甘胺酸肌胺酸氮甲基轉移酶之結構分析及活性調控機制之探討

Structural Analysis of Glycine Sarcosine N-methyltransferase from Methanohalophilus portucalensis Reveals Mechanistic Insights into the Regulation of Methyltransferase Activity

李易儒(Yi-Ru Lee)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2017年)

https://doi.org/10.6342/NTU201700284

摘要

甲基轉移酶在許多細胞生化運作過程中經常扮演著重要的角色，因此生物體中存在著多種調控甲基轉移酶活性的分子機制。對於參與在生物生合成路徑中的甲基轉移酶而言，負迴饋抑制是一種經常被採用的調控策略；以避免終產物的過度累積。然而目前為止文獻中尚無此類甲基轉移酶與終產物形成的複合體晶體結構被報導，因此終產物抑制甲基轉移酶活性的結構基礎仍待探討。本研究係藉由晶體結構解析、闡述一個來自嗜鹽古生菌 (高鹽甲烷太古生物Methanohalophilus portucalensis) 中的甘胺酸肌胺酸氮-甲基轉移酶 (簡稱MpGSMT) 的活性調控機轉。此結構是甘胺酸肌胺酸氮-甲基轉移酶蛋白家族中第一個被報導的晶體結構。MpGSMT負責催化菌體內生合成滲透壓保護劑甜菜鹼(betaine)的第一及第二個催化步驟，它是利用S-腺苷甲硫氨酸 (簡稱SAM) 做為甲基的提供者，將甘胺酸以及肌胺酸進行甲基化以合成肌胺酸與二甲基甘胺酸。生化分析指出此酵素的活性可受高鹽所刺激而提升、而終產物甜菜鹼則會以負迴饋方式抑制其活性。為了對於甲基轉移酶的調控機制有更深入的了解，我們解析了MpGSMT於不同形式的晶體結構，包括酵素本身的結構、酵素分別結合SAM或是S-(5'-腺苷)-L-高半胱氨酸 (簡稱SAH)的二重複合體結構、以及酵素結合SAH/肌胺酸和酵素結合SAH/二甲基甘胺酸的三重複合體結構。
MpGSMT整體結構與第一型SAM依賴型甲基轉移酶家族成員非常類似， 結構主體呈現一個典型的混合型β-平板摺疊形式；有七個β-strands，各strands之間以α-螺旋與loops連接。β-strands的排列順序為 β3-β2-β1-β4-β5-β7-β6，β1-β6以平行方式排列，而β7以反平行方式排列。β5羧基端之延伸序列形成獨立的功能區覆蓋在活性中心的上方，其組成包括一個α-螺旋以及四個反平行排列的β-strands。結合於活性中心的SAM與酵素之間形成多組交互作用；包含腺苷的鹼基與色胺酸115的側鏈形成推疊(stacking interaction)，D-核糖與天門冬胺酸88以及L-高半胱氨酸與丙胺酸67、亮胺酸132、天冬醯胺73、以及精胺酸43之間有氫鍵交互作用。受質肌胺酸結合區的空間的長、寬、高分別為7.7、7以及4 Å，位於蓋子結構與SAM結合的主結構之間。其中的天冬醯胺134、組胺酸138、精胺酸167、酪胺酸206、以及蛋胺酸218等胺基酸協助受質肌胺酸以最佳方位結合，以進行催化反應。結構分析進一步顯示結合於活性中心的SAH與受質肌胺酸之間的空間相當大，與一般的小分子氮-甲基轉移酶所具有較為窄小的活性中心有著顯著的不同。由於已知MpGSMT活性是需要被調控，因此推測儘管受質(肌胺酸)與輔酶(SAH)同時存在，此MpGSMT結構很可能仍處於非活化態的構形，推測需要額外的構型改變才能組合成一個有效的活性中心結構。此點也透過將輔酶(SAH)置換成甲基的提供者SAM，說明此構形的確處於非活化狀態，且不會引起甲基化反應。另外，此不活化的構型也透過浸泡晶體於甜菜鹼的水溶液中，並且經過結構解析的結果，確實觀察到有甜菜鹼的結合位，推測甜菜鹼可能透過穩定此非活化狀態而抑制MpGSMT酵素活性。此外，我們也模擬出一個MpGSMT處於有活性狀態的構形，以進一步了解活性中心可能的構型變化。比較MpGSMT非活化態的構形與處於有活性狀態的模型，我們發現N端第一個螺旋 (簡稱H1 helix) 在晶體結構中是遠離SAM結合的活性區塊，而在有活性的模型中此區域則形成一段延伸且包覆著活性中心的構形。突變此H1 helix區域，結果造成對鉀離子濃度原本刺激活性上升的敏感度降低了，此結果顯示N端區域與活性調控有關。
本研究是第一個生物生合成路徑中甲基轉移酶酵素與終產物形成複合體的蛋白結構，得以此推測負迴饋調控可能的機制。於研究過程中發現 N-端H1 helix區域對此小分子氮-甲基轉移酶的MpGSMT可能是一個重要的調控位點，它不僅是一個終產物甜菜鹼造成酵素活性受抑制的區域，也可能是高鹽得以提升酵素活性的關鍵調控位點。

