X-ray structures of (S)-enantioselective haloalkane dehalogenase DmmarA from Mycobacterium marinum reveal a new mode of homodimerization that is atypical for this enzyme family

K. Šnajdarová1, S. M. Marques1,2, J. Damborský1,2, D. Bednář1,2, M. Marek1,2

1Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, Bld. A13, 625 00 Brno, Czech Republic

2International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic

517805@mail.muni.cz

Haloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Their most important biotechnological applications include (i) biodegradation of pollutants such as 1,2-dichloroethane or 1,2,3-trichloropropane, (ii) decontamination of the warfare agent yperite, (iii) pollutant biosensing and (iv) HaloTag cell imaging [1, 2]. Atomic-level knowledge of both the inner organisation and supramolecular complexation of HLDs is thus crucial to understand their catalytic and non-catalytic functions.

Recently, database mining searches identified a new haloalkane dehalogenase, DmmarA, encoded in the genome of a waterborne pathogenic bacterium Mycobacterium marinum M. In our work, crystallographic structures of this (S)-enantioselective enzyme were determined at 1.6 and 1.85 Å resolution. The structures show a canonical αβα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of catalytic apparatus that exhibits a rare (S)-enantiopreference of this enzyme family. Additionally, the structures reveal a previously unobserved mode of homodimerization, which is predominantly mediated through unique L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering.

Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its atypical enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution. Our findings thus highlight key molecular features distinguishing the DmmarA enzyme from other HLD family members.

1. T. Koudelakova, S. Bidmanova, P. Dvorak, A. Pavelka, R. Chaloupkova, Z. Prokop, J. Damborsky, Biotechnology Journal, 8(1), (2013), 32–45.

2. S. M. Marques, M. Slanska, K. Chmelova, R. Chaloupkova, M. Marek, S. Clark, J. Damborsky, E. T. Kool, D. Bednar, Z. Prokop, JACS Au, 2(6), (2022), 1324–1337.

This work was supported by the Czech Science Foundation (22-09853S).