Li Xiangli, Geng Zhirong, Chang Jiayin, Song Xiaoli, Wang Zhilin
Biochimie. 2014 Dec;107 Pt B:396-405. doi: 10.1016/j.biochi.2014.10.014.
The functions of residues 57-RY-58, G60, L77, 80-GSGR-83, I101, T104, 134-GY-135, N155, V157 and 160-LV-161 in human arsenic (III) methyltransferase (hAS3MT) 5 Å around S-adenosylmethionine (SAM) have not been studied. Herein, sixteen mutants were designed by substituting these residues with Ala. Mutants G60A, G80A, I101A, N155A and L160A were completely inactive. Only MMA was detected when mutants R57A, Y58A, G82A and T104A were used as the enzymes, which suggested that their catalytic activities were seriously impaired compared with that of wild type (WT). The catalytic capacities of other mutants were also lower than that of WT-hAS3MT. The KM(SAM) values of mutants were 1.9–8.7 times that of WT, suggesting their affinities to SAM were weakened. As evidenced by the experimental data herein, earlier literature and the model of hAS3MT-SAM, 57-RYYG-60, G78, G80, G82 and 155-NCV-157 interacted with the methionine of SAM, and 101-IDMT-104 and 135-YIE-137 were associated with the nucleotide adenosine of SAM. Since C156 and L160 were the common residues between 5 Å around SAM and 5 Å around As, and C156S and L160A were inactive, we proposed that C156 and L160 functioned in the methyl transfer process. G78, G80 and G82 belonging to the consensus GxGxG were located in a loop connecting the first β-strand and α-helix in the Rossmann fold core. Y59, N155, C156 and L160 oriented S(+)-CH(3) during its approach to the arsenic lone pair, and further activated methyl transfer. G78, D102, M103, T104, I136 and N155 formed hydrogen bonds with SAM.
尚未对人砷（III）甲基转移酶（hAS3MT）中围绕S-腺苷甲硫氨酸（SAM）的5 Å范围内的57-RY-58、G60、L77、80-GSGR-83、I101、T104、134-GY-135、N155、V157和160-LV-161残基的功能进行研究。在此，通过将这些残基替换为丙氨酸设计了16个突变体。突变体G60A、G80A、I101A、N155A和L160A完全无活性。当使用突变体R57A、Y58A、G82A和T104A作为酶时，仅检测到一甲基砷，这表明与野生型（WT）相比，它们的催化活性严重受损。其他突变体的催化能力也低于WT-hAS3MT。突变体的KM（SAM）值是WT的1.9 - 8.7倍，表明它们对SAM的亲和力减弱。如本文的实验数据、早期文献以及hAS3MT-SAM模型所证明的，57-RYYG-60、G78、G80、G82和155-NCV-157与SAM的甲硫氨酸相互作用，101-IDMT-104和135-YIE-137与SAM的核苷酸腺苷相关。由于C156和L160是围绕SAM的5 Å和围绕砷的5 Å之间的共同残基，且C156S和L160A无活性，我们提出C156和L160在甲基转移过程中起作用。属于共有序列GxGxG的G78、G80和G82位于Rossmann折叠核心中连接第一个β链和α螺旋的环中。Y59、N155、C156和L160在S（+）-CH（3）接近砷孤对时使其定向，并进一步激活甲基转移。G78、D102、M103、T104、I136和N155与SAM形成氢键。
