Font Size: a A A

Biochemical Application Of Yeast3’,5’-Bisphosphate Nucleotidase

Posted on:2014-03-23Degree:MasterType:Thesis
Country:ChinaCandidate:Y YangFull Text:PDF
Abstract/Summary:PDF Full Text Request
Sulfur, an essential element for all organisms, plays its biological roles mainly with valence status of-2and+6. Sulfur activation is the key reaction of sulfur assimilation.Intracellular sulfate activating, first step for the assimilation of sulfate, includes biosynthesis of adenosine5’-phosphosulfate (APS) catalyzed by ATP sulfurylase (ATPS) and the subsequent phosphorylation of APS by adenosine5’-phosphosulfate kinase (APSK) to form3’-phosphoadenosine5’-phosphosulfate (PAPS), the universal sulfuryl group donor. Yeast3’,5’-bisphosphate nucleotidase (YND), also known as HAL2, can hydrolyze3’-phosphate from PAP to form AMP or from PAPS to form APS, had been found involved in sulfur metabolism and salt stress. It had also been found hydrolyzing3’-phosphate5’-phosphate adenosine (PAP) with submicromolar Km, which indicated the tight interactions between YND and PAP. The high affinity of ND to PAP and commercial availability for PAP agarose (the cross-linking product of activated agarose and the free amino group from N6-[(6-amino)octyl]-amino-adenosine-3’,5’-bisphosphate, Sigma-aldrich,#A3640) enabled affinity purification of YND which suggested YND could be used as a possible fusion affinity tag for protein purification.Affinity chromatography is one of the most popular methods for protein purification. Each tag method has its advantages and disadvantages, and combination of different tags and developing of new tags had been proposed and performed. In order to explore the feasibility of YND as a protein purification affinity tag, YND was characterized in detail. Firstly, YND was characterized for hydrolysis of PAP and binding to PAP analogue. Results demonstrated that KmPAp and KcatPAP were~0.3μM and~11s-1, and Kd were0.008and0.49μM in presence of Ca2+or Mg2+, respectively. Secondly, pH was also found to affect interactions between YND and PAP, with tightest binding with Kd8nM at pH7.5and8. Series of expression vectors including that for expression of fusion protease, were further constructed and E. coli APSK, as target protein, was prokaryotically expressed, purified and characterized. Thirdly, the purification protocol for YND tag was rationally designed based on nanomolar affinity to PAP agarose in presence of Ca2+, the reagent which could satisfy the metal requirement for PAP binding, prevent hydrolysis of immobilized PAP and could be chelated by EGTA easily. To simplify the tag method, ready to use expression vectors and protocols were established. By comparing resin cost, solubility, column flow rate, product purity and yield with other popular tags, YND tag was found to be an economical and efficient tag for prokaryotic protein expression and purification.It had been shown that expression/activity change of YND would regulate intracellular PAP concentration, which would affect cell growth and development by regulating activities of sulfotransferase, holoacyl carrier protein synthase and RNA processing enzymes. It had not been reported that how YND discriminate PAP from PAPS, the universal sulfuryl group donor and substrate of sulfotransferase. R group of G236may involve in determination of substrate selection based on the YND crystal structure. The affinity decreasing for PAPS was found with increasing the size of R group at residue236. And G236V with catalytic efficiency of PAPS5592times more efficient than that of PAP, was found to be PAP specific3’-nuleotidase. By coupling with myokinase, pyruvate kinase and lactate dehydrogenase, G236V could be used as a coupling enzyme for the analysis of sulfotransferases activity, which were commonly measured with radioactive compounds currently.Although the mechanism of APS phosphorylation by APSK had been reported in detail, it remains to be elucidated that if APS analogue-AMP could be phosphorylated by APSK. Structural analysis indicated that R68would form hydrogen bonds with oxygens of a phosphate and (3sulfate to stabilize the binding of APS. R68K mutation would weak the interaction to β sulfate by shortening R group, strengthen interaction to a phosphoate, and possibly discriminate APS from AMP. Preliminary studies here showed that AMP could be phosphorylated by APSK with product PAP. Our results clearly demonstrated that R68K had5times enhancement of catalytic efficiency and binding affinity than those for native APSK. These primary results lighted up for further construction of high efficient AMP3’-hydroxyl kinase.In conclusion, one YND based affinity purification system was established, and PAP specific3’nuleotidase and AMP3’-hydroxyl kinase, potentially coupling enzymes for assaying sulfotranftases and regenerating PAP for YND activity assay were created.
Keywords/Search Tags:Yeast ND, Affinity chromatography, Prokaryotic proteinexpression and purification
PDF Full Text Request
Related items