Structure-function relationships of ancestral haloalkane dehalogenases

         P. Babková^1,2, M. Marek^1, E. Šebestová^1, J. Brezovský^1,2, R. Chaloupková^1,2,

                                         J. Damborský^1,2


    ^1 Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic
   Compounds in the Environment RECETOX, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech
 Republic.^2 International Clinical Research Center, St. Anne's University Hospital Brno, Pekarska
                    53, 656 91 Brno, Czech Republic. Petra.Babkova01@gmail.com


Ancestral sequence reconstruction represents a powerful approach for empirical testing
structure-function relationships of the evolutionary intermediates of diverse proteins^[1]. We
employed the reconstruction to predict sequences of five ancestral haloalkane dehalogenases (EC
3.8.1.5, HLDs) from the subfamily HLD-II. HLDs are hydrolytic enzymes catalyzing conversion of
halogenated aliphatic hydrocarbons to their corresponding alcohols and halide anions^[2]. The genes
encoding inferred ancestral enzyme sequences were synthesized and expressed in Escherichia coli and
the resurrected ancestral enzymes AncHLD1-5 were experimentally characterized. All enzymes were
correctly folded, revealed significantly enhanced thermodynamic stability (ΔT[m] up to 24 °C) and
higher specific activities with preference for short multi-substituted halogenated substrates
compared to extant HLDs. The multivariate statistical analysis uncovered a shift in the substrate
specificity profiles of AncHLD1 and AncHLD2. The crystallization trials were performed by using
sitting-drop vapour-diffusion method at 23 °C. The crystals of AncHLD2, AncHLD3 and AncHLD5 grown
during the initial screening, were used for X-ray diffraction data collection and a complete data
sets were collected to a resolution of 1.7, 1.3 and 1.8 Å resolution, respectively. Obtained
microcrystals of AncHLD1 and AncHLD4 were optimized by variation of enzyme concentration, pH and
precipitant concentration. The structures of AncHLD2, AncHLD3 and AncHLD5 were solved by the
molecular replacement using the structure of DbjA (PDB ID 3AFI)^[3] as a search model. Similarly to
descendant HLDs, ancestral enzymes consists of two domains, the conserved main domain formed by
eight-stranded β-sheet with β2 lying in an antiparallel orientation with respect to the direction
of the β-sheet surrounded by 6 α-helices. The second variable cap domain consists of five
α-helices. During refinement of the crystal structure of AncHLD2, two chloride anions were detected
in the vicinity of the enzyme active site similarly as in the structure of modern HLD DbeA^[4]. The
CHES molecule was detected in the active site of AncHLD3 and large active site cavity was
identified in the structure of AncHLD5. Comparison of structures of ancestral enzymes with
structures of descendants accompanied by dynamic simulations will provide detailed insight into
their catalytic properties.


[1] Harms, M.J. and Thornton, J.W., Nat. Rev. Genet., 2013, 14, 559-571.

[2] Nagata, Y., et al., Appl. Environ. Microbiol., 1997, 63, 3707-3710.

[3] Prokop, Z., et al., Angew. Chem. Int. Ed. Engl., 2010, 49, 6111-6115.

[4] Chaloupkova, R., et al., Acta Cryst., 2014, D70, 1884-1897.