Mycobacterium tuberculosis AHAS is a potential target for the development of novel anti-tuberculosis agents. Silico analysis showed that conserved His84 and Gln86 residues lie in the catalytic dimer interface of M. tuberculosis AHAS. Mutational analyses of these invariants led to significant reduction in their activity with reduced affinity toward the substrate. Importantly, mutation of Gln86 to Trp abolished complete activity. Further, molecular dynamics simulation studies suggested that these residues are likely to play a key role in maintaining the G1u85 side chain in the required geometry with N1' atom of ThDP during catalysis. In addition, substitution of essential Glu85 by Ala, Asp, and Gin led to severe drop in catalytic activity with reduced affinity toward ThDP confirming its catalytic role in M. tuberculosis AHAS. (C) 2014 Elsevier Inc. All rights reserved.