Modulation of Enantioselectivity in Haloalkane Dehalogenase DbjA by Engineering of a Surface Loop

J. Brezovsky¹, Z. Prokop¹, Y. Sato^2,3,J. Florian ⁴, T. Mozga¹, R. Chaloupkova¹, T. Koudelakova¹, P. Jerabek¹, R. Natsume³, Y. Nagata², T. Senda⁵ and J. Damborsky¹

¹Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic;

²Department of Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan;

³Biomedicinal Information Research Center, Japan Biological Informatics Consortium, 2-42 Aomi, Koto-ku, Tokyo 135-0064, Japan;

⁴Department of Chemistry, Loyola University Chicago, Chicago, Illinois 60626, USA;

⁵Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, 2-42 Aomi, Koto-ku, Tokyo 135-0064, Japan

Email: jiri@chemi.muni.cz

The conversion of structurally and chemically simple chiral molecule 2‑bromopentane to 2‑pentanol is catalyzed by the novel haloalkane dehalogenase DbjA [1] with high enantioselectivity (E = 145), while other two closely related enzymes, DhaA and LinB, exhibit only low enantioselectivity (E = 7 and E = 16, respectively) with this substrate. The analysis of sequence of these enzymes identified a unique surface loop as possible determinant of this high enantioselectivity. Mutant enzyme DbjAΔ constructed by deletion of this extra loop has significantly lowered enantioselectivity with 2-bromopentane (E = 58). The high enatioselectivity was re-introduced in DbjAΔ+H139A (E = 120), carrying additional single-point mutation in the floppy residue H139.

In this study, we employed computer modeling to study a molecular basis for modulation of 2-bromopentane enantiodiscrimination by engineering of a surface loop. Free energies of binding were calculated using linear response analysis (LRA) [2] and the reactivities were estimated with populations of near attack configurations (NACs) for both enantiomers [3]. The calculations showed preference of (R)-enantiomer over (S)-enantiomer in all DbjA variants which is in correspondence with experimental observations. The calculated preferences arise mainly from the difference in reactivity since (S)-enantiomer occurs in NACs less frequently. The preference is further increased by tighter binding of (R)-enantiomer. Calculated data correctly reproduce observed changes in enantioselectivity in DbjAΔ and DbjAΔ+H139A. Deletion of the loop results in rotation of His139 towards the center of the active site pocket and reduced width of the pocket. Interactions with His139 displace (R)‑enantiomer from its reactive position and lead to significant drop in enantioselectivity of DbjAΔ mutant (NACs = 19.7 %, E_calc = 71 in DbjA versus NACs = 6.4 %, E_calc = 49 in DbjAΔ). Additional mutation of His139Ala reconstitutes width of the active site pocket, reactivity of (R)‑enantiomer and enantioselectivity of DbjAΔ+H139A to its original level (NACs = 24.1 %, E_calc = 79).

In summary, we show that the width of the active site pocket is important for enantioselective discrimination of structurally and chemically simple molecule 2-bromopentane by DbjA, and explain why DhaA and LinB with narrow active site pockets discriminate linear brominated alkanes poorly. Our study further demonstrates that enantioselectivity of enzymes can be modulated by the surface loop engineering which may have important implications for construction of new enantioselective biocatalysts.

[1] Y. Sato, M. Monincová, R. Chaloupková, Z. Prokop, Y. Ohtsubo, K. Minamisawa, M. Tsuda, J. Damborsky, Y. Nagata, Applied and Environmental Microbiology, 71, (2005), 4372-9.

[2] Y.Y. Sham, Z.T. Chu, H. Tao, A. Warshel, Proteins, 39, (2000), 393-407.

[3] S. Hur, K. Kahn, T.C. Bruice, Proceedings of the National Academy of Sciences of the United States of America, 100, (2003), 2215–221