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I02-Macromolecular Crystallography	Light-emitting dehalogenases: reconstruction of multi-functional biocatalysts Radka Chaloupkova , Veronika Liskova , Martin Toul , Klara Markova , Eva Sebestova , Lenka Hernychova , Martin Marek , Gaspar P. Pinto , Daniel Pluskal , Jitka Waterman , Zbyněk Prokop , Jiří Damborský Acs Catalysis DOI: 10.1021/acscatal.9b01031 Diamond Proposal Number(s): [4923] Show Abstract ▼ Abstract: To obtain structural insights into the emergence of biological functions from catalytically promiscuous enzymes, we reconstructed an ancestor of catalytically distinct, but evolutionarily related, haloalkane dehalogenases (EC 3.8.1.5) and Renilla luciferase (EC 1.13.12.5). This ancestor has both hydrolase and monooxygenase activities. Crystal structure revealed the presence of a catalytic pentad conserved in both dehalogenase and luciferase descendants, and a molecular oxygen bound in between two residues typically stabilizing a halogen anion. The differences in the conformational dynamics of the specificity-determining cap domains between the ancestral and descendant enzymes were accessed by molecular dynamics and hydrogen-deuterium exchange mass spectrometry. Stopped-flow analysis revealed that the alkyl-enzyme intermediate formed in the luciferase-catalyzed reaction is trapped by blockage of a hydrolytic reaction step. A single-point mutation (Ala54Pro) adjacent to one of the catalytic residues bestowed hydrolase activity on the modern luciferase by enabling cleavage of this intermediate. In conclusion: a single substitution next to the catalytic pentad may enable the emergence of promiscuous activity at enzyme class-level; and ancestral reconstruction has a clear potential for obtaining multi-functional catalysts.	Apr 2019

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I02-Macromolecular Crystallography

Light-emitting dehalogenases: reconstruction of multi-functional biocatalysts