Haloalkane dehalogenases can cleave a carbon-halogen bond in a broad range of halogenated aliphatic compounds. However, a highly conserved catalytic pentad composed of a nucleophile, a catalytic base, a catalytic acid, and two halide-stabilizing residues is required for their catalytic activity. Only a few family members, e.g., DsaA, DmxA, or DmrB, remain catalytically active while employing a single halide-stabilizing residue. Here, we describe a novel haloalkane dehalogenase, DsvA, from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017, possessing one canonical halide-stabilizing tryptophan (W125). At the position of the second halide-stabilizing residue, DsvA contains the phenylalanine F165, which cannot stabilize the halogen anion released during the enzymatic reaction by a hydrogen bond. Based on the sequence and structural alignments, we identified a putative second halide-stabilizing tryptophan (W162) located on the same α-helix as F165, but on the opposite side of the active site. The potential involvement of this residue in DsvA catalysis was investigated by the construction and biochemical characterization of the three variants, DsvA01 (F165W), DsvA02 (W162F), and DsvA03 (W162F and F165W). Interestingly, DsvA exhibits a preference for the (S)- over the (R)-enantiomers of β-bromoalkanes, which has not been reported before for any characterized haloalkane dehalogenase. Moreover, DsvA shows remarkable operational stability at elevated temperatures. The present study illustrates that protein sequences possessing an unconventional composition of catalytic residues represent a valuable source of novel biocatalysts.IMPORTANCE The present study describes a novel haloalkane dehalogenase, DsvA, originating from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017. We report its high thermostability, remarkable operational stability at high temperatures, and an (S)-enantiopreference, which makes this enzyme an attractive biocatalyst for practical applications. Sequence analysis revealed that DsvA possesses an unusual composition of halide-stabilizing tryptophan residues in its active site. We constructed and biochemically characterized two single point mutants and one double point mutant and identified the noncanonical halide-stabilizing residue. Our study underlines the importance of searching for noncanonical catalytic residues in protein sequences.

International Clinical Research Center St Anne's Universty Hospital Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Masaryk University Brno Czech Republic

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Janssen DB. 2004. Evolving haloalkane dehalogenases. Curr Opin Chem Biol 8:150–159. doi:10.1016/j.cbpa.2004.02.012. PubMed DOI

Damborsky J, Rorije E, Jesenska A, Nagata Y, Klopman G, Peijnenburg WJ. 2001. Structure-specificity relationship for haloalkane dehaloganases. Environ Toxicol Chem 20:2681–2689. doi:10.1002/etc.5620201205. PubMed DOI

Verschueren KHG, Seljée F, Rozeboom HJ, Kalk KH, Dijkstra BW. 1993. Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase. Nature 363:693–698. doi:10.1038/363693a0. PubMed DOI

Ollis DL, Cheah E, Cygler M, Dijkstra BW, Frolow F, Franken SM, Harel M, Remington SJ, Silman I, Schrag J, Sussman JL, Verschueren KHG, Goldman A. 1992. The alpha/beta hydrolase fold. Protein Eng 5:197–211. doi:10.1093/protein/5.3.197. PubMed DOI

Nardini M, Dijkstra BW. 1999. Alpha/beta hydrolase fold enzymes: the family keeps growing. Curr Opin Struct Biol 9:732–737. doi:10.1016/S0959-440X(99)00037-8. PubMed DOI

Holmquist M. 2000. Alpha/beta-hydrolase fold enzymes: structures, functions and mechanisms. Curr Protein Pept Sci 1:209–235. doi:10.2174/1389203003381405. PubMed DOI

Chovancova E, Kosinski J, Bujnicki MJ, Damborsky J. 2007. Phylogenetic analysis of haloalkane dehalogenases. Proteins 67:305–316. doi:10.1002/prot.21313. PubMed DOI

Bohac M, Nagata Y, Prokop Z, Prokop M, Monincova M, Tsuda M, Koca J, Damborsky J. 2002. Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis. Biochemistry 41:14272–14280. doi:10.1021/bi026427v. PubMed DOI

Verschueren KH, Kingma J, Rozeboom HJ, Kalk KH, Janssen DB, Dijkstra BW. 1993. Crystallographic and fluorescence studies of the interaction of haloalkane dehalogenase with halide ions. Studies with halide compounds reveal a halide binding site in the active site. Biochemistry 32:9031–9037. doi:10.1021/bi00086a008. PubMed DOI

Schindler JF, Naranjo PA, Honaberger DA, Chang CH, Brainard JR, Vanderberg LA, Unkefer CJ. 1999. Haloalkane dehalogenases: steady-state kinetics and halide inhibition. Biochemistry 38:5772–5778. doi:10.1021/bi982853y. PubMed DOI

