Exploring the functional diversity of naturally occuring halohydrin dehalogenase homologs featuring variations in HHDH-specific sequence motifs

卤代醇脱卤酶（HHDH）是短链脱氢酶/还原酶（SDR）超家族的成员，并且与SDR酶共享若干结构以及机制特征，尽管催化化学上非常不同的反应。我们最近鉴定了HHDH特异性序列基序，其映射到HHDH的亲核结合口袋（基序1，T-X 4-F/Y-X-G）和催化三联体（基序2，S-X12-Y-X3-R），这使得它们能够快速可靠地与SDR酶区分开。使用基于这些基序的简单数据库挖掘协议，我们已经显著增加了识别的HHDH酶的数量，从仅仅少数到现在超过70种高度多样化的酶，其中一些还显示新的特征和功能。已经获得了63个额外的序列，它们与已知的HHDH高度同源，但在序列基序1和2中携带不同的变异。因此，本项目的总体目标是阐明这些天然存在的基序变化对酶功能，性能和结构的影响。一方面，我们将研究编码的蛋白质的潜在HHDH活性的基础上，他们的显着同源性已知的HHDH。我们推测，至少有一些携带基序变异的序列仍将编码功能性HHDH酶。此外，我们假设基序1中的序列变异，映射到HHDH的亲核结合口袋，甚至可能实现新的生物催化功能。通过与已知HHDH的突变体的结构和功能比较，我们的目标是更好地了解HHDH特异性序列基序1中保守残基的结构和功能作用。此外，我们的目标是调查也不显示HHDH活性的HHDH同系物，更详细。在这里，特别是蛋白质，其特征在于仅交换催化酪氨酸（基序2），同时携带完整的基序1，将是高度感兴趣的。虽然明显缺乏HHDH活性，我们假设这些蛋白质仍然能够结合HHDH典型底物。结合研究的基础上，在硅片上分析的基因组背景以及结构的调查，我们的目标是阐明这些HHDH同系物的潜在生理功能。总的来说，该项目推进了对天然存在的HHDH同源物的功能多样性的基本理解，其特征在于序列基序变异。在这方面，该项目的进一步目标是分配功能到SDR超家族迄今为止尚未确定的成员。

Halohydrin dehalogenases (HHDHs) are members of the short-chain dehydrogenase/reductase (SDR) superfamily and share several structural as well as mechanistic features with SDR enzymes albeit catalyzing chemically very different reactions. We recently identified HHDH-specific sequence motifs mapping to the nucleophile-binding pocket (motif 1, T-X4-F/Y-X-G) and catalytic triad (motif 2, S-X12-Y-X3-R) of HHDHs, which enables their fast and reliable discrimination from SDR enzymes. Using a simple database mining protocol based on these motifs, we have significantly increased the number of recognized HHDH enzymes from a mere handful to now more than 70 highly diverse enzymes, of which several display also new features and functionalities.When performing conventional BLASTP searches in sequence databases in combination with phylogenetic analyses, 63 additional sequences have been obtained that are highly homologous to known HHDHs but carry distinct variations in sequence motifs 1 and 2. Hence, the overall aim of this project is to elucidate the impact of those naturally occurring motif variations on enzyme functionality, performance and structure. On the one hand, we will study the encoded proteins for their potential HHDH activity based on their significant homology to known HHDHs. We hypothesize that at least some of those sequences carrying motif variations will still encode functional HHDH enzymes. Moreover, we suppose that sequence variations in motif 1, mapping to the nucleophile binding pocket of HHDHs, might even enable new biocatalytic functionalities. By structural and functional comparison with generated mutants of known HHDHs, we aim to gain a better understanding of the structural and functional roles of conserved residues present in HHDH-specific sequence motif 1. In addition, we aim to investigate also those HHDH homologs that do not display HHDH activity, in more detail. Here, especially proteins featuring only an exchange of the catalytic tyrosine (motif 2) while carrying an intact motif 1, will be of high interest. Though obviously lacking HHDH activity, we hypothesize that these proteins are still able to bind HHDH-typical substrates. Based on binding studies, in silico analyses of the genomic context as well as structural investigations, we aim to shed some light on the potential physiological functions of those HHDH homologs. Overall, this project advances the fundamental understanding of the functional diversity of naturally occurring HHDH homologs featuring sequence motif variations. In this regard, this project further aims to assign function to hitherto uncharacterized members of the SDR superfamily.