3Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, United Kingdom
Ancestral sequence reconstruction allows resurrection of ancient enzymes based on the sequences predicted by a phylogenetic analysis. Starting from an alignment of modern sequences, the phylogenetic tree is inferred, and statistical methods are used to predict the most likely ancestral sequences at the internal nodes of the tree. Genes that encode the inferred ancestral sequences can then be synthesized and expressed in cultured cells; allowing the structure, function and biophysical properties of each resurrected protein to be experimentally characterized [1,2]. The main objective of this project is to provide structural insights into catalysis of resurrected ancestor of two functionally distinct enzymes, hydrolytic haloalkane dehalogenase (HLD) LinB from Sphingobium japonicum UT26 [3,4] and luciferase Rluc from Renilla reniformis [5,6], belonging to oxidoreductases. Design, construction and activity testing of the resurrected ancestral enzyme ancHLD-RLuc revealed correct folding, enhanced thermodynamic stability up to 20 °C compared to the present-day enzymes and the most importantly promiscuous hydrolase and oxidoreductase activity. The ancestral enzyme was successfully crystallized by the sitting-drop vapour diffusion method. Two different crystal forms of ancHLD-RLuc were obtained at 292 K in Morpheus, JSCG (Molecular Dimensions, UK) and Wizard (Rigaku Reagents, USA) screens. The crystals size and shape was further optimized by varying the buffer pH and PEG concentration. Diffraction data for optimized crystals of ancHLD-RLuc grown from different crystallization conditions were collected to resolutions ranging from 1.5 to 1.9 Å. The crystals belonged to the orthorhombic space group P21212 and monoclinic space group P1211. Structure of the ancestral enzyme was solved by molecular replacement using the coordinates of RLuc [6]. X-ray structural analysis revealed that ancHLD-RLuc can adopt two different conformations: monomeric conformation, very similar to that of LinB and RLuc monomers, and domain-swapped dimer conformation which has never been detected before for any HLD member or RLuc. Both monomer and dimer conformations of ancHLD-RLuc were identified in solution together with small portion of tetramer, confirming that the domain-swapped dimer is not a crystallization artefact. The structural data, together with kinetic and molecular modelling data, will provide insight into structural basis of dual hydrolase and oxidoreductase activity of resurrected ancestor.