Krooshof GH, Ridder IS, Tepper AW, Vos GJ, Rozeboom HJ, Kalk KH, Dijkstra BW, Janssen DB. 1998. Kinetic analysis and X-ray structure of haloalkane dehalogenase with a modified halide-binding site. Biochemistry 37:15013–15023. doi:10.1021/bi9815187. PubMed DOI

Schanstra JP, Kingma J, Janssen DB. 1996. Specificity and kinetics of haloalkane dehalogenase. J Biol Chem 271:14747–14753. doi:10.1074/jbc.271.25.14747. PubMed DOI

Pikkemaat MG, Ridder IS, Rozeboom HJ, Kalk KH, Dijkstra BW, Janssen DB. 1999. Crystallographic and kinetic evidence of a collision complex formed during halide import in haloalkane dehalogenase. Biochemistry 38:12052–12061. doi:10.1021/bi990849w. PubMed DOI

Prokop Z, Monincova M, Chaloupkova R, Klvana M, Nagata Y, Janssen DB, Damborsky J. 2003. Catalytic mechanism of the haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26. J Biol Chem 278:45094–45100. doi:10.1074/jbc.M307056200. PubMed DOI

Kennes C, Pries F, Krooshof GH, Bokma E, Kingma J, Janssen DB. 1995. Replacement of tryptophan residues in haloalkane dehalogenase reduces halide binding and catalytic activity. Eur J Biochem 228:403–407. doi:10.1111/j.1432-1033.1995.0403n.x. PubMed DOI

Carlucci L, Zhou E, Malashkevich VN, Almo SC, Mundorff EC. 2016. Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea. Protein Sci 25:877–886. doi:10.1002/pro.2895. PubMed DOI PMC

Chrast L, Tratsiak K, Planas-Iglesias J, Daniel L, Prudnikova T, Brezovsky J, Bednar D, Kuta Smatanova I, Chaloupkova R, Damborsky J. 2019. Deciphering the structural basis of high thermostability of dehalogenase from psychrophilic bacterium Marinobacter sp. ELB17. Microorganisms 7:498. doi:10.3390/microorganisms7110498. PubMed DOI PMC

Fung HK, Gadd MS, Drury TA, Cheung S, Guss JM, Coleman NV, Matthews JM. 2015. Biochemical and biophysical characterisation of haloalkane dehalogenases DmrA and DmrB in Mycobacterium strain JS60 and their role in growth on haloalkanes. Mol Microbiol 97:439–453. doi:10.1111/mmi.13039. PubMed DOI

Pati A, Sikorski J, Nolan M, Lapidus A, Copeland A, Glavina Del Rio T, Lucas S, Chen F, Tice H, Pitluck S, Cheng J-F, Chertkov O, Brettin T, Han C, Detter JC, Kuske C, Bruce D, Goodwin L, Chain P, D'haeseleer P, Chen A, Palaniappan K, Ivanova N, Mavromatis K, Mikhailova N, Rohde M, Tindall BJ, Göker M, Bristow J, Eisen JA, Markowitz V, Hugenholtz P, Kyrpides NC, Klenk H-P. 2009. Complete genome sequence of Saccharomonospora viridis type strain (P101). Stand Genomic Sci 1:141–149. doi:10.4056/sigs.20263. PubMed DOI PMC

Greiner-Mai E, Korn-Wendisch F, Kutzner HJ. 1988. Taxonomic revision of the genus Saccharomonospora and description of Saccharomonospora glauca sp. nov. Int J Syst Bacteriol 38:398–405. doi:10.1099/00207713-38-4-398. DOI

Sayers EW, Barrett T, Benson DA, Bolton E, Bryant SH, Canese K, Chetvernin V, Church DM, Dicuccio M, Federhen S, Feolo M, Fingerman IM, Geer LY, Helmberg W, Kapustin Y, Krasnov S, Landsman D, Lipman DJ, Lu Z, Madden TL, Madej T, Maglott DR, Marchler-Bauer A, Miller V, Karsch-Mizrachi I, Ostell J, Panchenko A, Phan L, Pruitt KD, Schuler GD, Sequeira E, Sherry ST, Shumway M, Sirotkin K, Slotta D, Souvorov A, Starchenko G, Tatusova TA, Wagner L, Wang Y, Wilbur WJ, Yaschenko E, Ye J. 2012. Database resources of the National Center for Biotechnology Information: update. Nucleic Acids Res 40:D13–D25. doi:10.1093/nar/gkr1184. PubMed DOI PMC

Jesenska A, Monincova M, Koudelakova T, Hasan K, Chaloupkova R, Prokop Z, Geerlof A, Damborsky J. 2009. Biochemical characterization of haloalkane dehalogenases DrbA and DmbC, representatives of a novel subfamily. Appl Environ Microbiol 75:5157–5160. doi:10.1128/AEM.00199-09. PubMed DOI PMC

Christenson JK, Robinson SL, Engel TA, Richman JE, Kim AN, Wackett LP. 2017. OleB from bacterial hydrocarbon biosynthesis is a β-lactone decarboxylase that shares key features with haloalkane dehalogenases. Biochemistry 56:5278–5287. doi:10.1021/acs.biochem.7b00667. PubMed DOI

Drienovska I, Chovancova E, Koudelakova T, Damborsky J, Chaloupkova R. 2012. Biochemical characterization of a novel haloalkane dehalogenase from a cold-adapted bacterium. Appl Environ Microbiol 78:4995–4998. doi:10.1128/AEM.00485-12. PubMed DOI PMC

Kodicek M, Karpenko V. 2000. Physical biochemistry, p 283–294. Academia, Prague.

