The effect of post translational phosphorylation on S-adenosylhomocysteine hydrolases from halophilic methanoarchaeon - Methanohalophilus portucalensis FDF1T

• Main Authors: Wen-Chieh Chen, 陳玟潔
• Other Authors: Mei-Chin Lai
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/77813752820957093991

碩士 === 國立中興大學 === 生命科學系所 === 104 === S-adenosylhomocysteine hydrolase (SAHH; E.C.3.3.1.1), could hydrolyze the inhibitor of SAM-dependent methyltransferase, S-adenosylhomocysteine (SAH), which plays a critical role in maintaining homeostasis of the balance of S-adenosyl-L-methionine (SAM) and SAH for all living cells. The halophilic methanogen Methanohalophilus portucalensis FDF1T could survive in 3 M salt stress environment by internal accumulation, through uptake or de novo synthesis, of organic compatible glycine betaine. Two copies of SAHH in its betaine synthesis cluster were found and phylogenetic analysis revealed one was bacterial/eukaryotic origin, SAHH1, and the other was archaeal native, SAHH2. Since the post translational regulation of methanoarchaea was unknown, the phosphoproteomic analysis was investigated and showed that there are six phosphorylated sites in SAHH1 and none in SAHH2. Results in this study showed the MpSAHH1 formed octamer structure instead of commonly tetramer structure. The site directed mutagenesis on phosphorylated Ser224 residue in recombinant overexpressed SAHH1 were replaced by Ala and Asp residue to mimic the dephosphorylated and phosphorylated states, respectively. The kinetic analysis revealed that substrate SAH affinity declined for 130-fold and hydrolytic velocity decreased for 30-fold in phospho-mimic type MpSAHH1. The structural modeling analysis suggested that phospho-mimic S224D decreased the distance between Ser224 and Arg467 within C-terminal domain of neighbor subunit, and indirectly affect the stronger interaction between product Ado and SAH-binding site Lys223 with SAH affinity declined. Furthermore, S224D indirectly affect the weakly interaction between cofactor binding site Asn288 and cofactor NAD+ with protein hydrolytic velocity declined. Above these analysis results suggested that the phosphorylated Ser224 may negatively affect hydrolytic activity of MpSAHH1.