Analysis of in vivo function of predicted isoenzymes:a metabolomic approach

Isoenzymes occur in all organisms. Often, they are regulated with respect to environmental perturbations to assure metabolic flexibility. In bioinformatics-driven functional genome annotations, the presence of isoenzymes is frequently predicted. It is desirable to verify the functions of putative isoenzymes experimentally for a correct estimation of the metabolic capacities in a given organism. Using metabolome analysis, we investigated two knockout mutants of putative shikimate dehydrogenases (SDH) in Corynebacterium glutamicum (C. glutamicum). Here, we show that different metabolic responses to defined gene deactivations of the putative SDHs clearly indicate different functions of these enzymes. Our investigation revealed that the gene product of open reading frame Cg1835 is the essential SDH in C. glutamicum, whereas it is more likely that the gene product of the open reading frame Cg1283 belongs to the SDH-like enzyme family (SDH-L). The results of the metabolome analysis are verified with two independent methods. First, by in vitro characterization of kinetic constants after heterologous expression, and second, by measurement of SDH activity in raw cell extracts.