Investigation of the molecular determinants of the substrate- and stereoselectivity of dihydroxyacetone- dependent aldolases and transaldolases

二羟基丙酮依赖性醛缩酶和转醛缩酶的底物选择性和立体选择性的分子决定因素的研究

• 批准号: 201065813
• 负责人: Professor Dr. Georg Sprenger, since 8/2014
• 金额: --
• 依托单位: Institut für Mikrobiologie (IMB)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/201065813?language=en
• 关键词: Investigation; molecular; determinants; substrate; stereoselectivity

C-C bonding enzymes, such as aldolases and transaldolases, catalyse the formation of a C-C bond with concomitant control of the stereo configuration at the newly formed stereo centre(s). Aldolases with novel stereo- and substrate selectivities are highly desired for application in biocatalysis, e.g. enantiopure synthesis of pharmaceuticals. Our understanding of the mechanism behind the high stereoselectivity of C-C bonding enzymes is still quite limited. In the proposed project, transaldolase B (TalB) and fructose-6-phosphate aldolase (FSA) of Escherichia coli are used as model systems to gain insights into the molecular determinants of their substrate- and stereoselectivities. TalB transfers a dihydroxyacetone moiety from a ketose donor onto an aldehyde acceptor whereas FSA catalyses an aldol addition using dihydroxyacetone (not DHAP!) as donor. The wealth of structural and mechanistic studies on TalB and FSA allows the prediction of the function of active site residues and the effect of Anne Samland substrate- and stereoselectivity of aldolases their replacements. TalB variants exhibiting a novel stereoselectivity (3S,4S) and broadened donor and acceptor specificity will be generated. Furthermore, TalB will be completely converted to a dihydroxyacetone-dependent aldolase. In parallel, FSA will be converted into a transaldolase and the two trajectories will be compared to get insights into the mechanism determining the different reaction types (lyase and transferase) as well as the evolution of FSA from transaldolases. Innovative screening and selection assays will be developed for screening of mutant libraries. The structural model of the active site will be improved by crystallisation of the new variants and determination of the structures of TalB or FSA in complex with substrates or inhibitors.

C-C键合酶（例如醛缩酶和转醛酶）催化C-C键的形成，同时控制新形成的立体中心的立体构型。具有新颖的立体选择性和底物选择性的醛缩酶在生物催化，例如药物的对映体纯合成中的应用是高度期望的。我们对C-C键合酶高立体选择性背后的机制的理解仍然非常有限。在拟议的项目中，转醛醇酶B（TalB）和果糖-6-磷酸醛缩酶（FSA）的大肠杆菌被用作模型系统，以深入了解其底物和立体选择性的分子决定因素。TalB将二羟基丙酮部分从酮糖供体转移到醛受体上，而FSA使用二羟基丙酮（不是DHAP！）催化羟醛加成。作为捐赠者。对TalB和FSA的丰富结构和机制研究使得可以预测活性位点残基的功能以及Anne Samland底物的影响-以及其替代品醛缩酶的立体选择性。将产生表现出新的立体选择性（3S，4S）和加宽的供体和受体特异性的TalB变体。此外，TalB将完全转化为二羟基丙酮依赖性醛缩酶。同时，FSA将被转化为转醛醇酶，并将比较两种轨迹，以了解决定不同反应类型（裂解酶和转移酶）的机制以及FSA从转醛醇酶的演变。将开发创新的筛选和选择测定用于筛选突变体文库。活性位点的结构模型将通过新变体的结晶和与底物或抑制剂复合的TalB或FSA的结构的测定来改进。

项目成果

Acid–Base Catalyst Discriminates between a Fructose 6‐Phosphate Aldolase and a Transaldolase

• DOI: 10.1002/cctc.201500478
• 发表时间: 2015-10
• 期刊: ChemCatChem
• 影响因子: 4.5
• 作者: L. Stellmacher;T. Sandalova;S. Leptihn;G. Schneider;G. Sprenger;A. K. Samland

Conservation of structure and mechanism within the transaldolase enzyme family

• DOI: 10.1111/j.1742-4658.2011.08467.x
• 发表时间: 2012-03-01
• 期刊: FEBS JOURNAL
• 影响因子: 5.4
• 作者: Samland, Anne K.;Baier, Shiromi;Sandalova, Tatyana
• 通讯作者: Sandalova, Tatyana