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

PLP 依赖性转氨酶超家族内结构功能关系的研究

• 批准号: 277430376
• 负责人: Professor Dr. Uwe T. Bornscheuer
• 金额: --
• 依托单位: Arbeitsgruppe Biokatalyse
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/277430376?language=en
• 关键词: Investigation; structure; function; relationships; within

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.

在基因组测序时代，精确预测编码蛋白的生理功能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特异性（即III类转氨酶）。然而，底物特异性只能以有限的方式预测。为了进行可靠的注释，需要详细了解结构-功能关系以及在蛋白质序列水平上推导这些因子的能力。PLP依赖性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，我们最近在我们先前的工作中鉴定了20（R）-选择性胺转氨酶（ATA）的平台。最近，我们成功地确定了（R）-ATA的晶体结构，并可以阐明四种以前未表征的PLP依赖性酶的功能，这些酶的结构已经存放在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-功能关系。初步序列分析显示，III类转氨酶中>12种不同酶活性的底物特异性与氨基酸序列模式相关（通过关注活性位点）。通过机器人辅助的生化表征的大量进一步的蛋白质，我们将（i）确定和实验确认的因素，根据我们的假设，确定底物特异性，（ii）发现进一步迄今未知的因素和（iii）确定新的尚未探索的酶活性在这些亚家族。通过实验性诱变研究，将缬氨酸转氨酶转化为（R）-ATA，反之亦然，我们将更深入地了解PLP折叠IV类中的结构-功能关系。为了了解底物结合以及蛋白质的灵活性如何决定催化过程中蛋白质的底物特异性，我们将通过计算机模拟预测突变并通过实验表征相应的酶变体。总之，我们期望更好地理解反应的分子原因和底物特异性，这对于更精确地注释PLP依赖性转氨酶非常有用。

项目成果

Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades

• DOI: 10.1002/cssc.201802580
• 发表时间: 2019-02-21
• 期刊: CHEMSUSCHEM
• 影响因子: 8.4
• 作者: Aumala, Ville;Mollerup, Filip;Master, Emma R.
• 通讯作者: Master, Emma R.

Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases

• DOI: 10.1007/s00253-015-7275-9
• 发表时间: 2016-05-01
• 期刊: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
• 影响因子: 5
• 作者: Steffen-Munsberg, Fabian;Matzel, Philipp;Hoehne, Matthias
• 通讯作者: Hoehne, Matthias

Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae

• DOI: 10.1002/cbic.201700257
• 发表时间: 2017-10-18
• 期刊: CHEMBIOCHEM
• 影响因子: 3.2
• 作者: Matzel, Philipp;Krautschick, Lukas;Hoehne, Matthias
• 通讯作者: Hoehne, Matthias

In Silico Based Engineering Approach to Improve Transaminases for the Conversion of Bulky Substrates

• DOI: 10.1021/acscatal.8b03900
• 发表时间: 2018-10
• 期刊: ACS Catalysis
• 影响因子: 12.9
• 作者: M. Voss;D. Das;M. Genz;Anurag Kumar;N. Kulkarni;J. Kustosz;Pravin Kumar;U. Bornscheuer;M. Höhne

Enzyme Cascade Reactions for the Biosynthesis of Long Chain Aliphatic Amines from Renewable Fatty Acids

• DOI: 10.1002/adsc.201801501
• 发表时间: 2019-03-15
• 期刊: ADVANCED SYNTHESIS & CATALYSIS
• 影响因子: 5.4
• 作者: Lee, Da-Som;Song, Ji-Won;Park, Jin-Byung
• 通讯作者: Park, Jin-Byung