以X-射線晶體學探討嗜鹽性甲烷古生菌 Methanohalophilus portucalensis 甘胺酸肌胺酸甲基轉移酶之結構與功能

Abstract

當微生物面臨外在環境劇烈的變化，如鹽濃度的改變所造成的滲透壓力，它們能夠藉由累積細胞內的滲透壓調節物質（又可稱為相容性溶質）以適應高滲透壓的外在環境。嗜鹽性甲烷古生菌 Methanohalophilus portucalensis FDF1T 可以生長在鹽濃度 1.2-2.9 M 的環境中，在高鹽環境下M. portucalensis FDF1T 不僅能自行合成相容性溶質，例如 glycine betaine、 β-glutamine、 β-glutamate、和 Nε-acetyl-β-lysine 等小分子，並且會從外在環境中吸收這些物質或其合成前驅物，而吸收的同時伴隨著鉀離子的進入，其在細胞內部累積可以減少水分的流失和幫助細胞膨壓的維持。在這些滲透壓調節物質中， glycine betaine (N, N, N-trimethylglycine) 簡稱為 betaine，為最廣泛使用的調節物質之一。 Betaine 在細菌、古生菌、真菌、植物、和動物中具有重要的滲透壓調節保護的功能。 Betaine 除了可以藉由 choline 去氫酶經過兩步驟的催化反應生合成，在一些中度或極度嗜鹽性甲烷古生菌中，亦可藉由將 glycine 進行三次的甲基轉移生合成。如 M. portucalensis 中有兩個甲基轉移酶參與此催化反應，一個為 glycine sarcosine methyltransfease (GSMT)，將 glycine 甲基化形成 sarcosine，再將 sarcosine 甲基化形成 dimethylglycine；另一個為 sarcosine dimethylglycine methyltransferase (SDMT)，將 sarcosine 甲基化形成 dimethylglycine，最後將 dimethylglycine 甲基化形成 betaine。 本篇論文的主要目的是藉由 X-射線晶體學解析 M. portucalensis 的 MpGSMT 之蛋白結構，利用蒸氣擴散結晶技術，我們成功地獲得 MpGSMT 的晶體，在此篇論文中，我們會解析出兩個晶體結構，分別為 ligand-free MpGSMT 和MpGSMT-AdoMet (S-Adenosyl-L-methionine) 複合體的結構。MpGSMT 的晶體結構解析度為 1.9 Å，其結構包含一個保留性的結構核心（AdoMet 結合的 Rossmann 摺疊構型功能區）和一個受質結合的輔助功能區，此輔助功能區在甲基轉移酶中扮演辨識受質的角色。 MpGSMT 的結構和受質同為 glycine 的 glycine N-methyltransferase (GNMT) 非常的相似，因此我們認為 MpGSMT 應屬於小分子類的甲基轉移酶。將此 MpGSMT 晶體浸泡入含有 glycine 和 AdoMet 的溶液，經由 X- 射線繞射分析也成功得到了 MpGSMT-AdoMet 複合體結構，其解析度為 2.88 Å。然而我們尚未觀察找到 glycine 在 MpGSMT 中的結合位置，但有趣地是，當 AdoMet 一結合到 MpGSMT 上， His138 會轉向活化區，由此推測 His138 可能為催化反應中關鍵的胺基酸之一。

Microorganisms need to adapt rapidly to extreme variations in salinity, temperature or osmolarity to survive. To avoid dehydration or swelling, the cells adjust their intracellular solute pool in response to environmental changes. Many organisms have developed similar strategies to counteract high osmolarity through the intracellular accumulation of osmolytes, which are often referred to as compatible solute. The halophilic methanoarchaeon Methanohalophilus portucalensis strain FDF1T can grow optimally over the salt range of 1.2-2.9 M. Under the hypersaline environment, M. portucalensis FDF1T not only synthesizes low molecular weight compatible solutes such as glycine betaine, β-glutamine, β-glutamate, and Nε-acetyl-β-lysine, but also uptakes osmolytes and theirs precursors from surrounding environment. Intracellular accumulation of these organic molecules along with potassium ions minimizes water loss and helps to maintain the cellular turgor pressure. Among these osmolyts, glycine betaine (N, N, N-trimethylglycine), often referred to simply as betaine, is one of the most widely adopted compatible solutes. Betaine plays a crucial osmoprotective function in bacteria, archaea, fungi, plants, and animal cells. Betaine is synthesized from choline by oxidation. A choline dehydrogenase has been shown to catalyze the two-step reaction of choline to betaine in many microbes. Moreover, the moderately and extremely halophilic methanoarchaea also synthesize betaine de novo for use as a compatible solute. Two methyltransferase genes involved in betaine biosynthesis are present in M. portucalensis. One of the gene products (GSMT) catalyzed the methylation reactions of glycine and sarcosine to sarcosine and dimethlglycine, respectively, whereas the other one (SDMT) catalyzed the methylations of sarcosine and dimethylglycine to dimethylglycine and betaine. The goal of my thesis research is to determine the structure of GSMT from M. portucalensis by X-ray crystallography. Using vapor-diffusion crystallization technique, we have successfully obtained crystals of MpGSMT. Here we report two crystal structures: native MpGSMT and MpGSMT in complex with S-Adenosyl-L-methionine (SAM/AdoMet). The crystal structure of the native MpGSMT was solved at a resolution of 1.9 Å. The MpGSMT structure consists the consensus structural core, the AdoMet-binding Rossmann fold domain, and a substrate-binding auxiliary domain that are inserted throughout the methyltransferase fold and appear to play roles in substrate recognition. Rossmann fold domain comprises a seven-stranded β sheet with a central topological switch-point and a characteristic reversed β hairpin at the carboxyl terminal end of the sheet. This sheet is flanked by α helices to form a doubly wound open αβα sandwich. Substrate-binding auxiliary domain comprises a four-stranded anti-parallel β sheet. The MpGSMT structure is similar to that of glycine N-methyltransferase (GNMT), whose substrate is also glycine. Our study revealed that MpGSMT structure resembles those seen in the small molecular methlytransferase superfamily. We also obtained MpGSMT-AdoMet binary complex structure by soaking the cofactor into the pre-grown crystals with glycine-AdoMet-containing buffer. The crystal structure of the MpGSMT-AdoMet was solved at a resolution of 2.88 Å. However, the binding site of glycine has not yet been observed. Interestingly, His138 faces toward active site upon AdoMet binding, we speculate that His138 may be a key catalytic residue.