Crystallization of resurrected ancestral enzymes of haloalkane dehalogenase DbjA and DbeA

       P. Babková^1, E. Šebestová^1, J. Brezovský^1,2, J. Damborský^1,2, R. Chaloupková^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 is a powerful approach allowing the resurrection of ancient
enzymes based on sequences predicted by a phylogenetic analysis (Skovgaard et al., 2006). In this
project, the sequences of representative members of haloalkane dehalogenase subfamily II were
selected as targets for prediction of the common ancestor of haloalkane dehalogenase DbjA (Sato et
al., 2007) and DbeA (Chaloupkova et al., 2014), AncHLD1, and additional ancestors corresponding to
the deeper nodes of the branch leading towards the present-day enzymes, AncHLD2-5. The genes
encoding predicted sequences were synthesized, expressed in Escherichia coli and the resurrected
ancestral enzymes AncHLD1-5 were experimentally characterized. The ancestral HLDs exhibited
significantly enhanced thermodynamic stability (ΔT[m] up to 24 °C) and higher specific activities
with a preference for short multi-substituted halogenated substrates compared to extant enzymes.
All crystallization trials were performed by using the sitting-drop vapor-diffusion method at 23
°C. The crystals of AncHLD2, 3 and 5 grew during the initial screening and no further optimization
of the crystallization conditions was necessary. The triangular prism shaped crystals of AncHLD2
with dimensions 0.5 x 0.09 x 0.08 mm grew in condition No. 42 of JCSG consisting of 0.02 M
magnesium chloride, 0.1 M Tris pH 8.5 and 20% (w/v) PEG 8000. The hexagonally shaped crystals of
AncHLD3 with dimensions 0.2 x 0.1 x 0.04 mm appeared in condition No. 16 of the Wizard classic
consisting of 100 mM potassium phosphate/sodium phosphate pH 6.2 and 2.5 M sodium chloride. The
trigonal shaped crystals of AncHLD5 with average dimension 0.11 x 0.05 x 0.31 mm were observed in
condition No. 73 of PEG suite containing 0.2 M magnesium acetate and 20 % (w/v) PEG 3350. These
crystals were used for collection of X-ray diffraction data and complete diffraction data sets were
collected at 1.66, 1.26 and 1.25 Å resolution for AncHLD2, AncHLD3, and AncHLD5, respectively.
Obtained microcrystals of AncHLD1 and AncHLD4 were further optimized by variation of enzyme
concentration, pH and precipitant concentration. Optimized crystals of AncHLD4 appeared within
three days from the drop composed of 9.5 % (w/v) mix of PEG 1000, PEG 3350 and MPD, 0.1 M
MOPS/HEPES-Na pH 7.0. On-going structural analysis of ancestral enzymes will provide insight into
their unique catalytic properties.

References

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Acta Cryst. D70, 1884-1897.

Sato Y., Natsume, J., Tsuda, M., Damborsky, J., Nagata, Y. & Senda, T. (2007) Acta Cryst. F63,
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Skovgaard, M., Kodra, J. T., Gram, D. X., Knudsen, S. M., Madsen, D. & Liberles D. A. (2006). J.
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