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.

发散进化的酒石酸脱氢酶 (TDH) 在单个活性位点表现出多种催化活性；来自恶臭假单胞菌(pTDH)的酶的结构和生化特征已得到充分表征。芽孢杆菌分离株中出现与 TDH 相关的有氧代谢 L-酒石酸盐的能力，以及ycsA编码的蛋白质序列与 pTDH的有限相似性，使得芽孢杆菌TDH 成为一种有趣的酶，具有可能的催化多样性以及进化意义。本研究通过动力学、结构和分子对接分析，探讨了枯草芽孢杆菌168 (168bTDH) 和地衣芽孢杆菌DSM-13 (429bTDH) 的 TDH 的底物相互作用。从大肠杆菌BL21(DE3)细胞的不溶性组分中纯化得到的异源表达的bTDH ，可以显着催化L-酒石酸盐和内消旋酒石酸盐作为正向反应的底物。与 pTDH 不同，bTDH 使用二羟基富马酸底物遵循 S 形动力学，明显且更有效地催化逆反应；168bTDH 的能力高出约 4 倍。通过分子对接预测它们的结合能，进一步证实了相对底物特异性，同时揭示了活性位点上参与蛋白质-配体相互作用的主要残基。使用 Ramachandran 图分析验证的动力学分析和同源性模型预测了 bTDH 的二聚体性质。总的来说，这些结果凸显了系统发育上最近的 bTDH 的独特催化潜力，为介导有效的对映选择性酶促生物转化提供了重要的蛋白质工程靶点。