RESUMO
Divergently evolved Tartrate dehydrogenase (TDH) exhibits multiple catalytic activities at a single active site; the enzyme from P. putida (pTDH) being structurally and biochemically well-characterized. Occurrence of TDH-associated ability to aerobically metabolize L-tartrate in Bacillus isolates and limited resemblance of ycsA-encoded protein sequences with pTDH rendered Bacillus TDH as an intriguing enzyme with possible catalytic diversity as well as evolutionary significance. The present study explores substrate interactions of TDHs from B. subtilis 168 (168bTDH) and B. licheniformis DSM-13 (429bTDH) through kinetic, structural and molecular docking-based analysis. Heterologously expressed bTDHs, purified from insoluble fractions of E. coli BL21(DE3) cells, could significantly catalyze L-tartrate and meso-tartrate as substrates in forward reaction. Unlike pTDH, bTDHs distinctly and more efficiently catalyzed the reverse reaction using dihydroxyfumarate substrate following sigmoidal kinetics; the ability being ~ 4 fold higher in 168bTDH. Their binding energies predicted from molecular docking, further substantiated the relative substrate specificities, while revealing major residues involved in protein-ligand interactions at active site. The kinetic analysis and homology modelling validated using Ramachandran Plot analysis predicted a dimeric nature for bTDH. Collectively, the results highlight unique catalytic potential of phylogenetically recent bTDHs, offering an important protein engineering target to mediate efficient enantioselective enzymatic biotransformations.