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

卤丙烷脱卤酶（HHDHs）是短链脱氢酶/还原酶（SDR）超家族的成员，虽然催化的化学反应非常不同，但与SDR酶在结构和机理上有许多相同的特征。我们最近发现了hhdh特异性序列基序映射到hhdh的亲核结合袋（motif 1, T-X4-F/Y-X-G）和催化三联体（motif 2, S-X12-Y-X3-R），这使得它们能够快速可靠地与SDR酶区分。使用基于这些基序的简单数据库挖掘协议，我们已经将识别的HHDH酶的数量从少数几个增加到现在的70多个高度多样化的酶，其中一些酶还显示出新的特性和功能。结合系统发育分析，在序列数据库中进行常规BLASTP搜索时，获得了63个与已知HHDHs高度同源的序列，但序列基序1和2存在明显差异。因此，该项目的总体目标是阐明这些自然发生的基序变化对酶的功能，性能和结构的影响。一方面，我们将根据编码蛋白与已知HHDHs的显著同源性来研究其潜在的HHDH活性。我们假设至少其中一些携带基序变异的序列仍将编码功能性的HHDH酶。此外，我们假设基序1的序列变化，映射到HHDHs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突变体的结构和功能比较，我们旨在更好地了解hhdh特异性序列motif 1中存在的保守残基的结构和功能作用。此外，我们的目标是更详细地研究那些不显示HHDH活性的HHDH同源物。在这里，特别是具有催化酪氨酸交换（基序2）而携带完整基序1的蛋白质将引起高度关注。虽然明显缺乏HHDH活性，但我们假设这些蛋白仍然能够结合HHDH典型底物。基于结合研究，基因组背景的计算机分析以及结构调查，我们的目标是阐明这些HHDH同源物的潜在生理功能。总体而言，该项目促进了对自然发生的HHDH同源物功能多样性的基本理解，这些同源物具有序列基序变化。在这方面，本项目进一步旨在赋予SDR超家族中迄今尚未确定的成员功能。

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.