the STH strain as in the others, almost exactly the same. So that hypothesis failed.

The other hypothesis was that SthA might increase the yield of ethanol (by simultaneously reducing the production of glycerol). In actuality, all three strains produced similar levels of these compounds, and ironically the strain with SthA produced a little less ethanol (11% less) and a little more glycerol, if these are even real differences. The authors attempted to explain this by pointing out that the cell biomass produced in strain TN21 was slightly lower also (13%), so it seemed that this strain was less efficient in general.

What they did notice, though, was that TN21 produced a lot of a compound called 2-oxoglutarate. About 23 times more than the other strains. This compound is normally consumed by oxidizing NADPH, so an overabundance of it indicates a lack of NADPH; the transhydrogenase may be converting all of it to NADH.

This is the opposite of what the authors wanted to happen, but such is the nature of science. They modeled what they thought was happening in the cells energy-wise, and found that there was so much more NADPH than NADH in the cells to begin with that it's no wonder which way the reaction went. I'm not sure why they expected anything different, considering that the same thing happens in bacteria. But in science, you gotta try! Good on them for publishing negative results.

Still, the transhydrogenase enzyme seems like it could be useful for some things, depending on the reaction you want to happen. I will have to investigate further.

Citation: Nissen, T. L., Anderlund, M., Nielsen, J., Villadsen, J. & Kielland-Brandt, M. C. Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool. Yeast 18, 19–32 (2001).

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