Drexler, Lukas and Fuertges, Torben F. and Rudack, Till and Sterner, Reinhard (2025) On the Origin of Substrate Specificity of Enzymes from the Amidohydrolase Superfamily. WILEY-V C H VERLAG GMBH, WEINHEIM.
Full text not available from this repository. (Request a copy)Abstract
The sequencing of numerous genomes has led to the identification of open reading frames for millions of enzymes, many of which use unknown substrates. Hence, the identification of both primary and promiscuous activities remains a major challenge for enzyme research. Here, we identified the mechanistic basis of substrate specificity for members of the amidohydrolase superfamily (AHS). Comprehensive analyses of two AHS classes revealed that catalysis proceeds either via 1,4 or 1,6 nucleophilic conjugate addition mediated by a glutamine that is located at two different positions within the active site thereby shaping substrate scope in these enzymes. These different enzymatic properties result in an inverted enantioselectivity for fleeting chiral intermediates, which are transient chiral species on the reaction pathway from an achiral substrate to an achiral product. Moreover, we demonstrated that catalysis focuses on conserved core structures rather than on all moieties of a given substrate, which increases the degree of promiscuity and evolvability in these enzymes. Using site-directed mutagenesis, we showed that an enzyme specialized in a specific nucleophilic conjugate addition can both readily adapt to secondary substrates and accommodate novel substrates by few amino acid exchanges. Hence, our study reveals mechanistic principles that underly substrate specificity, promiscuity, and enantioselectivity.
| Item Type: | Other |
|---|---|
| Uncontrolled Keywords: | 3-DIMENSIONAL STRUCTURE; CATALYTIC MECHANISM; CYTOSINE; DEAMINATION; AMMELINE; AMINOHYDROLASE; DEGRADATION; HYDROLYSIS; EVOLUTION; GUANINE; Amidohydrolase superfamily; Enantioselectivity; Enzyme catalysis; Enzyme promiscuity; Fleeting chiral intermediates |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 26 May 2026 09:38 |
| Last Modified: | 26 May 2026 09:38 |
| URI: | https://pred.uni-regensburg.de/id/eprint/65883 |
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