Grabarse W, Vaupel M, Vorholt J A, Shima S, Thauer R K, Wittershagen A, Bourenkov G, Bartunik H D, Ermler U
Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043, Marburg, Max-Planck-Institut für Biophysik, Heinrich-Hoffmann-Strasse 7, 60528, Frankurt, Germany.
Structure. 1999 Oct 15;7(10):1257-68. doi: 10.1016/s0969-2126(00)80059-3.
The reduction of carbon dioxide to methane in methanogenic archaea involves the tetrahydrofolate analogue tetrahydromethanopterin (H(4)MPT) as a C(1) unit carrier. In the third step of this reaction sequence, N(5)-formyl-H(4)MPT is converted to methenyl-H(4)MPT(+) by the enzyme methenyltetrahydromethanopterin cyclohydrolase. The cyclohydrolase from the hyperthermophilic archaeon Methanopyrus kandleri (Mch) is extremely thermostable and adapted to a high intracellular concentration of lyotropic salts.
Mch was crystallized and its structure solved at 2.0 A resolution using a combination of the single isomorphous replacement (SIR) and multiple anomalous dispersion (MAD) techniques. The structure of the homotrimeric enzyme reveals a new alpha/beta fold that is composed of two domains forming a large sequence-conserved pocket between them. Two phosphate ions were found in and adjacent to this pocket, respectively; the latter is displaced by the phosphate moiety of the substrate formyl-H(4)MPT according to a hypothetical model of the substrate binding.
Although the exact position of the substrate is not yet known, the residues lining the active site of Mch could be tentatively assigned. Comparison of Mch with the tetrahydrofolate-specific cyclohydrolase/dehydrogenase reveals similarities in domain arrangement and in some active-site residues, whereas the fold appears to be different. The adaptation of Mch to high salt concentrations and high temperatures is reflected by the excess of acidic residues at the trimer surface and by the higher oligomerization state of Mch compared with its mesophtic counterparts.
产甲烷古菌中二氧化碳还原为甲烷的过程涉及四氢叶酸类似物四氢甲烷蝶呤（H(4)MPT）作为C(1)单位载体。在该反应序列的第三步中，N(5)-甲酰基-H(4)MPT被亚甲基四氢甲烷蝶呤环水解酶转化为亚甲基-H(4)MPT(+)。嗜热古菌坎氏甲烷嗜热菌（Mch）的环水解酶具有极高的热稳定性，并适应细胞内高浓度的促溶盐。
通过单同晶置换（SIR）和多波长反常散射（MAD）技术相结合，使Mch结晶并以2.0 Å分辨率解析其结构。同源三聚体酶的结构揭示了一种新的α/β折叠，由两个结构域组成，它们之间形成一个大的序列保守口袋。在该口袋内及相邻位置分别发现了两个磷酸根离子；根据底物结合的假设模型，后者被底物甲酰基-H(4)MPT的磷酸部分取代。
尽管底物的确切位置尚不清楚，但可以初步确定Mch活性位点的内衬残基。将Mch与四氢叶酸特异性环水解酶/脱氢酶进行比较，发现其在结构域排列和一些活性位点残基上存在相似性，而折叠方式似乎不同。Mch对高盐浓度和高温的适应性体现在三聚体表面酸性残基的过量以及与中温对应物相比Mch更高的寡聚化状态上。
