Identification of a Unique Radical S-Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii
Identification of a Unique Radical S-Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii
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DOI：
10.1128/jb.01903-14
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发表时间：
2014-09-01
影响因子：
3.2
通讯作者：
White, Robert H.
中科院分区：
文献类型：
--
作者：
Allen, Kylie D.;Xu, Huimin;White, Robert H.
甲烷蝶呤（MPT）及其类似物是特定微生物产甲烷和甲基化所需的辅酶。pterin环C-7和C-9的甲基将MPT与所有其他含pterin的天然产物区分开来。然而，负责添加这些甲基的酶尚未被确定。本研究表明，在产甲烷菌jannaschii中，由MJ0619基因编码的一个假定的自由基s -腺苷- l-蛋氨酸（SAM）酶超家族成员可能是这种缺失的甲基化酶。当MJ0619在大肠杆菌中异源表达时，检测到多种甲基化的蝶呤，这与MJ0619催化7,8-二氢-6-羟甲基蝶呤在C-7和C-9位点的甲基化一致，7,8-二氢-6-羟甲基蝶呤是叶酸和MPT生物合成的常见中间体。MJ0619中存在的两个典型自由基SAM CX3CX2C基序中的第一个基序中的Cys77位点定向突变没有抑制C-7甲基化，而另一个自由基SAM氨基酸基序中的Cys102位点突变导致C-7甲基化缺失，这表明第一个基序可能参与C-9甲基化，而第二个基序则是C-7甲基化所必需的。进一步的实验表明，C-7甲基不是由蛋氨酸衍生的，甲基化不需要钴胺素。当表达MJ0619的大肠杆菌细胞以氘标记的醋酸盐作为唯一碳源生长时，翼蛋白上的甲基主要被三个氘标记。基于这些结果，我们提出这种古菌自由基SAM甲基化酶采用了一种以前未被表征的甲基化机制，使用亚甲基四氢叶酸作为甲基供体。
Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-L-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX3CX2C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.