Overexpression, Purification And Enzymatic Characterization Of Two Soluble Pyridine Nucleotide Transhydrogenases

The soluble pyridine nucleotide transhydrogenase (STH or UdhA) is an energy-independent flavoprotein that catalyzes the reversible transfer of 4B hydride between NAD(H) and NADP(H). STH is exclusively found in Gammaproteobacteria and a few Gram-positive bacteria, which plays significant roles in regulating the homeostasis of two cofactors. This enzyme has been widely utilized in the cofactor regeneration in industrial production through genetic and metabolic engineering.The enzymatic properties of STH have not been completely identified yet. In this work, two soluble pyridine nucleotide transhydrogenases from Salmonella typhimurium (StSTH) and Escherichia coli (EcSTH) were overexpressed and purified, and their enzymatic properties were then characterized in detail for the first time, including optimal pH, optimal temperature, activators, inhibitors, and kinetic parameters. The comprehensive information of STH may provide the fundament for the further investigation of its crystal structure, physiological roles, evolution and applications.StSTH and EcSTH showed similar enzymatic characteristics. The optimal pHs of StSTH and EcSTH were 7.2 and 7.5, and optimal temperatures were 32°C and 35°C, respectively. Both of them had a half-time of 5 hours at 50°C and were very stable at 4°C. Two STHs were found to be metal-independent. However, the enzyme activities were inhibited by most metal ions (Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺ and Cu²⁺) and strongly activated by adenine nucleotides(ATP, ADP and AMP).The kinetic studies showed that StSTH catalyzed NADPH and thio-NAD⁺ to NADP⁺ and thio-NADH via a ping-pong mechanism, the K_m and apparent K_m (K_m^app) for substrates of StSTH were very similar. The K_m and K_m^app for NADPH were 69.5μM and 68.5μM, respectively. The K_m and K_m^app for thio-NAD⁺ were 169μM. The turnover numbers (k_cat or k_cat^app) were over the range from 119-198 s^-1. The K_m^app for NADPH and thio-NAD⁺ of EcSTH were 68.3μM and 133.2μM, and the apparent turnover numbers (k_cat^app) were 167.9 s^-1 and 259.5 s^-1, respectively. Although the affinities of StSTH and EcSTH for NADPH were higher than that for thio-NAD⁺, the activities of them were inhibited by high concentration of NADPH.