The high catalytic efficiency of enzymes under reaction conditions is one of the main goals in biocatalysis. Despite the dramatic progress in the development of more efficient biocatalysts by protein design, the search for natural enzymes with useful properties remains a promising strategy. The pyridoxal 5'-phosphate (PLP)-dependent transaminases represent a group of industrially important enzymes due to their ability to stereoselectively transfer amino groups between diverse substrates; however, the complex mechanism of substrate recognition and conversion makes the design of transaminases a challenging task. Here we report a detailed structural and kinetic study of thermostable transaminase from the bacterium Thermobaculum terrenum (TaTT) using the methods of enzyme kinetics, X-ray crystallography and molecular modeling. TaTT can convert L-branched-chain and L-aromatic amino acids as well as (R)-(+)-1-phenylethylamine at a high rate and with high enantioselectivity. The structures of TaTT in complex with the cofactor pyridoxal 5′-phosphate covalently bound to enzyme and in complex with its reduced form, pyridoxamine 5′-phosphate, were determined at resolutions of 2.19 Å and 1.5 Å, and deposited in the Protein Data Bank as entries 6GKR and 6Q8E, respectively. TaTT is a fold type IV PLP-dependent enzyme. In terms of structural similarity, the enzyme is close to known branched-chain amino acid aminotransferases, but differences in characteristic sequence motifs in the active site were observed in TaTT compared to canonical branched-chain amino acid aminotransferases, which can explain the improved binding of aromatic amino acids and (R)-(+)-1-phenylethylamine. This study has shown for the first time that high substrate specificity towards both various l-amino acids and (R)-primary amines can be implemented within one pyridoxal 5′-phosphate-dependent active site of fold type IV. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further biochemical and bioinformatic studies to understand the sequence-structure-function relationship in these enzymes.

在反应条件下酶的高催化效率是生物催化的主要目标之一。尽管通过蛋白质设计在开发更有效的生物催化剂方面取得了巨大进步，但是寻找具有有用特性的天然酶仍然是一个有前途的策略。吡咯醛5'-磷酸（PLP）依赖性转氨酶代表一组工业上重要的酶，因为它们能够在不同的底物之间立体选择性地转移氨基。然而，底物识别和转化的复杂机制使得转氨酶的设计成为一项艰巨的任务。在这里，我们报告从细菌耐高温转氨酶的详细结构和动力学研究Thermobaculum terrenum（TATT）使用酶动力学，X射线晶体学和分子建模的方法。TaTT可以高速率和高对映选择性地转化L-支链和L-芳族氨基酸以及（R）-（+）-1-苯基乙胺。以2.19Å和1.5Å的分辨率测定TaTT的结构，该结构与共价键合酶的辅因子吡ido醛5'-磷酸络合物及其还原形式的吡ido胺5'-磷酸络合物，并沉积在蛋白质数据库中分别作为条目6GKR和6Q8E。花边是IV型折叠PLP依赖性酶。就结构相似性而言，该酶与已知的支链氨基酸氨基转移酶非常接近，但与典型的支链氨基酸氨基转移酶相比，在TaTT中观察到了活性位点的特征序列基序的差异，这可以解释其结合性的提高。芳香族氨基酸和（R）-（+）-1-苯基乙胺。这项研究首次表明，对各种l-氨基酸和（R伯胺可以在折叠型IV的一个吡ido醛5'-磷酸盐依赖性活性位点内实施。这些结果补充了我们对转氨酶催化多样性的认识，并表明需要进一步的生化和生物信息学研究以了解这些酶的序列-结构-功能关系。