Investigation of the structure function relationships within the superfamilies of PLP-dependent transaminases

在基因组测序时代，对编码蛋白质的生理功能的准确预测是破译生物体代谢性能的重要前提。尽管有现代生物信息学方法，但通常只能预测反应的特异性(即III类转氨酶)。然而，底物的特异性只能以有限的方式进行预测。为了能够进行可靠的注释，需要详细了解结构与功能的关系，以及在蛋白质序列水平上推导这些因子的能力。PLP依赖的酶催化一系列令人印象深刻的反应。在PLP折叠I类和IV类的超家族中，我们最近在之前的工作中发现了一个由20(R)选择性氨基转移酶(ATA)组成的平台。最近，我们成功地测定了AN(R)-ATA的晶体结构，并可以阐明四种先前未描述的PLP依赖酶的功能，其结构已保存在PDB-蛋白质数据库中。在这些研究的基础上，本研究项目的目的是研究属于亚家族III类和PLP折叠IV类转氨酶的酶的结构与功能的关系。初步的序列分析表明，GT；12类转氨酶中不同酶的底物专一性与氨基酸序列模式相关(通过关注活性部位)。通过机器人辅助对更多蛋白质的生化表征，我们将(I)识别并实验确认根据我们的假设决定底物特异性的因素，(Ii)进一步发现迄今未知的因素，以及(Iii)在这些亚家族中鉴定新的但尚未探索的酶活性。通过实验突变研究，通过将Valine转氨酶转移到a(R)-ATA中，反之亦然，我们将对PLP折叠IV类中的结构-功能关系有更深入的了解。为了了解底物结合以及蛋白质的灵活性如何决定蛋白质在催化过程中的底物特异性，我们将通过计算机模拟来预测突变并从实验上表征相应的酶变体。总之，我们希望对反应的分子原因和底物特异性有更好的理解，这对于更准确地解释PLP依赖的转氨酶非常有用。

In the era of genome sequencing the precise prediction of the physiological function of the encoded proteins is an important prerequisite to decipher the metabolic performance of an organism. Despite modern bioinformatic methods, usually only the reaction specificity (i.e. class III transaminase) can be predicted. The substrate specificity however can only be predicted in a limited fashion. To allow for a reliable annotation a detailed understanding of the structure-function relationships is required as well as the ability to derive these factors at the level of protein sequences.PLP-dependent enzymes catalyze an impressive array of reactions. In the superfamilies of the PLP fold classes I and IV we recently identified in our previous work a platform of 20 (R)-selective amine transaminases (ATA). Recently, we succeeded in the determination of the crystal structure of an (R)-ATA and could elucidate the function of four previously uncharacterized PLP-dependent enzymes of which the structures were already deposited in the pdb-protein database. On the basis of these investigations it is the aim of this research project to investigate the structure-function relationships of the enzymes belonging to the subfamily class III and to the PLP fold class IV transaminases. Preliminary sequence analyses revealed that the substrate specificity of >12 different enzyme activities within the class III transaminases correlate with amino acid sequence patterns (by focussing on the active site). By means of a robot-assisted biochemical characterizations of a multitude of further proteins we will (i) identify and experimentally confirm the factors, which according to our hypothesis, determine the substrate specificity, (ii) discover further so far unknown factors and (iii) identify novel yet unexplored enzyme activities in these subfamilies. By transfer of a valine transaminase into a (R)-ATA and vice versa through experimental mutagenesis studies we will gain a much deeper understanding of the structure-function relationship in the PLP fold class IV. In order to understand how substrate binding as well as the flexibility of the protein determines the substrate specifity of the protein during catalysis, we will predict via computer simulations mutations and characterize the corresponding enzyme variants experimentally. Altogether we expect a significanlty better understanding of the molecular reasons for the reaction and substrate specificity, which are very useful for a more precise annotation of PLP-dependent transaminases.