Enzymatic synthesis of beta-hydroxy-alpha-amino acids based on recombinant D- and L-threonine aldolases
Enzymatic synthesis of beta-hydroxy-alpha-amino acids based on recombinant D- and L-threonine aldolases
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DOI：
10.1021/ja9720422
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发表时间：
1997-12-10
影响因子：
15
通讯作者：
Wong, CH
中科院分区：
文献类型：
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作者：
Kimura, T;Vassilev, VP;Wong, CH
利用酶促法合成β -羟基- α -氨基酸，克隆大肠杆菌l -苏氨酸醛缩酶（LTA; EC 2.1.2.1）和米黄病菌d -苏氨酸醛缩酶（DTA）编码基因，通过引物定向聚合酶链反应在大肠杆菌中过表达。研究了纯化的重组酶的动力学、特异性、稳定性、添加剂要求、温度分布和pH依赖性。DTA需要镁离子作为辅助因子，而LTA不需要金属离子。这些酶在DMSO浓度高达40%的条件下表现良好，并观察到DMSO诱导lta催化反应速率加快。这两种酶都使用吡哆醛磷酸辅酶来激活甘氨酸与各种醛反应。LTA的动力学控制产物为红-羟基- α - l -氨基酸与脂肪醛和与芳香醛的三种异构体。另一方面，DTA与脂肪醛和芳香醛形成三羟基- α - d -氨基酸作为动力学控制产物，但非对映选择性低于LTA。在最佳条件下，利用这些酶在制备尺度上立体选择性地合成了几种对羟基-a-氨基酸衍生物（3-羟基亮氨酸、γ -苄基氧基苏氨酸、γ -苄基氧基甲基苏氨酸和多肟酸）。此外，DTA和磷酸酶的串联使用也使得磷酸d -异丙苏氨酸和d -苏氨酸的合成和分离成为可能。
To exploit the enzymatic method for the synthesis of beta-hydroxy-alpha-amino acids, the genes coding for the Escherichia coli L-threonine aldolase (LTA; EC 2.1.2.1) and Xanthomonus oryzae D-threonine aldolase (DTA) were cloned and overexpressed in E. coli through primer-directed polymerase chain reactions. The purified recombinant enzymes were studied with respect to kinetics, specificity, stability, additive requirement, temperature profile, and pH dependency. DTA requires magnesium ion as a cofactor, while LTA needs no metal ions. These enzymes work well in the presence of DMSO with concentration up to 40%, and DMSO-induced rate acceleration of LTA-catalyzed reaction was observed. Both enzymes use pyridoxal phosphate coenzyme to activate glycine to react with a wide range of aldehydes. LTA gave erythro-beta-hydroxy-alpha-L-amino acids with aliphatic aldehydes and the three isomer with aromatic aldehydes as kinetically controlled products. On the other hand, DTA formed threo-beta-hydroxy-alpha-D-amino acids as kinetically controlled products with aliphatic and aromatic aldehydes but the diastereoselectivity was lower than that of LTA. Under optimal conditions, several P-hydroxy-a-amino acid derivatives (3-hydroxyleucines, gamma-benzyloxythreonines, gamma-benzyloxymethylthreonines, and polyoxamic acids) have been stereoselectively synthesized on preparative scales using these enzymes. Also, the tandem use of DTA and phosphatases has made possible the synthesis and separation of D-allo-threonine phosphate and D-threonine.