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Enzymatic Analysis on Sulfate Activating Complex from Thermobifida Fusca and Oligomerziation Analysis on Sulfate Activating Complex from Rodobacter Sphaeroides

Author: LiHongYan
Tutor: SunMeiHao
School: Zhejiang Normal University
Course: Botany
Keywords: Thermobifida fusca Sulfate activating complex APS kinase phosphorylation
CLC: Q93
Type: Master's thesis
Year: 2013
Downloads: 12
Quote: 0
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Sulfur as one of the essential elements involved in lots of metabolic pathways. Sulfur containing cmpounds play important roles in maintening cellular redox status, regulating gene expression, detoxifying heavy metals, defensing on pathogenic bacteria and pests. Sulfur in soil exists mainly in the form of sulfates and sulfites. Once sulfate enters in cells, ATP sulfuryalse (ATPS) and adenosine5’-phosphosulfate kinase(APSK) catalyze the activation of sulfate into adenosine5’-phosphosulfate (APS) and3’-phospho-adenosine5’-phosphosulfate (PAPS), which could be further reduced or transferred to form sulfated molecules. The synthesis of APS is thermodynamically unfavorable. It had been shown that cells evolve to form sulfate activating complex (SAC) to facilitate the activation of sulfate. SAC from Rhodobacter sphaeriodes channels APS from ATPS site to APSK site directly to possibly promoting biosynthesis of PAPS.Thermobifida fusca is a class of prokaryotes living in a relatively high temperature environment, and homologue of channeling SAC from R. sphaeroides was found in its genome. SAC from T. fusca (tfSAC) was purified and its activity was analyzed. Results showed the absence of ATPS activity and the presence of APSK activity for tfSAC. tfSAC APSK activity is not changed significantly after68hr incubation at room temperature or10min incubation at temperatures under60℃. Incubation at61℃for10min reduced its activity to80%of control. SAC from R. sphaeroides (rsSAC) lost its APSK activity after43℃treatment for10min. Analyzing the modeled structure, it was found the missing of two loops at the putative ATPS domain, and adding back of these two loops did not recover its ATPS acitivty. This result suggested a new SAC type ATPS*-APSK could be added into the APSK type SAC, and tfSAC could be used in relatively high temperature coupling reaction systems.To analyze effect of rsSAC oligomerization on its channeling property, mutants37DRY, SA214E forming inclusion bodies and SACMONO-APSK were purified and analyzed. It was found that SA214E lost the APSK activity, while37DRY, mutant missing of two a helixes lost ATPS activity. SACMONO-APSK, created by adding APSK domain to the C terminus of SAC whose activity of APSK domain was disabled, had comparable activities for ATPS and APSK and provided a good material for further analysis of substrate channeling mechanism and identifying the channeling path.We speculated that rsSAC has the same mechanism as E. coli APSK for the formation of phosphoralated enzyme intermediate, and by indentifying the residue account for the formation of E-P in ecAPSK to facilitate revealing of the E·P in rsSAC. With the mutants of S12A, S37A, S42A, S109A and S201A, and Weastern Blot using antibody for anti phosphorylated serine. We found positive results for all mutants, while S42A lost its APSK activity which suggested that this S42is critical for its activity.The completion of the work, laid a foundation for our future research on the channeling mechanism of rsSAC and analysis for E-P of APSK.

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