Evaluating the role of transhydrogenases in lipid D/H ratios

Abstract:

We investigate the importance of transhydrogenases in determining lipid D/H ratios using the model organism Methylobacterium extorquens.

Transhydrogenases are a class of oxidoreductase enzymes that are responsible for transferring reducing power between intracellular pools of pyrimidine nucleotides. At least three classes of transhydrogenases have been described: i) the proton translocating transhydrogenase PntAB, which is known to carry an extremely large hydrogen isotope effect; ii) the soluble energy-independent transhydrogenase UdhA, which is present in many proteobacteria; iii) the electron-bifurcating transhydrogenase NfnAB, present in many anaerobic bacteria and archaea.

In wild-type Methylobacterium grown on C1 compounds such as methanol or methylamine, NADPH is produced by methylene tetrahydromethanopterin (H₄MPT) dehydrogenase during the cytoplasmic oxidation of formaldehyde to formate. Lipids produced under these conditions are are enriched in deuterium relative to media water by ~80 permil. We compare this to an engineered mutant (EM) in which the H₄MPT pathway has been inactivated and replaced by the non-orthologous glutathione-dependendent pathway from Paracoccus denitrificans that produces NADH but not NADPH. In the EM strain, NADPH is limiting. To obtain sufficient reducing power for biosynthesis, pntAB is upregulated and transhydrogenase activity is increased. Lipids in this strain are depleted in deuterium relative to media water by ~200 permil. In several evolved strains derived from EM, transhydrogenase expression is higher than in EM, correlating with restoration of NAD(H) and NADP(H) pools towards the wild type, and lipids in these strains are enriched in deuterium relative to the EM.

We compare these results to a pntAB deletion mutant which has been complemented with pntAB on an inducible promoter, allowing a direct comparison between transhydrogenase expression levels, NADPH/NADP ratios, and lipid D/H ratios.