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Instruments / Technical Area	Publication Details	Published
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
I03-Macromolecular Crystallography	Structures of hyperstable ancestral haloalkane dehalogenases show restricted conformational dynamics Petra Babkova , Zuzana Dunajova , Radka Chaloupkova , Jiri Damborsky , David Bednar , Martin Marek Computational And Structural Biotechnology Journal DOI: 10.1016/j.csbj.2020.06.021 Diamond Proposal Number(s): [11175] Open Access Show Abstract ▼ Abstract: Ancestral sequence reconstruction is a powerful method for inferring ancestors of modern enzymes and for studying structure-function relationships of enzymes. We have previously applied this approach to haloalkane dehalogenases (HLDs) from the subfamily HLD-II and obtained thermodynamically highly stabilized enzymes (ΔTm up to 24°C), showing improved catalytic properties. Here we combined crystallographic structural analysis and computational molecular dynamics simulations to gain insight into the mechanisms by which ancestral HLDs became more robust enzymes with novel catalytic properties. Reconstructed ancestors exhibited similar structure topology as their descendants with the exception of a few loop deviations. Strikingly, molecular dynamics simulations revealed restricted conformational dynamics of ancestral enzymes, which prefer a single state, in contrast to modern enzymes adopting two different conformational states. The restricted dynamics can potentially be linked to their exceptional stabilization. The study provides molecular insights into protein stabilization due to ancestral sequence reconstruction, which is becoming a widely used approach for obtaining robust protein catalysts.	Jun 2020
I03-Macromolecular Crystallography	Computational enzyme stabilization can affect folding energy landscapes and lead to catalytically enhanced domain-swapped dimers Klara Markova , Antonin Kunka , Klaudia Chmelova , Martin Havlasek , Petra Babkova , Sérgio M. Marques , Michal Vasina , Joan Planas-Iglesias , Radka Chaloupkova , David Bednar , Zbynek Prokop , Jiri Damborsky , Martin Marek Acs Catalysis 562, 12864 - 12885 DOI: 10.1021/acscatal.1c03343 Show Abstract ▼ Abstract: The functionality of an enzyme depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (Tm = 73.5 °C; ΔTm > 23 °C) affected the protein folding energy landscape. The introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded soluble domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations might (i) activate cryptic hinge-loop regions and (ii) establish secondary interfaces, where they make extensive noncovalent interactions between the intertwined protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from small-angle X-ray scattering (SAXS) and cross-linking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain swapping occurs exclusively in vivo. Crucially, the swapped-dimers exhibited advantageous catalytic properties such as an increased catalytic rate and elimination of substrate inhibition. These findings provide additional enzyme engineering avenues for next-generation biocatalysts.	Oct 2021

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