Deinococcus radiodurans N-acylamino acid racemase 之結構-功能關係與蛋白質工程

Structure-Function Relationship and Protein Engineering of Deinococcus radiodurans N-acylamino Acid Racemase

邱偉鈞(Wei-Chun Chiu)

指導教授 ： 王雯靜

國立清華大學/生命科學院/生命科學系/博士(2006年)

摘要

N-acylamino acid racemase (NAAAR)為催化N-acylamino acid消旋化作用之酵素，在製藥工業上與aminoacylase共同作用可生產enantiopure α-amino acid，極具工業上之應用價值。 本論文在第一章先介紹NAAAR之應用價值及與enolase superfamily的關連性，第二章即討論以MAD（multiple-wavelength anomalous diffraction）的方式解出此酵素之晶體結構達解析度1.3 Å，對此重要酵素之晶體結構進行詳細之分析，此結構在一個unit cell中為一個結構相當緊密的homooctamer，具有與enolase superfamily相同特徵的bi-domain構造，一為capping domain，一為(β/α)7β barrel domain。 為研究此酵素之催化機制與結構同源性，本論文在第三章中進一步解出NAAAR-Mg2+、NAAAR-NAQ-Mg2+與NAAAR-NAM-Mg2+結構，使吾人能確定Lys170-Asp195-Glu220-Asp245-Lys269為負責催化受質N-acylamino acid 之1,1-proton exchange反應最重要之殘基。 分析受質周邊參與作用之胺基酸，可將受質結合區域分成四個區域：catalytic site (C site)、metal-binding site (M site)、side-chain-binding region (S region)與lid region (L region)。 比較其他enolase superfamily可發現C site與M site具有高度的保守性，為此類酵素最重要之催化平台，負責摘取各種不同受質上的α-proton，推測負責受質辨識的S region與L region則呈現多變的結果，也顯示此種酵素在功能上經過演化而造成多變的特色。 為驗證受質結合位置周邊胺基酸的重要性與功能，第四章即以定點突變的方式製備突變株S142A、K170A、K170R、K269A、K269R、M298A、L299A、 D322N、D322A與Y329A，並測試其生化活性，結果顯示Lys170與Lys269確實為重要催化殘基，經突變後酵素會完全失去活性，而Ser142、Asp322與Tyr329經突變後呈現部分失活或完全喪失活性，在催化時應扮演穩定enolic intermediate的角色，而Met298與Leu299經過突變後可分別維持2 %–10 %的活性，顯示S region對於催化反應的影響應著重於基質的選擇性上。 在NAAAR的蛋白質工程上，為增加其在製藥工業上的價值，第五章即以設計增加inter-及intra-subunit S-S bonds的方式以提高其熱穩定性，並與另一多倍體酵素N-carbamoyl-D-amino acid amidohydrolase互相比較其形成inter-subunit S-S bonds後對酵素之thermostability與thermoactivity的影響有何差異。 以定點突變之方式製備兩株NAAAR之inter-subunit S-S bonds突變株：A68C-D72C與P60C-Y100C，及兩株intra-subunit S-S bonds突變株：E149C-A182C與V265C。晶體結構顯示經過突變之突變株在四級結構上並無太大的改變，但在突變株apo-A68C-D72C的結構中lid loop構造呈現穩定狀態，顯示增加inter-subunit S-S bonds會使得NAAAR結構獲得穩定，並且增加NAAAR的thermostability。相較於NAAAR野生株的酵素活性，突變株A68C-D72C在高溫下維持與野生株相似的kcat/Km比值，而同樣形成inter-subunit S-S bond的D-NCAase突變株A302C在高溫下卻有高於野生株4.2倍的kcat/Km比值，進一步以molecular dynamic simulation確定其原因為增加的inter-subunit S-S bond可有效的降低active sites在高溫下的波動而提高了催化活性。 本論文的最後一個章節中，以增加NAAAR的酵素催化活性為目的，根據前述章節分析，NAAAR的hydrophobic cavity應對其酵素的催化與受質的結合具有重要的影響，吾人即於高度hydrophobic特性之L1 region與L2 region選擇這兩個區域中帶有hydrophilic胺基酸：Thr28、Gln33、Tyr62進行突變，將之突變成phenylalanine，結果發現突變株Q33F之催化活性較野生株提高了四倍（Q33F之kcat/Km比值較野生株高四倍），而突變株Y62F僅可進行L form → D form單方向的消旋化作用。

