Expression, Purification And Enzymatic Characterization Of Cysteine Desulfhydrase CDes

Hydrogen sulfide (H2S), as a gasotransmitter, had become a hot topic of in biology and medicine in recent years. Keeping pace with the research on the regulatory mechanisms of H2S participating in physiological processes in plants and mammals, the study on structure, enzyme activity and enzyme kinetics of the generation key enzymes of endogenous H2S is flourishing. In mammals, the research on the structure and characteristic of endogenous H2S generation enzymes CBS (cystathionine β-synthase; EC 4.2.1.22) and CSE (Cystahionine y-lyase; EC 4.4.1.1) were systemic reported. LCD (L-cysteine desulphydrase; EC 4.4.1.1) and DCD (D-cysteine desulphydrase; EC 4.4.1.15), collectively referred to as CDes. CDes’function was the most explicit in catalyzing the generation process of endogenous H2S in plant. They catalysed L-cysteine or D-cysteine to generate H2S, ammonia and pyruvate. Recently, the studys of H2S regulating plant development and responsing abiotic stress resistance make a spurt of progress. However, further exploration on the enzymatic properties of CDes is limited.In this study, we analyzed these enzymes catalyze the production of H2S in plants, the results were as follows:1. In this experiment, the AtLCD and AtDCD gene in Arabidopsis were cloned into prokaryotic expression vectors pET28a, and the recombinant DNA were transformed into wild-type Escherichia coli strain BL21 (DE3), named BL21 (DE3)/pET28a-AtLCD and BL21 (DE3)/pET28a-AtDCD.2. The AtLCD and AtDCD gene were induced expression by isopropyl-β-D-thiogalactoside (IPTG) in BL21 (DE3)/pET28a-AtLCD and BL21 (DE3)/pET28a-AtDCD strains. The optimized induced expression conditions for the AtLCD and AtDCD protein were induced by 0.1 mmo/L IPTG at 30℃ for 3 h and 0.1 mmo/L IPTG at 20℃ for 6 h, respectively. AtLCD and AtDCD were purified through Ni-AKTA column. The optimized imidazole concentration of elution hybridprotein and target proteins were: 0-500 mmol/L and 500 mmol/L for AtLCD,0-200 mmol/L and 200-500 mmol/L for AtDCD.3. The enzymatic properties of AtLCD was studied after determining the cysteine desulfhydrase activity of the purified protein. The results showed that the optimal pH value and temperature of the AtLCD were 9.5 and 37℃, respectively. Under the optimum conditions, the Km value and Vmax of the AtLCD for hydrolysis of L-Cysteine were 1.572 mmol/L and 1.52 nmol/ (mg·min). Mg2+, Fe3+ and EDTA inhibited the enzyme activity of AtLCD lightly, while Ba2+, Ca2+ and Co2+ enhanced it, (and the effect of Co2+ was obviously). Moreover, the recombination AtLCD activity was inhibited by SDS and hydroxylamine.