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Light-emitting dehalogenases: reconstruction of multi-functional biocatalysts

DOI: 10.1021/acscatal.9b01031 DOI Help

Authors: Radka Chaloupkova (Masaryk University) , Veronika Liskova (Masaryk University) , Martin Toul (Masaryk University) , Klara Markova (Masaryk University) , Eva Sebestova (Masaryk University) , Lenka Hernychova (Masaryk Memorial Cancer Institute) , Martin Marek (Masaryk Memorial Cancer Institute) , Gaspar P. Pinto (Masaryk Memorial Cancer Institute) , Daniel Pluskal (Masaryk University) , Jitka Waterman (Diamond Light Source) , Zbyněk Prokop (Masaryk University; St. Anne's University Hospital Brno) , Jiří Damborský (Masaryk University; St. Anne's University Hospital Brno)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: April 2019
Diamond Proposal Number(s): 4923

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.

Journal Keywords: catalytic promiscuity; ancestral reconstruction; haloalkane dehalogenase; monooxygenase; luciferase; emergence of biological function

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I02-Macromolecular Crystallography