Publication details

Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts

Authors

CHALOUPKOVÁ Radka LIŠKOVÁ Veronika TOUL Martin MARKOVÁ Klára ŠEBESTOVÁ Eva HERNYCHOVÁ Lenka MAREK Martin RANGEL PAMPLONA PIZARRO PINTO José Gaspar PLUSKAL Daniel WATERMAN Jitka PROKOP Zbyněk DAMBORSKÝ Jiří

Type Article in Periodical
Magazine / Source ACS Catalysis
MU Faculty or unit

Faculty of Science

Citation
Web Full Text
Doi http://dx.doi.org/10.1021/acscatal.9b01031
Keywords catalytic promiscuity; ancestral reconstruction; haloalkane dehalogenase; monooxygenase; luciferase; emergence of biological function
Description 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. Its crystal structure solved to 1.39 angstrom resolution 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. Thus, a single substitution next to the catalytic pentad may enable the emergence of promiscuous activity at the enzyme class level, and ancestral reconstruction has a clear potential for obtaining multifunctional catalysts.
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