Identification Of The Enzymatic Properties And Functional Alteration Of Glucose-6-Phosphate Dehydrogenase And Malic Enzyme From Yarrowia Lipolytica

Glucose-6-phosphate dehydrogenase?G6PD?and malic enzyme?ME?are two key enzymes involved in the lipid accumulation in oleaginous Yarrowia lipolytica.G6PD is the first key enzyme of pentose phosphate pathway?PPP?,catalyzing the oxidation of glucose-6-phospate to 6-phosphoglucono-?-lactone,a rate-limiting reaction,with the reduction of NAD?P?⁺.ME is a key enzyme to regulate the malate metabolism,which catalyzes the oxidative decarboxylation of L-malate to pyruvate,NAD?P?H and CO₂,requiring a divalent cation.These two enzymes are the major sources of NADPH for biosynthesis and the maintenance of the antioxidant system.In current study,Y.lipolytica G6PD?YlG6PD?and ME?YlME?were expressed and purified to homogeneity,and their enzymatic properties were characterized in detail.Furthermore,their coenzyme specificities were altered via site-directed mutagenesis.The experimental results were as follows:1 Expression,purification and enzymatic identification of YlG6PDIn this work,YlG6PD was successfully expressed in Escherichia coli and purified to homogeneity by Co²⁺affinity chromatography.YlG6PD was found to be219 kDa homotertamer as assessed by SDS-PAGE and gel filtration chromatography.The optimum pH and temperature for YlG6PD catalysis were determined to be pH 8.5and 47.5°C,respectively.Heat-inactivation study showed that YlG6PD retained approximately 50%activity after 20 min of incubation at 48°C.Kinetic analysis indicated that YlG6PD was a NADP⁺-dependent enzyme,and showed no detectable activity in the presence of NAD⁺.Also,the K_m values of YlG6PD for glucose-6-phosphate and NADP⁺were 261?M and 34.12?M,respectively.With respect to NADP⁺,NADPH was a competitive inhibitor of YlG6PD?K_i=56.04?M?.2 Expression,purification and enzymatic characterization of YlMEIn this study,YlME was over-expressed in E.coli and purified to homogeneity through Co²⁺affinity chromatography.The subunit molecular weight of YlME was approximately 69 kDa as measured by SDS-PAGE.The molecular weight of native YlME was approximately 144 kDa using the gel filtration chromatography.Thus,YlME was a homodimeric protein.The optimum pH and temperature for YlME catalysis were determined to be pH 5.8 and 25°C,respectively.Heat-inactivation experiment revealed that YlME lost almost 50%of the original activity after incubation at 33°C for 20 min,indicating a poor thermal stability.Kinetics studies showed that YlME was a NAD⁺-dependent enzyme with a 38-fold preference for NAD⁺over NADP⁺.The K_m values of YlME for NAD⁺and NADP⁺were 672.30?M and 2180?M,respectively.Interestingly,YlME was allosterically regulated by its substrate malate with a S_0.5.5 value of 58.20 mM and a Hill coefficient of 2.16.3 Alteration of coenzyme specificities of YlG6PD and YlMEBased on the structure-based protein sequence alignments,four mutant enzymes of YlG6PD?R52D,R52D/S53Q,R52D/S53Q/Y93V and R52D/S53Q/Y93V/A124S?were constructed.Kinetics studies revealed that all four mutants failed to utilize NADP⁺as a coenzyme and the best catalytic efficiency of R52D/S53Q/Y93V/A124S for NAD⁺was 25.6 s^-1M^-1.Then,a double mutant enzyme?M388V/P391K?of YlME was constructed.The affinity for NAD⁺of M388V/P391K was decreased by 4.77 fold,and the affinity for NADP⁺of this mutant enzyme was increased by 3.6 fold.The K_m values of M388V/P391K for NAD⁺and NADP⁺were 3207?M and 605?M,respectively.Therefore,M388V/P391K displayed a 5-fold affinity for NADP⁺over NAD⁺.The further alteration of the coenzyme specificity of YIME is in progress.In this study,the detailed enzymatic characterization and functional alteration of YlG6PD and YlME,two key enzymes in the central metabolism of Y.lipolytica,were carried out.The importance of the amino acids involved in the coenzyme binding of these two enzymes is revealed,and the biochemical information of them is enriched.This work would provide usefully scientific basis for the metabolism engineering of yeast.