Affiliation:
1. Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Kamenice 5, Bld. C13 625 00 Brno Czech Republic
2. International Clinical Research Center St. Anne's University Hospital Brno Pekarska 53 656 91 Brno Czech Republic
3. Structure and Instability of Genomes (StrInG) Museum of National Natural History INSERM CNRS Alliance Sorbonne University 43 Rue Cuvier - CP N° 26 75231 Paris Cedex 05 France
Abstract
AbstractEnvironmentally friendly industrial and biotech processes greatly benefit from enzyme‐based technologies. Their use is often possible only when the enzyme‐catalytic mechanism is thoroughly known. Thus, atomic‐level knowledge of a Michaelis enzyme‐substrate complex, revealing molecular details of substrate recognition and catalytic chemistry, is crucial for understanding and then rationally extending or improving enzyme‐catalyzed reactions. However, many known enzymes sample huge protein conformational space, often preventing complete structural characterization by X‐ray crystallography. Moreover, using a cognate substrate is problematic since its conversion into a reaction product in the presence of the enzyme will prevent the capture of the enzyme‐substrate conformation in an activated state. Here, we outlined how to deal with such obstacles, focusing on the recent discovery of a Renilla‐type bioluminescence reaction mechanism facilitated by a combination of engineered ancestral enzyme and the availability of a non‐oxidizable luciferin analogue. The automated ancestral sequence reconstructions using FireProtASR provided a thermostable enzyme suited for structural studies, and a stable luciferin analogue azacoelenterazine provided a structurally cognate chemical incapable of catalyzed oxidation. We suggest that an analogous strategy can be applied to various enzymes with unknown catalytic mechanisms and poor crystallizability.
Funder
Grantová Agentura České Republiky
Technology Agency of the Czech Republic
Subject
Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Catalysis