Studies On The Mechanism Of Substrate Channeling In Sulfate Activating Complex Of Rhodobacter Sphaeroides

Sulfur is an essential macronutrient for all kinds of living things. Mammals can absorb sulfur nutrients from diet directly, and plants and mircro-organisms usually absorb sulfate from the surrounding environment as the primary source of sulfur. The first step of intracellular sulfate activation, transferring of adenylyl moiety of ATP onto sulfate to produce adenosine 5'-phosphosulfate (APS) and pyrophosphate is an extremely unfavorable reaction catalyzed by ATP sulfurylase (ATPS, EC 2.7.7.4). It had been demonstrated that ATPS boosts APS synthesis by forming sulfate activating complexes (SACs) with enzyme(s) involving in sulfate assimilation. SACs have been found in mammals, plants and microorganisms.Consisted of ATPS and APS kinase acitivities, SAC from Rhodobacter sphaeroides has APS channeling property, and it is the only channeling SAC reported to date. In order to study the channeling mechanism, three studies were performed based on its crystal structure:1) Oligomerization of SAC; 2) Function of electrostatic interactions in APS moving; 3) Phosphorylation of APS.In order to get different SAC oligomers and lay foundations for further analysis APS channel, mutants were constructed, overexpressed, purified and characterized according the crystal structure of SAC and using site-directed mutagenesis method. Results indicated that the oligomerization states have not been altered in the mutants we currently constructed.To study the functions of electrostatics in APS channeling, charged amino acids in the putative channel were mutated to non-charged or reverse-charged amino acids. Mutants were constructed, purified and characterized. Results indicated the KD Loop is essential for ATPS activity and mutagenesis of K231 and D234 lead to dramatically reduction or loss of ATPS activity. E109 and K438 mutants decreased the APSK catalytic efficiency about 90%. Other mutants we studied have little effects on ATPS and APSK activity.It had been demonstrated that freshly prepared SAC could phosphorylate APS in the absence of ATP. In order to reveal which phosphorylated residue contributes the phosphoryaltion of APS, several serine residues in APSK active sites (S405, S407 and S410) near the 3'-OH group of APS were mutagenized to alanines, and corresponding proteins were further analyzed by Western blot. Results demonstrated that mutation of all three residues have no effect on Western blot results, and S405 and S407 mutants have similar wild type APSK activity. Whereas, S410A APSK activity was not detected, which indicated the importance of this residue for APSK activity.All work done here lay a solid foundation for further ananylis of APS channeling mechanism.