關鍵字

晶體結構 ； 消旋酶 ； 蛋白質工程 ； 熱穩定性

並列摘要

N-acylamino acid racemase (NAAAR) that catalyzes racemization of N-acylamino acid is valuable to produce enantiopure α-amino acids in couple with an aminoacylase. The pupose of this investigation is to study the structure-function relationship of NAAAR and to increase the industrial value by protein engineering. Chapter 1 describes the significance and background of the thesis. Chapter 2 focused on results and discussion of crystal structural determination, the overall structural descriptions, and structural comparisons. The crystal structure of NAAAR was solved to 1.3 Å using multiwavelength anomalous diffraction (MAD) method. The structure consist of a homooctamer in which each subunit has an architecture characteristic of enolases with a capping domain and a (β/α)7β barrel domain. In chapter 3, we solved the lignaded structures of NAAAR-Mg2+, NAAAR-N-acetyl-L-glutamine-Mg2+ and NAAAR-N-acetyl-D-methionine-Mg2+. Based on these structures, the active site region contains Lys170, Asp195, Glu220, Asp245, Lys269 are identified critical residues: Lys170 and Lys269 are two catalysts to abstract an α-proton of a carboxylate, while Asp195, Glu220, and Asp245 are Mg2+-binding residues. Four subsites for substrate binding are also identified: catalytic site (C), metal-binding site (M), side-chain-binding region (S), and a flexible lid region (L). Structural comparisons of various members of the enolase superfamily reveal a highly conserved catalytic and metal-binding sites among enolases. Moreover, L region and S site involved in the substrate recognition are less conserved, suggesting a consequence of divergence into functionally distinct enzymes. In Chpater 4, variants of NAAAR (S142A, K170A, K170R, K269A, K269R, M298A, L299A, D322N, D322A and Y329A) were prepared by site directed mutagenesis, and then tested their catalytic efficiency to determine the importance for efficient catalysis. Four variants (K170A, K170R, K269A and K269R) had no enzyme activity, confirming the important roles of Lys170 and Lys269 in catalysis. In Chapter 5, we futher investigated the effects of introducing potentially stabilizing S-S bridges in these different multimeric enzymes. Cysteines predicted to form inter- or intra-subunit disulfide bonds were introduced by site-directed mutagenesis. Inter-subunit S-S bonds were formed in two NAAAR variants (A68C-D72C and P60C-Y100C). Intra-subunit S-S bonds were formed in two additional NAAAR variants (E149C-A182C and V265C). Crystal structures of NAAARs variants show limited deviations from the wild-type overall tertiary structure. An apo A68C-D72C subunit differs from the wild-type enzyme, in which it has an ordered lid loop, resembling ligand-bound NAAAR. All mutants with inter-subunit bridges had increases in thermostability. Compared with the wild-type enzyme, A68C-D72C NAAAR showed similar kcat/Km ratios, whereas mutant D-NCAases demonstrated increased kcat/Km ratios at high temperatures (A302C: 4.2 fold at 65 °C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the fine-tuned catalytic site as temperature increases, achieving enhanced activity. In the last chapter, we focused on increasing enzyme activity. According the analysis of prior chapters, the hydrophobic property in the catalytic cavity is very important to enzyme catalyst and substrate binding. We replaced residues of Thr28, Gln33 and Tyr62 by phenylalanine in the L1 and L2 regions that are highly hydrophobic in catalytic cavity. The results showed Q33F mutant was 4-fold more activity than wild-type, and Y62F mutant catalyzed the single way of L form → D form racemization only.

並列關鍵字

crystal structure ； racemase ； protein engineering ； thermostability

參考文獻

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