Regulation Mechanism Of APS Kinase Activity In Rice A Dissertation

Sulfur is an essential element for plant growth and development.In nature,the sulfur assimiliation pathway varies in different organisms.In plants,sulfate is transported into cells from the outside enviroment,activated by ATP sulfurylase to adenosine-5'-phosphate(APS),which can be reduced by APS reductase(APSR)to sulfite or be further phosphorylated by APS kinase to form 3'-phospho-adenosine 5'-phosphoryl sulfate(PAPS),the sole sulfuryl group donor in cells for sulfation.Obviously,APSR and APSK compete APS for the sulfur first and secondary metabolism,which is critical for plant normal development.Although sulfated compounds are widely distributed in plant,little is known about the enzyme APSK which provides the sulfate donor PAPS in vivo.Four APSK had been identified in Arabidopsis.Based on the localization and functions,APSK1 and APSK2 are believed play vital roles in sulfur metabolism.APSK1 participates in the regulation of first and secondary sulfur metabolism,and oxidative stress will accelerate formation of disulfide bond between C86 and C119,which will down-regulate its activity.Based on sequence analysis,APSK1 form Oryza sativa(osAPSK)was found containing the corresponding cysteines(C36 and C69).Previous studies in our laboratory showed that oxidative stress reduced wild type osAPSK activity and double mutation of C36A and C69A relieved this oxidative effect.Sequence and modeled structural analysis showed that C77,C168,C212 were also susceptible to be oxidized and may have a role in its activity regulation.Here,using osAPSK as template,we constructed mutant of C77A,C168A and C212A,respectively.Kinetic analysis was performed with the prokaryotic expressed mutants under reducing and oxidizing environments.In presence of DTT,the kcat of C77A,C168A and C212A were reduced to 21.3%,9.1%and 9.9%,Km reduced to 2.6%,12.2%,20.2%,to the wild type enzymes,respectively.And Kiaps was found increased significantly.While under oxidative conditions,kcat of C77A,C168A and C212A were reduced to 36.2%,22.6%and 38.2%,Km reduced to 1.8%,6.1%and 5.9%,Kiaps increased to 328%,745%and 655%of the wild type enzyme,respectively.All these indicated that except C36 and C69 play vital roles in its activity regulation,C77A,C168A and C212A might also involve the activity regulation,especially under oxidative conditions.Further structural analysis might provide further insights.Many enzymes will lose their activity under high temperature conditions,but most of the thermophilic enzymes can still maintain a high catalytic activity in this environment,so the research and development of thermophilic enzymes is currently a hot topic in the market.It is the most direct and reliable source of thermotropic enzymes currently available.Sulfate forms a branching point in the secondary metabolic transformation pathway.Some pathogenic bacteria,such as M.tuberculosis,reduce APS to sulfite mainly through APS reductase,providing the main route for the synthesis of various metabolites from sulfur.In contrast,other bacteria,fungi and humans use APSK and APS reductases in turn to convert APS to PAPS in the primary sulfur assimilation pathway and then further metabolize to sulfite by PAPS reductase.In our previous studies,we isolated one moderate thermophile Aeribacillus pallidus,which excrets antibacterial compounds with optimum growth temperature of 50?.Its genome had been sequenced(unpublished).In this study,we prokaryotically expressed,purified and characterized APSK from A.pallidus(apAPSK).Kinetic data suggest its optimum Ph is 8.0,which is consistent with plant APSKs.Optimum temperature is 25?,although its optimum growth temperature is 50?.This also indicated that under the optimum growth temperature,its activity is inhibited.KmAPS and KcatAPS are 10.25 ?M/L and 62.87s-1,respectively,which are larger than those of plant,while there is no much difference between their Vm/Km.the conservation of APSK function in A.pallidus also suggests APSK is vital for its growth and development.