Ancestral sequence reconstruction: understanding shifts in substrate specificity of GMC oxidoreductases
Abstract
Wider research context / theoretical framework. The glucose-methanol-choline (GMC) superfamily of FAD-dependent oxidoreductases comprises a range of oxidases and dehydrogenases acting on various electron donor substrates such as sugars or alcohols. This large superfamily of oxidoreductases is defined by a common fold and by a close phylogenetic relationship, and includes industrially relevant enzymes such as aryl-alcohol oxidases (AAO) and pyranose dehydrogenases (PDH), forming one clade of this superfamily, or glucose oxidases (GOx) glucose dehydrogenases (GDH) constituting another GMC clade. Hypotheses / research questions / objectives. We hypothesis that the AAO/PDH as well as the GOx/GDH clades of GMC oxidoreductases have a common ancestor each, which most probably was a generalist biocatalyst, and that the substrate specificities of these individual enzymes were introduced slowly during evolution. AAOs are mainly active on aromatic alcohols and show pronounced activity with oxygen, while PDHs oxidize various sugars and show negligible activity with oxygen. Both GOx and GDH oxidize glucose, and while the reactivity with oxygen has been tuned up significantly for the former enzyme compared to free FAD, it is almost abolished in GDH. We are interested in structure/function relationships of these enzymes, i.e., how amino acid residues in the direct vicinity of the FAD modulate the reactivity with oxygen (increase or decrease it compared to free FAD), and how the transition from an alcohol-oxidizing to a sugar-oxidizing enzyme proceeded during evolution. Approach / methods. We will use ancestral sequence reconstruction (ASR) to recreate common ancestors at different internal nodes of the AAO/PDH as well as the GOx/GDH phylogenetic trees using GRASP (Graphic Representation of Ancestral Sequence Predictions), which had been developed by our partners from the University of Queensland, Brisbane. We will then study the biochemical and biophysical properties of these ancestral enzymes. Level of originality / innovation. Oxygen reactivity of flavin-dependent enzymes is still poorly understood. It is not clear at present how the polypeptide environment of the isoalloxazine modulates this activity. Several studies have focused on this topic, yet none aimed at understand how oxygen reactivity was introduced into oxidoreductases during evolution. Hence using ASR is a novel approach to study this important topic in flavin enzymology.
Publikationen
Project staff
Dietmar Haltrich
Univ.Prof. Dipl.-Ing. Dr.techn. Dietmar Haltrich
dietmar.haltrich@boku.ac.at
Tel: +43 1 47654-75211
Project Leader
01.06.2020 - 31.12.2024
Georg Schütz
Dipl.-Ing. Dr. Georg Schütz
georg.schuetz@boku.ac.at
Tel: +43 1 47654-35012, 75213
Project Staff
01.06.2020 - 31.12.2024
Leander Sützl
Dipl.-Ing. Dr. Leander Sützl
leander.suetzl@boku.ac.at
Tel: +43 1 47654-75214
Project Staff
01.10.2023 - 30.06.2024