Charactering And Site-Directed-Mutagenesis Of The S-Adenosylmethionine Synthetase Gene From Bacillus

S-adenosylmetionine is an important intermediate in vivo, which is involved inmany biochemistry reactions such as DNA methelation, protein methelation,polyamine synthesis. In vivo, S-adenosylmetionine was synthesized byS-adenosylmetionine synthetase (EC2.5.1.6) from ATP and MET. With the primerdesigned according to SAM synthetase gene sequence of the reported Bacillusthuringiensis genome, eight SAM synthetase gene fragments were amplified from 12strains by PCR technique. The fragment was inserted into the expression vectorpGEX-6p-1, and eight rEcombinant expression plasmids (pGEX-sam2, pGEX-sam5,pGEX-sam7, pGEX-sam8, pGEX-sam9, pGEX-sam10, pGEX-sam11, pGEX-sam12)were obtained. Sequence analysis showed that the homologues of sam2, sam5, sam 7,sam 8, sam 9, sam10, sam11, sam12 and Bacillus thuringiensis is more than 97%,while the homologues of sam2, sam5, sam 7, sam 8, sam 9, sam10, sam11, sam12 andE. coli SAM synthetase gene is more than 63 %. Eight rEcombinant plasmids weretransformated into E. coli DH5a, sam2, sam5, sam7, sam10 and sam11 wereexpressed in E. coli DH5a successfully. SAM2, SAM5, SAM7, SAM10 and SAM11were purified by GST affinity chromatography. SDS-PAGE analysis showed that themolecular weight of these proteins is 43KD. The activity of SAM2, SAM5, SAM7,SAM10 and SAM11 in vitro was analysed with paper chromatography and HPLC.The analysis showed that SAM2, SAM5, SAM7, SAM10 and SAM11 can catalysethe formation of S-adenosylmetionine from ATP and MET in vitro. The activity ofSAM7 is four times higher than SAM2, SAM5, SAM10 and SAM11. SAM7m wasobtained by site-directe mutagenesis which converted G104 of SAM7 to I104 ofSAM7m. sam7m was also successfully expressed in E. coli DH5a. The activityanalysis showed that SAM7m lost the activity of catalyzing the formation of SAMfrom ATP and L-methionine by paper chromatography, which indicates that G104 isrelated with its activity center.