Protein engineering studies of thermostable D-amino acid transaminase

我们发现芽孢杆菌YM-1的D-氨基酸氨基转移酶（D-AAT）和大肠杆菌的支链L-氨基酸氨基转移酶（BCAT）催化重表面氢转移。这些酶彼此之间显示出显著的序列同源性，但与所有其他氨基转移酶不具有序列同源性。D-AAT的三维结构表明，D-AAT的催化残基的拓扑位置与AspAT相反，晶体学结果也表明D-AAT的整体折叠与AspAT及其他结构相似的转氨酶的折叠有很大不同。基于这些发现，我们建立了一种简单的测定辅酶C-4'氢转移立体专一性的方法。我们还开发了一种通过D-AAT和2-氧代己酸测定除D-脯氨酸外的常见游离D-氨基酸的通用程序：正亮氨酸的形成表示存在一些D-氨基酸，其身份可以通过处理后敏感氨基酸的相应减少来确定。我们发现，D-AAT是失活的孵育与D-天冬氨酸，D-谷氨酸和D-丙氨酸，最好的底物，和失活是伴随着缓慢释放的cx-羧基这些氨基酸。结合吡哆醛-P的Lys-145不参与失活，因为K145 Q和K145 N突变酶也被失活。我们研究了Leu-201的催化作用，Leu-201是活性位点附近的残基，与结合的吡哆醛-P相互作用。L201 A和L201 W突变体酶表现出异常的动力学行为。这表明Leu-201在D-AAT反应中调节辅因子的功能。

We found that D-amino acid aminotransferase (D-AAT) of Bacillus sp.YM-1 and branched-chain L-amino acid aminotransferase (BCAT) of E.coli catalyze the re-face hydrogen transfer. These enzymes show a significant sequence homology with each other, but does not with all other aminotransferases. The three-dimensional structure of D-AAT demonstrated that the topographical situation of the catalytic residue of D-AAT is opposite to that of AspAT.The result of the crystallography also showed that the overall fold of D-AAT is quite different from those of AspAT and other aminotransferases, whose structures resemble each other. We established a simple method for determination of the stereospecificity of C-4' hydrogen transfer of the coenzyme based on these findings. We developed also a general procedure to determine the common free D-amino acids except D-proline by means of D-AAT and 2-oxohexanoate : the formation of norleucine denotes the presence of some D-amino acid (s) whose identity can be established by a corresponding decrease in the susceptible amino acid (s) after treatment. We found that D-AAT is inactivated by incubation with D-aspartate, D-glutamate and D-alanine, the best substrates, and that the inactivation is accompanied by the slow release of the cx-carboxylg group of these amino acids. Lys-145, which binds pyridoxal-P,is not involved in the inactivation since K145Q and K145N mutanat enzymes are also inactivated. We studied the catalytic role of Leu-201, the residue in the vicinity of the active site to interact with the bound pyridoxal-P.The L201A and L201W mutant enzymes showed anomalous kinetic behavior. These show that Leu-201 regulates the function of cofactor during the reaction of D-AAT.