酵母菌第三型－四異戊二烯焦磷酸合成酵素之結構與功能研究

Structural and Functional Studies of Type-III geranylgeranyl Pyrophosphate Synthase from Saccharomyces cerevisiae

張道欣(Tao-Hsin Chang)

指導教授 ： 梁博煌

國立臺灣大學/生命科學院/生化科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01187

摘要

類異戊二烯族 (isoprenoid) 為廣泛分布於自然界中的化合物，由一類異戊二烯轉移酵素 (trans-prenyl transferase) 所催化合成，並且亦是以異戊二烯焦磷酸 (isopentenyl pyrophosphate) 為骨架所構成的聚合物。本研究之酵母菌第三型－四異戊二烯焦磷酸合成酵素 (geranylgeranyl pyrophosphate synthase) 可催化一個異戊二烯焦磷酸與法呢基焦磷酸 (farnesyl pyrophosphate) 反應產生四異戊二烯焦磷酸 (geranylgeranyl pyrophosphate)，此產物是四異戊二烯化 (geranylgeranylated)蛋白質、胡蘿蔔素 (carotenoid)、細胞膜脂質等－這些在生物體內重要分子的前趨物。不過藉由不同的一類異戊二烯轉移酵素，所合成出長度具多樣性的直鏈產物，分別在生物體內亦扮演不同的生理角色，因此此群酵素必須非常精準地調控其直鏈產物的長短。但是酵母菌第三型－四異戊二烯焦磷酸合成酵素不論從胺基酸系列比對，還是以已知結構的法呢基焦磷酸合成酵素為藍圖，皆無法推論出其可能調控直鏈產物的機制，而此即為本論文探討重點之一；此外，近年已有文獻報導以此纇酵素作為治療癌症的重要標的物之一。在本論文中，首度解出此酵素立體結構，為兩個相同單元體 (monomer) 所組成的二聚體 (dimer)，每一個單元體由15個α螺旋 (helix) 並以環狀物 (loop) 所連接構成，其催化中心由兩個DDXXD motif所構成；同時意外發現鎂離子和活性中心兩個重要的胺基酸形成配位鍵，之後藉由動力學與螢光光譜的分析，證實鎂離子在催化反應中的聚合反應(condensation reaction) 扮演重要角色，而似乎不影響受質和酵素的親合。然於過去的研究中，指出合成約10到25碳數的短碳鏈一類異戊二烯轉移酵素，利用位於第一個DDXXD motif前面第四或第五個的芳香族胺基酸來調控其直鏈產物的長短；不過自胺基酸序列的比對中，卻發現第三型－四異戊二烯焦磷酸合成酵素沒有此特徵。因此利用酵素立體結構與定點突變，對調控產物鏈長之反應機制做進一步的探討，由研究結果證實第三型－四異戊二烯焦磷酸合成酵素的產物鏈長由Tyr107和His108這兩個重要殘基來調控。在立體結構的比較中，發現酵母菌第三型－四異戊二烯焦磷酸合成酵素其N端第一個α螺旋和之後的環狀物，其構形與方向和其他一類異戊二烯轉移酵素相異；而兩個單體間界面 (interface) 的高度保留殘基，為不具有極性的胺基酸Met111和也和多數一類異戊二烯轉移酵素利用芳香族胺基酸的π-π交互作用促進二元體的形成機制不同。本論文證實酵素N端的結構和兩個單體間界面共同決定二元體的形成；但為何具有與二元體相同活性區域的單元體，會失去酵素活性? 因此利用定點突變製造與無活性單元體△(1–17)，擁有相同螢光的特質但保留活性的二聚體W15F做進一步的探討，之後再由螢光光譜分析和阻流反應分析儀 (stopped-floe) 來分析兩者之間的差異，自結果可初步推論單元體似乎仍具有與受質的結合能力但無法進行催化反應中的聚合反應；亦自實驗中發現酵母菌第三型－四異戊二烯焦磷酸合成酵素和兩個不同受質－異戊二烯焦磷酸和法呢基焦磷酸－的結合沒有先後順序。最後希望藉由本研究，對酵母菌第三型－四異戊二烯焦磷酸合成酵素在立體結構、反應機制和動力學有進一步的了解。