關鍵字

甘胺酸肌胺酸氮甲基轉移酶 ； S-腺苷甲硫氨酸(SAM) ； 甜菜鹼 ； 高鹽甲烷太古生物Methanohalophilus portucalensis ； X射線晶體學 ； 肌胺酸 (sarcosine) ； N, N-二甲基甘氨酸 (DMG)

並列摘要

Methyltransferases play crucial roles in many cellular processes, and various regulatory mechanisms have evolved to control their activities. For methyltransferases involved in biosynthetic pathways, regulation via feedback inhibition is a commonly employed strategy to prevent excessive accumulation of the pathways’ end products. To date, no biosynthetic methyltransferases have been characterized by X-ray crystallography in complex with their corresponding end product. To understand the structural basis by which feedback regulation of methyltransferase activity is acheved, we characterized the crystal structures of the glycine sarcosine N-methyltransferase from the halophilic archaeon Methanohalophilus portucalensis (MpGSMT). As the first enzyme in the biosynthetic pathway of the osmoprotectant betaine, MpGSMT catalyzes N-methylation of glycine and sarcosine, and its activity is feedback-inhibited by the end product betaine. Here, we report the crystal structures of MpGSMT in its apo state, cofactor S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)-bound binary complexes, and in complexes with cofactor and substrate/product. This work represents the first structural elucidation of the GSMT methyltransferase family.
The overall structure of MpGSMT exhibits a classical mixed β-sheet fold common to all class I SAM-dependent methyltransferase (SAM-MTase). The seven β-strands are sandwiched by three helices on each side and are arranged in the order of β3-β2-β1-β4-β5-β7-β6, where β7 is the only strand showing anti-parallel packing. A lid structure comprises a helix and a 4-strand anti-parallel β-sheet protrudes from β5 to shield the active site cavity from solvent. Multiple sets of interactions were identified between MpGSMT and the bound SAH: stacking interaction between the adenine ring and the side chain of Trp115; hydrogen bonds between the D-ribose and Asp88; and L-homocysteine with Ala67, Leu132, Asn73 and Arg43. The substrate binding pocket is ~7.7 Å long, ~7 Å wide, and ~4 Å deep and is located between the lid structure and the mixed β-sheet domain. Residues Asn134, His138, Arg167, Tyr206 and Met218 are involved in orienting sarcosine for catalysis. A structural analysis revealed that, despite the simultaneous presence of both substrate (sarcosine) and cofactor (S-adenosyl-L-homocysteine; SAH), the enzyme was likely crystallized in an inactive conformation, as additional structural changes are required to complete the active site assembly. Consistent with this interpretation, the bound SAH can be replaced by the methyl donor S-adenosyl-L-methionine without triggering the methylation reaction. Furthermore, the observed conformational state was found to harbor a betaine-binding site enclosed by the H1 helix, H2 helix and αB helix, suggesting that betaine may inhibit MpGSMT activity by trapping the enzyme in an inactive form. In addition, we obtained a structural model of MpGSMT in its catalytically active state by homology modeling using the closely related glycine N-methyltransferase (PDBid: 1NBH) as the template. Comparison of catalytically inactivated structure and the active state model, the H1 helix region was found to locate away from the SAM binding fold in the crystal structure, whereas it forms an extend loop structure that covers the active site in the active state model. Mutagenesis of the H1 helix region resulted in enhancement of basal catalytic activity but reduces the enzyme’s response to potassium concentration, suggesting that the N-terminal region may be involved in the regulation of MpGSMT activity.
Taken together, we resolved the structure of a catalytically inactive state of MpGSMT and discovered that the H1 helix region may play an important regulatory module that not only participates in the betaine-mediated feedback inhibition but also in salt-induced activation.

並列關鍵字

Glycine sarcosine N-methyltransferase ； S-Adenosyl methionine (SAM) ； betaine ； Methanohalophilus portucalensis ； X-ray crystallography ； Dimethylglycine

參考文獻

62 Craig, S. A. Betaine in human nutrition. The American journal of clinical nutrition 80, 539-549 (2004).

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