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

• Applicant: Professorin Dr. Anett Schallmey
• Subject Area: Biochemistry
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 437641034

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.

• DFG Programme: Research Grants