具有D-氨基酸合成能力的内消旋-二氨基庚二酸脱氢酶的催化机理及蛋白质工程改造

The meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH) is the first meso-DAPDH which has an unusual broad substrate profile. The enzyme catalyzes the asymmetric reductive amination of some 2-keto acids to produce D-amino acids, and shows the highest activity toward pyruvic acid. Recently, the structural studies have shed some insight into the enzyme activity toward pyruvic acid by identifying an additional smaller substrate entrance tunnel, but it is still not clear why StDAPDH has an unusual broad substrate profile. In this project, we are trying to address this question from two different points of view. Firstly, bioinformatics analysis on the protein sequences and structures, and catalytic studies will be performed for the meso-DAPDH enzymes with available sequence in the databases. These meso-DAPDHs will be categorized into two subfamilies according to their substrate profiles. By combining with site-mutagenesis, such studies will provide insights into the subfamily-specific amino acid residues determining protein functions. Secondly, based on the crystal structure of StDAPDH, amino acid virtual mutation and mutant library construction will be carried out with an aim to identify the key amino acid residues, which are critical for the enzyme-substrate interaction and enzyme activity. Finally, with the guidance of the above studies, StDAPDH will be rationally designed and engineered with a series of valuable D-amino acids as synthesis target.

来源于Symbiobacterium thermophilum的内消旋-二氨基庚二酸脱氢酶（StDAPDH）是该家族中首个被发现的具有较宽底物谱的酶，该酶催化多种2-酮酸不对称还原胺化生成相应的D-氨基酸，其中对丙酮酸的活性最好。最近通过其晶体结构解析从底物结合通道的角度解释了该酶对丙酮酸活性较高的原因，但是尚不清楚其具有独特活性的原因。本项目拟从两个角度探索这个问题。首先对基因数据库中该家族酶进行蛋白质序列比对及活性鉴定，进行亚家族分类，通过分析比较并结合定点突变，从进化过程的角度来探索它们一级结构的特征，寻找决定蛋白功能的亚家族特异性位点；其次，在StDAPDH晶体结构的基础上从酶与底物相互作用的角度，通过氨基酸虚拟突变选择目标残基，构建突变体库，探索影响其三级结构、进而影响其催化性质的关键因素。最后，在此基础上以一系列有价值的D-氨基为合成目标，对StDAPDH进行理性设计和改造。

来源于Symbiobacterium thermophilum的内消旋-二氨基庚二酸脱氢酶（StDAPDH）是内消旋-二氨基庚二酸脱氢酶（meso-DAPDH）中第一个已知的、可以催化2-氧代酸发生不对称还原胺化反应生成D-氨基酸的成员。搞清楚这个家族中StDAPDH和其他酶的催化机理对于认识和改造该酶具有十分重要的作用。本项目从基因组数据库中获取了该家族的941条氨基酸序列，随后通过系统进化发育分析及活性分析确定了meso-DAPDH可以分成两种类型。I型对内消旋-二氨基庚二酸（meso-DAP）表现出较高的专一活性，而II型表现出明显的可逆胺化活性，并且具有宽底物谱。其中，StDAPDH属于II型。四级结构分析显示插入/缺失碱基或者四级结构的丢失导致meso-DAPDH家族出现分支。来源于Corynebacterium glutamicum 的meso-DAPDH（CgDAPDH）作为I型中的代表，通过与StDAPDH结构对比，显示它们具有相同的折叠。根据序列和保守性分析，我们发现StDAPDH中的两个氨基酸残基R35和R71在II型中具有较高保守性，同时，也与亚型I明显不同。定点突变实验证实，非活性位点R71可以作为与StDAPDH的底物偏好相关的残基。为了解释其机理，我们利用分子动力学模拟的技术手段进行了分析，结果表明R71位点的突变破坏了其与Y205之间的cation-π相互作用，而这一相互作用仅存在于催化胺化反应时的蛋白构象中。同时，我们将该位点在CgDAPDH中的等位氨基酸A69进行了突变，证实当CgA69突变成StDAPDH中的R之后，对2-酮酸的催化效率均有所提高。这些结果加深了目前对meso-DAPDH家族的理解，而且为用于D-氨基酸生物合成的meso-DAPDH的发现和工程改造提供了坚实基础。