關鍵字

一類異戊二烯轉移酵素 ； 四異戊二烯焦磷酸合成酵素 ； X光晶體繞射 ； 定點突變

並列摘要

Geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes the condensation reaction of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C20 geranylgeranyl pyrophosphate, which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether linked lipid. For short-chain trans-prenyltransferases synthesizing C10–C25 products, bulky residues generally occupy the 4th or 5th position upstream from the first DDXXD motif to block further elongation of the final products. However, type-III GGPPs in eukaryotes lack any large residue at these positions. In this study, the first structure of homodimeric type-III GGPPs from Saccharomyces cerevisiae has been determined to 1.98-Å resolution. Each subunit is composed of 15 alpha-helices joined by connecting loops and is arranged with alpha-helices around a large central cavity. An elongated hydrophobic crevice surrounded by D, F, G, H, and I alpha-helices contains two DDXXD motifs at the top for substrate binding with one Mg2+ coordinated by Asp75, Asp79, and four water molecules. It is sealed at the bottom with three large residues of Tyr107, Phe108, and His139. Compared to the major product C30 synthesized by mutant H139A, the products generated by mutant Y107A and F108A are predominantly C40 and C30, respectively, suggesting the most important role of Tyr107 in determining the product chain length.
Distinct from other known structures of trans-prenyltransferases, the N-terminal 17 amino acids (9-amino acid helix A and the following loop) of the yeast GGPPs protrude from the helix core into the other subunit and contribute to the tight dimer formation. Deletion of the first 9 or 17 amino acids caused the dissociation of dimer into monomer and these two mutants of △(1–9) and △(1–17) showed a 300-fold decrease and abolished in enzyme activity, respectively. Unlike other trans-prenyltransferases usingπ-πstacking interactions to form dimer, we also identified Met111 residue on the highly conserved helix F in the interface between two subunits of GGPPs to be the essential for dimer formation. Consequently, the replacement Met111 with Glu resulted in a shift form dimer to monomer for the M111E mutant enzyme and about 3.5-fold decrease in catalytic activity. The replacement of Met111 with Phe to create a hydrophobic dimer interface brought the monomeric △(1–9) mutant to a partial tetramer and 15-fold increase in catalytic activity compared with △(1–9). These results suggest the N-terminal helix and the critical residue in the interface region may both contribute to the dimer formation.
To investigate the differences in the catalytic properties between dimer and monomer, the site-directed mutagenesis, fluorescence assay, and stopped-flow experiments were performed. One of two fluorescent Trp resides in GGPPs primary sequence, Trp15, was changed to Phe to create W15F to monitor the fluorescence change of Trp148 during the substrate binding and reaction. The monomeric △(1–17) also containing Trp148 served for comparison. Similar protein intrinsic fluorescence change was observed upon addition of FPP and IPP for the W15F and △(1–17). According to this preliminary study on fluorescence spectrophotometer assay and stopped-flow experiments, the monomer without catalytic activity may lose the performance on the condensation reaction, one step of the catalytic reaction. Based on the fluorescence measurements, GGPPs bind with FPP and IPP in a random binding order. The Mg2+ concentration dependence of the catalytic rate by GGPPs shows that the activity is maximal at [Mg2+] = 5 mM, but drops significantly when [Mg2+] = 50 mM.
In summary, our results provide a thorough understanding of S. cerevisiae type-III GGPPs in its structure, mechanism and kinetics, in terms of product chain length determination, dimer formation, substrate induced protein conformational change, and the role of Mg2+ ion in catalysis.

並列關鍵字

trans-prenyltransferase ； geranylgeranyl pyrophosphate synthase ； X-ray crystallography ； multiple isomorphous replacement ； site-directed mutagenesis

參考文獻

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