Genomics and proteomics continue to deliver a wealth of information about genes and their putative proteins, but experimental functional validation remains a great challenge.
Genomics and proteomics continue to deliver a wealth of information about genes and their putative proteins, but experimental functional validation remains a significant challenge. For instance, hundreds of genes from Mycobacterium tuberculosis are predicted to encode enzymes that are involved in biosynthesis and degradation of cofactors, amino acids, carbohydrates and lipids, but very few of these have had their function revealed and validated.
In addition, several proteins encoded in the mycobacterial genome do not share any obvious homology with proteins seen in other organisms. Approximately 35 per cent of these “orphan” proteins are predicted to be essential and therefore could become potential targets for drug discovery, if their function was known.
We employ high-resolution mass spectrometry-based metabolomic methods such as activity-based metabolic profiling (ABMP) and global metabolomic profiling (GMP) to uncover substrates and products of orphan enzymes, thereby annotating their catalytic function and metabolic role. In some cases, these enzymes will indicate the existence of completely new pathways, which have escaped identification for over 100 years.
For example, we have just identified a previously unknown enzyme involved in three metabolic pathways in M. tuberculosis. Rv2498c encodes an unusual carbon-carbon bond lyase enzyme that acts on (R)-hydroxymethylglutaryl-CoA, (S)-citramalyl-CoA and (S)-malyl-CoA (Figure 1). Therefore, this new enzymatic activity is important in leucine catabolism, itaconate detoxification and core carbon metabolism in M. tuberculosis and related species.
In addition to finding new enzymatic functions and metabolic pathways, we are studying how metabolite-driven control of enzymatic activity and metabolic pathways takes place in M. tuberculosis. Allosteric regulation by small molecule ligands is a highly-efficient, evolutionary conserved regulatory mechanism that has been poorly investigated in M. tuberculosis and represents another untapped source for novel antimicrobial drug discovery.
Figure 1: Reactions catalysed by Rv2498c in M. tuberculosis. It is noteworthy that (R)-HMG-CoA (top reaction) was not thought to exist in M. tuberculosis and is the only stereoisomer found in this bacterium. The middle reaction, which leads to breakdown of (S)-citramalyl-CoA (CML-CoA), was not thought to exist in M. tuberculosis and it demonstrates for the first time that M. tuberculosis can degrade the host-derived antibacterial metabolite itaconate.