離胺酸消旋酵素（lysine racemase, Lyr），是負責離胺酸在兩種鏡像異構物互相轉換的酵素。目前Lyr已知有極大的潛力可應用於轉基因植物上，以Lysine為篩選因子，建立新型的非抗生素篩選機制，以俾應對世界上日漸高漲，對於抗生素基因使用於各項轉基因作物的疑慮。在本篇研究中，我們解出了來自Proteus mirabilis BCRC10725的Lyr蛋白質立體結構，這是世界上第一個被解析建立的Lyr結構。除此之外，我們還建立了來自Pseudomonas putida DSM84的廣受質胺基酸消旋酵素（broad-specificity amino acid racemase, Bar）之立體結構，此結構同樣是世界上第一個建立的Bar 蛋白結構。雖然Lyr的基因序列與丙胺酸消旋酵素（alanine racemase, Alr）有極高的相似度，但Lyr卻顯示出對於離胺酸（lysine）具有高度的消旋活性，另外還有較弱的精胺酸（arginine）消旋活性，除此之外對於其他胺基酸就完全不具活性。相對地，Bar除了對離胺酸有最高的活性之外，還表現出了非常顯著的廣受質特性。Bar的分子立體模型是利用SeMet-Bar蛋白質晶體以及multiwavelength anomalous dispersion （MAD）的方法所建構的，而Lyr便以Bar蛋白結構為模版，進行molecular replacement（MR）的方式去建構其分子模型。Lyr和Bar都具有與Alr類似的蛋白結構摺疊特性：在N端區域是由八段（α螺旋/ β平板）所組成的筒狀結構，其中含有輔因子pyridoxal 5’-phosphate（PLP）結合在其中，而C端區域則主要是由β平板所組成的，並且都形成二聚體（dimer）的堆疊模式。在Lyr與Bar的立體結構之活性區域內都發現有PLP的存在，並且都與蛋白質上專一的lysine殘基（在Lyr上是Lys74，Bar上則為Lys75）形成特殊的aldimine linkage而以protonated Schiff base的形式存在。另外，在Lyr與Bar的結構內都可發現到兩個高度保留的殘基（在Lyr上是Lys74, Tyr299’，而在Bar上則為Lys75, Tyr301’）（’代表在dimer中，位於chain B的位置），這兩個殘基是參與消旋化反應並扮演catalytic bases的重要角色。藉由受質結合位置與各反應殘基在結構上的比較與分析，可協助釐清Lyr, Bar與Alr為何在受質專一性方面有如此顯著的差異性。在本篇研究中，發現Lyr和Bar的10號α螺旋上有兩個關鍵胺基酸殘基（在Lyr上是Thr391, Ser395，而在Bar則為Ala393, Tyr396）皆指向活化區域，且對於受質專一性方面扮演很重要的角色。藉由定點突變法的實驗，發現Lyr的雙重點突變蛋白（T391Y-S394Y）會大幅降低對於lysine的消旋化活性，最終相較於野生型Lyr蛋白只殘留6％的活性。另外在Lyr與Bar的立體結構中，還發現到有兩個重要的胺基酸殘基位置（分別是Lyr上的Arg173, Asn174與Bar上的Arg174, Asn175）對於酵素活性方面有顯著的重要性。除此之外，運由電腦分子模擬的方法，我們分別建立了Lyr與Bar含有不同受質的分子模型，並藉此分析判斷Lyr與Bar在受質選擇性方面的機制差異。我們更建立了一個 Bar-lysine 的複合物蛋白質晶體結構，經過立體結構上的比對，發現此晶體複合物結構與電腦模擬所得之 Bar-lysine 複合物模型具有相當高的立體位置相似度，足以證明本篇文章所使用的電腦模擬方法是相當可靠的。整合本篇所得之結果，可以提出一個Lyr和Bar都適用的可逆消旋反應之酵素機制，並找出數個對於受質專一性與酵素活性具有關鍵重要性的殘基位置，對於了解PLP-dependent amino acid racemase酵素反應的分子機制上有很大的助益。最終，本研究對於設計特殊受質專一性之消旋脢的蛋白質工程提供了結構上的理論基礎，可以更有效地提高對於轉基因植物之非抗生素篩選機制的運作效率，加強此新穎的轉基因植物篩選方法之實質應用性。

Lysine racemase, an enzyme that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the crystal structures of a novel lysine racemase (Lyr) from Proteus mirabilis BCRC10725 and a broad-specificity amino acid racemase (Bar) from Pseudomonas putida DSM84. Lyr showed a level of high acitivity towards lysine and weaker activity towards arginine, whereas the lyr gene was found to share highest identity with the putative alanine racemases. In contrast, the Bar protein presented not only the highest activity towards lysine but also remarkably broad substrate specificity. The structural model of Lyr was established by molecular replacement method within the template: Bar structure, which was solved by using the multiwavelength anomalous dispersion (MAD) method of SeMet-Bar crystal. Both structures demonstrate the fold of alanine racemase, which consists of an N-terminal domain with eight-stranded α/β barrels containing the pyridoxal 5’-phosphate (PLP) cofactor and a C-terminal domain primarily composed of β-strands. Both active sites of Lyr and Bar show that the PLP cofactor forms an aldimine linkage to Lys74 and Lys75 in Lyr and Bar, respectively, as a protonated Schiff base. Two conserved residues are presented in both Lyr (Lys74, Tyr299’) and Bar (Lys75, Tyr301’) to serve as the catalytic bases for racemization. Structural comparison and analysis of substrate binding sites and active residues would assist to clarify the mechanism of variant substrate selectivity. Two key residues (Thr391, Ser394 in Lyr and Ala393, Tyr396 in Bar), which are both located at -helix 10 and point toward the active site, are discovered to play an important role in the substrate specificity. The racemization activity toward lysine of the Lyr double-mutant (T391Y-S394Y) reduces to only 6% compared with the wild-type protein. In addition, two important residues are also investigated in both Lyr and Bar (Arg173, Asn174 and Arg174, Asn175, respectively) and are all in charge of the enzyme activity. Moreover, molecular modeling using different substrates with Lyr or Bar structure assists to elucidate the difference of substrate specificity between Lyr and Bar. A Bar-lysine liganded crystal structure is also established here and helps to prove that the docking approach used in this study is dependable according to the structural similarity between docking model and crystal structure of Bar-lysine complex. Together, our results suggest a proposed mechanism for the reversible racemization reaction of both enzymes described here and point out the key residues responsible for the substrate specificity and enzyme activity. This work provides a structural foundation for the design of racemases with pre-determined substrate specificity and assists in improving the reliability of the non-antibiotic selection system for transgenic plants.

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