S-Adenosyl-L-methionine(SAM)is the most common methyl donor in a multitude of cellular methyla-tion reactions. Numerous methyltransferases transfer the methyl group from SAM to their respective biological acceptors, forming S-adenosyl-L-homocysteine(SAH). SAH is hydrolyzed to adenosine and L-homocysteine by S-adenosyl-L-homocysteine hydrolase(SAHH, EC 3.3.1.1). SAHH is an essential enzyme in all living cells, and has been explored as a potential drug target for many bacteria and parasites. The inhibition of SAHH activity can result in the accumulation of SAH and reduce the S-adenosylmethionine(SAM):SAH ratio in cells. In that case, SAHH can act as a potent feedback inhibitor, blocking the SAM-dependent methylation that is required for the metabolism of a wide variety of biological compounds such as nucleic acids, proteins, phospholipids, and other small molecules. SAHH is found in animals, plants, fungi, and other microorganisms. Antarctic ice microalgae with special characteristics to withstand the extreme environment characterized by low temperatures and ice, high levels of dissolved oxygen, and strong seasonal changes in light intensity have been investigated in recent years. To further understand the intrinsic mechanisms by which the Antarctic sea ice alga Chlamydomonas sp. ICE-L responds to ecological and environmental factors, we cloned and analyzed its SAHH. A cDNA encoding S-adenosyl homocysteine hydrolase(designated as ICE-LSAHH)was cloned from this alga by RT-PCR and RACE-PCR methods. The ICE-LSAHH full-length cDNA sequence was 1844 bp, including a 5'-terminal untranslated region(UTR)of 36 bp, a 3'-terminal UTR of 344 bp with a poly(A)tail, and an open reading frame(ORF)of 1461 bp encoding a polypeptide of 487 amino acids. The predicted molecular weight(MW)of ICE-LSAHH was 53 kD with an estimated pi of 5.16. Using SignalP 3.0 and TMHMM Server v. 2.0 software, it was predicted that the ICE-LSAHH protein did not contain a signal peptide or a transmembranous region, suggesting that it is not a secretory protein. To further analyze the evolutionary relationship among SAHH enzymes, a molecular phylogenetic tree was constructed using ClustalX 2.0 and Mega 5.0 software. The reliability of branching was tested by bootstrap re-sampling(l 000 pseudo-replicates). In the phylogenetic tree, members of the SAHH protein formed clear subgroups of plants, animals, algae, and bacteria. The Chlamydomonas sp. ICE-LSAHH evolved in parallel with those from Dunaliella salina, Chlamydomonas reinhardtii, and Chlorella vahabilis. Sequence analysis with the BLAST algorithm showed that the ICE-LSAHH gene shared 62%-85% sequence identity with other SAHHs. The highest sequence identity was 85% with the SAHH gene of C. Reinhardtii. Amino acid sequence alignment between ICE-LSAHH and other SAHHs showed that the ICE-LSAHH protein exhibited high sequence homology with other SAHHs. The three-dimensional structure of ICE-LGPx was determined using SWISS-MODEL workspace and PyMOL Viewer software. The 3D molecular model showed that the ICE-LSAHH subunit has 12 P-sheets and 20 a-helices, and consists of three domains: a substrate-binding domain, an NAD-binding domain, and a C-terminal domain. In conclusion, SAHH is a conserved protein that is found in the cytoplasm of the ice alga Chlamydomonas sp. ICE-L.%S-腺苷同型半胱氨酸水解酶(S-adenosyl-L-homocysteine hydrolase,SAHH)是一种细胞内广泛存在的酶,在调节生物体转甲基化反应中占据重要地位.本研究通过RT-PCR及RACE-PCR技术克隆了南极冰藻(Ch lamydomonassp.ICE-L)S-腺苷同型半胱氨酸水解酶全序列,命名为ICE-LSAHH.ICE-LSAHH全长1844 bp,包括5’非编码区36 bp,3 ’非编码区344 bp,含有一个较长的poly(A)尾.开放阅读框1461bp,编码487个氨基酸.根据推导的氨基酸序列预测其分子量为53.0 kD,理论等电点为5.16.SignalP 3.0、TMHMM Server v.2.0、NetNGlyc l.0和NetPhos2.0预测结果显示,ICE-LSAHH蛋白不存在信号肽与跨膜区,含有1个N-糖基化位点及23个磷酸化位点.利用ClastalX 2.0及MEGA 5.0软件,以邻位相连法对南极冰藻及其他物种SAHH构建系统进化树,结果显示,ICE-LSAHH与杜氏盐藻(Dunaliella salina)、莱茵衣藻(Chlamydomonas reinhardtii)、小球藻(Chlorella variabilis)等有较近亲缘关系.利用SWISS-MODEL程序和PyMOL Viewer软件成功模拟了ICE-LSAHH蛋白亚基的三维结构,该结构包括20个α-螺旋和12个β-折叠,且存在3个结构域:N端底物结合域、NAD结合域和C端域.可见,SAHH基因存在于南极冰藻并进行了有效的表达,该蛋白定位于细胞质,进一步证实SAHH是一个进化上比较保守的蛋白.
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