Study of the Active Site Residues of MetA Enzyme From M. Tuberculosis, a Potential Drug Targeting Enzyme

Mycobacterium tuberculosis is known to cause tuberculosis also called phthisis or phthisis pulmonalis. It is a global widespread, top infectious disease killer in the world. Therefore, new anti-TB drugs are the need of the hour. They need to be superior to those available today in their speed of action, safety, and tolerability. Earlier studies showed that knocking out the metA gene encoding homoserine-acetyltransferase (MetA) inactivated methionine and S-adenosylmethionine (SAM) biosynthesis in-vivo. Cell death of Mtb ΔmetA is more effective than the other enzyme mutants involved in tryptophan, pantothenate, leucine and biotin biosynthesis. This new way of killing pathogens suggests that this enzyme is a potent target in the development of anti-tuberculosis drugs. In order to design original drugs against TB, we focused on the active site residues of homoserine-acetyltransferase encoded by metA (Rv3341) gene from M. tuberculosis. Similarity search of MetA peptide showed highest homology to homoserine-acetyltransferase from M. abscessus (77%). It also showed about 25 to 36% similarity to various esterases and hydrolases. Multiple sequence alignment of related acetyltransferases (ATs) and hydrolases with MetA showed that Ser, Asp, and His amino acids are conserved across species of ATs and hydrolases and are involved in catalytic triad formation. The 3D structure of MetA was generated based on PDB ID 5W8P as a template using Swiss Model server. In the generated model, these three amino acids (S157, D320, and H350) are in close proximity, illustrating the genuine active site for this protein. In order to study the role of amino acids involved in active site formation, we prepared catalytic triad mutants such as S157A, D320A, and H350A using site-directed mutagenesis. All three mutants were purified by affinity chromatography and their activities were measured and compared with wild type MetA enzyme activity. D320A MetA mutant showed the least activity in comparison to S157A and H350A MetA mutants. MetA activity was checked in presence of EDTA (ethylenediaminetetraacetic acid), PMSF (phenylmethylsulfonyl fluoride), and Coumarin. 10 mM EDTA did not show any effect on the activity of enzyme while 10 mM PMSF completely inhibits enzyme activity. This indicated that serine is one of the residues present in the catalytic triad formation, and is involved in the reaction. Coumarin showed an inhibitory effect on MetA enzyme when it was used from 0 to 25 µM of coumarin concentration. Beyond 25 µM of coumarin concentration, activity of MetA starts getting restored. Therefore, based on our results, S157, D320, and H350 residues are important for catalytic activity and are involved in the active site formation. Inhibition of MetA by PMSF, strongly supports the fact that it is a serine hydrolase. Based on PMSF structure, we could try to synthesize or design some more chemicals for future applications to achieve potent drugs against MetA of M. tuberculosis.