Functions Of The Double Domain 3-Phosphate Glycerol Dehydrogenase(GPDH) Isoenzyme In Dunaliella Salina

Dunaliella salina has been widely concerned and studied since 1960s because of its extraordinary adaptability to high salt environment.Dunaliella can tolerate such a high salt concentration,mainly because it can produce a lot of glycerol to deal with the extracellular osmotic pressure.3-phosphate glycerol dehydrogenase(GPDH)is considered to be the main rate limiting enzyme in glycerol synthesis pathway,which plays an important role in the adaptation of Dunaliella to abiotic stress.Previous studies have found that GPDH in Dunaliella has a special double domain(GPDH-Ser B),the classical GPDH domain located at the C-terminal,which can reduce the substrate dihydroxyacetone phosphate(DHAP)to glycerol 3-phosphate(G-3-P)in the presence of coenzyme NADH/NADPH.The other one is the Ser B domain located at the N terminal can catalyze the production of glycerol and inorganic phosphate(Pi)from G-3-P due to it plays the role of 3-phosphoglycerin phosphatase(GPP).We found four NAD(P)H-dependent GPDH genes can encode GPDH-Ser B double domain by sequencing the transcriptome of Dunaliella.We found GPDH1 and GPDH4 in the cytoplasm,GPDH2 and GPDH3 in the chloroplast through the prediction of chloroplast transport peptides.The purpose of this study is to distinguish the properties and double domain functions of these four GPDH isozymes,and to lay a foundation for the subsequent study of their different roles in cell,and to provide some help for exploring the mechanism of rapid glycerol synthesis in Dunaliella under hypertonic stress.Based on this,the following studies were carried out in this paper:1.Soluble expression of four fusion proteins(GPDH1,GPDH2,GPDH3 and GPDH4)was achieved at 16?.Four kinds of proteins with high purity were obtained by nickel ion affinity chromatography and molecular exclusion chromatography.The pure proteins also met the requirements of the subsequent enzyme activity determination and enzyme kinetics related tests.In addition,it was preliminarily speculated that GPDH1 in the form of monomer,GPDH3 in the form of tetramer,and GPDH4 in the form of hexamer existed in the solution.2.According to the results of the studies on the functions of GPDH isoenzymes,we found that both GPDH2 and GPDH3 possessed protein activity of classical GPDH domain and the Ser B domain,indicating that they could directly use the substrate DHAP to catalyze the formation of glycerol and inorganic phosphate in two steps.However,GPDH1 and GPDH4 cannot catalyze the production of G-3-P by DHAP due to the classical GPDH domain is inactive,while its can catalyze the production of glycerol and inorganic phosphate by G-3-P because of the Ser B domain is active.3.The enzymatic properties of the classical GPDH domain of GPDH2 and GPDH3 were studied.The results showed that different salt concentrations(NaCl,Na₂HPO₄ and MgCl₂)showed different degrees of inhibitory effect on the enzyme activities of GPDH2 and GPDH3,and the inhibitory effect increased with the increase of salt concentration.In addition,the results of enzyme kinetics test showed that GPDH3 showed obvious affinity for NADPH,which indicated that it was more inclined to synthesize glycerol in Dunaliella cells by using NADPH produced by photosynthesis,while GPDH2 showed no bias for coenzyme NADH and NADPH.Furthermore,we found that using GPDH2 could make the reaction system get higher catalytic efficiency by comparing the catalytic efficiency constant k_cat/K_m.4.Real-time quantitative PCR results showed that the expression levels of GPDH1,GPDH2,GPDH3 and GPDH4 of Dunaliella treated with high concentration of NaCl were up-regulated within 2 h,but down-regulated to a certain extent under low concentration of NaCl stress.The expression of GPDH decreased significantly with high phosphate.In addition,with the increase of treatment time and the decrease of nitrogen dosage,the expression of the four kinds of GPDH were up-regulated.The results showed that there was no significant difference in the expression levels of the four types of GPDH under different stress conditions.In conclusion,the function of four GPDH isozymes in Dunaliella was studied in this paper,and they were preliminarily distinguished.The results provided some help for the subsequent study of their roles in Dunaliella and elucidating the molecular mechanism of the salina in response to hypertonic stress.