Evolution of plant secondary metabolism: The promiscuity of short chain dehydrogenases/reductases involved in tropinone reduction

该项目的目标是研究短链脱氢酶/还原酶(SDR)的催化能力和进化能力，并获得有关其固有功能的信息。托品酮还原是由SDR进行的托烷生物碱生物合成的一个步骤。产自十字花科(十字花科)托烷生物碱形成的Cochlearia office inalis的一个托品酮还原酶在拟南芥中有16个同源基因，由于序列同源性为50%，被注释为“可能的托品还原酶”。然而，刺参并不积累托烷生物碱。在其他十字花科基因组中保守的大量托品酮类还原酶基因表明，这些SDR形成了次生产物酶进化的储存库。我们将研究十字花科胡萝卜酮还原酶类SDR形成或不形成托烷生物碱的催化特性。SDR酶模型用于通过在电子药效团中搜索并与已知的十字花科代谢产物进行比较来识别配基。有希望的配体将在体外进行测试。因此，代谢灵活性和催化冗余性被定义，并且可以预期SDR的天然功能的提示。以托烷为底物进行测试，以区分接受托烷的SDR和决定托品酮结合的残基。SDR是突变的，以实现或增强托品酮还原和测试模型指导的体外酶进化。

The goal of the project is to study short-chain dehydrogenases/reductases (SDR) for catalytic capacities and evolvability and to gain information on their native function. Tropinone reduction is a step in tropane alkaloid biosynthesis performed by SDR. A tropinone reductase from tropane alkaloid-forming Cochlearia officinalis (Brassicaceae) has 16 orthologs in A. thaliana annotated as “putative tropine reductase” due to sequence identity of >50%. However, A. thaliana does not accumulate tropane alkaloids. Numerous tropinone reductaselike genes conserved in other Brassicaceae genomes suggest that those SDR form a reservoir for the evolution of secondary product enzymes. We will investigate catalytic characteristics of tropinone reductase-like SDR from Brassicaceae that form or do not form tropane alkaloids. SDR enzyme models serve for ligand identification by in silico pharmacophore search and comparison with known Brassicaceae metabolites. Promising ligands will be tested in vitro. Thus, metabolic flexibility and catalytic redundancy are defined, and hints for the native function of the SDR can be expected. Tropanes are tested as substrates to distinguish tropane-accepting SDR and residues decisive for tropinone binding. SDR are mutated to achieve or enhance tropinone reduction and test model-guided in vitro enzyme evolution.