L-KDO 醛縮酶之晶體結構與基質鏡像選擇性

Abstract

藉由噬菌體呈現技術，與L 型糖分子專一性結合的L 型胜肽可被篩選出，其對映的D 型胜肽可利用化學方法合成，並用於標靶細胞表面的天然D 型糖分子。這些D 型胜肽或許可發展為許多疾病的候選新藥，而此策略需要非天然的鏡像L 型糖分子。近期的定向演化研究顯示，大腸桿菌D 型唾液酸醛縮酶可藉由八個點突變，而轉變成L 型KDO 醛縮酶，此酵素比D 型唾液酸醛縮酶更容易選擇L 型糖分子做為基質。在此論文研究中，為了探討基質鏡像選擇性的基礎機制，L 型KDO 醛縮酶和D 型唾液酸醛縮酶的晶體結構已解出，其解析度分別為1.98-Å 和1.47-Å。這兩種酵素皆為四聚體分子，並且每一個單體含有一個(α/β)8 桶狀結構。由結構訊息可見所有點突變皆距離催化中心很遠，除了V251I，其位置很靠近桶狀結構之開口，並且亦靠近可形成Schiff 氏鹼的離胺酸。第251 號的纈胺酸突變成 異白胺酸會造成糖分子鍵結凹槽變狹窄，並且產生更多空間障礙。除此之外，L 型KDO 醛縮酶-羥基丙酮酸(產物類似物)複合物的晶體結構也已解出，此結構有益於更精確地將基質L 型KDO 和D 型唾液酸擺放於L 型KDO 醛縮酶的催化中心。很有趣地，只有L 型KDO 才可以符合此酵素之糖分子鍵結凹槽的狹窄空間。此外，根據已解出的D 型唾液酸醛縮酶-L 型阿拉伯糖複合物之晶體結構，推測在催化L 型KDO 分解的過程中，D 型唾液酸醛縮酶之寬廣的糖分子鍵結凹槽可能會讓產物L 型阿拉伯糖滑落至鄰近的洞穴中，而導致催化活性降低。進一步的酵素活性與結構分析可見，位在第251 號的纈胺酸之單一點突變即足以影響酵素選擇L 型糖分子，此現象和之前結構所觀察到的結果一致。基於上述的實驗結果，可證實KDO 醛縮酶和唾液酸醛縮酶的基質選擇性可受到糖分子鍵結凹槽的大小不同而改變。

L-form peptides that bind specifically to L-sugars can be prepared by phage display. The corresponding D-form peptides which can be synthesized chemically can then be used to target natural D-form sugars on cell-surface. Such D-form peptides may be developed as drug candidates for various diseases. The strategy requires the unnatural, enantiomeric L-sugars. In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase which showed a preferred enantioselectivity toward L-sugars. In this study, the crystal structures of L-KDO aldolase at 1.98-Å resolution and D-sialic acid aldolase at 1.47-Å resolution have been determined in order to investigate the underlying molecular basis of substrate enantioselectivity. Both enzymes are tetramers with each subunit consisting of a (α/β)8 barrel. The structural information indicated that all mutations are away from the catalytic center, except for V251I which is near the opening of the (α/β)8-barrel and is proximal to the Schiff base-forming Lys165. The V→I substitution causes the sugar-binding pocket to become relatively narrow, thus creating a large steric hindrance for the sugar binding. The crystal structure of L-KDO aldolase in complex with hydroxypyruvate (a product analogue) has also been solved to allow the precise docking of the substrates L-KDO and D-sialic acid into the active site. It is interesting that only L-KDO can be properly accommodated in the narrow binding pocket. Moreover, the crystal structure of D-sialic acid aldolase in complex with L-arabinose was determined, which points out that the wide sugar-binding pocket may allow the product L-arabinose to slip into the neighboring cavity during catalysis of the L-KDO cleavage, resulting in a decreased cleavage activity. Further enzymatic and structural analyses indicated that single mutations on V251 are sufficient to invert the enantioselectivity toward L-sugar, in good agreement with the structural observations described above. Based on these results, we propose that the substrate specificity of sialic acid / KDO aldolase can be regulated by changing the size of sugar-binding pocket.