Koudelakova T, Bidmanova S, Dvorak P, Pavelka A, Chaloupkova R, Prokop Z, Damborsky J. 2013. Haloalkane dehalogenases: biotechnological applications. Biotechnol J 8:32–45. doi:10.1002/biot.201100486. PubMed DOI

Fortova A, Sebestova E, Stepankova V, Koudelakova T, Palkova L, Damborsky J, Chaloupkova R. 2013. DspA from Strongylocentrotus purpuratus: the first biochemically characterized haloalkane dehalogenase of non-microbial origin. Biochimie 95:2091–2096. doi:10.1016/j.biochi.2013.07.025. PubMed DOI

Buryska T, Babkova P, Vavra O, Damborsky J, Prokop Z. 2017. A haloalkane dehalogenase from a marine microbial consortium possessing exceptionally broad substrate specificit. Appl Environ Microbiol 84:e01684-17. doi:10.1128/AEM.01684-17. PubMed DOI PMC

Vanacek P, Sebestova E, Babkova P, Bidmanova S, Daniel L, Dvorak P, Stepankova V, Chaloupkova R, Brezovsky J, Prokop Z, Damborsky J. 2018. Exploration of enzyme diversity by integrating bioinformatics with expression analysis and biochemical characterization. ACS Catal 8:2402–2412. doi:10.1021/acscatal.7b03523. DOI

Koudelakova T, Chovancova E, Brezovsky J, Monincova M, Fortova A, Jarkovsky J, Damborsky J. 2011. Substrate specificity of haloalkane dehalogenases. Biochem J 435:345–354. doi:10.1042/BJ20101405. PubMed DOI

Prokop Z, Sato Y, Brezovsky J, Mozga T, Chaloupkova R, Koudelakova T, Jerabek P, Stepankova V, Natsume R, van Leeuwen JG, Janssen DB, Florian J, Nagata Y, Senda T, Damborsky J. 2010. Enantioselectivity of haloalkane dehalogenases and its modulation by surface loop engineering. Angew Chem Int Ed Engl 49:6111–6115. doi:10.1002/anie.201001753. PubMed DOI

Chaloupkova R, Prokop Z, Sato Y, Nagata Y, Damborsky J. 2011. Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature. FEBS J 278:2728–2738. doi:10.1111/j.1742-4658.2011.08203.x. PubMed DOI

Hasan K, Fortova A, Koudelakova T, Chaloupkova R, Ishitsuka M, Nagata Y, Damborsky J, Prokop Z. 2011. Biochemical characteristics of the novel haloalkane dehalogenase DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. Appl Environ Microbiol 77:1881–1884. doi:10.1128/AEM.02109-10. PubMed DOI PMC

Westerbeek A, Szymański W, Wijma HJ, Marrink SJ, Feringa BL, Janssen DB. 2011. Kinetic resolution of α-bromoamides: experimental and theoretical investigation of highly enantioselective reactions catalyzed by haloalkane dehalogenases. Adv Synth Catal 353:931–944. doi:10.1002/adsc.201000726. DOI

Liskova V, Stepankova V, Bednar D, Brezovsky J, Prokop Z, Chaloupkova R, Damborsky J. 2017. Different structural origins of the enantioselectivity of haloalkane dehalogenases toward linear β-haloalkanes: open-solvated versus occluded-desolvated active sites. Angew Chem Int Ed Engl 56:4719–4723. doi:10.1002/anie.201611193. PubMed DOI

Damborsky J, Kuty M, Nemec M, Koca J. 1997. A molecular modeling study of the catalytic mechanism of haloalkane dehalogenase: 1. Quantum chemical study of the first reaction step. J Chem Inf Comput Sci 37:562–568. doi:10.1021/ci960483j. DOI

Nagata Y, Miyauchi K, Damborsky J, Manova K, Ansorgova A, Takagi M. 1997. Purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26. Appl Environ Microbiol 63:3707–3710. doi:10.1128/AEM.63.9.3707-3710.1997. PubMed DOI PMC

Keuning S, Janssen DB, Witholt B. 1985. Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. J Bacteriol 163:635–639. doi:10.1128/JB.163.2.635-639.1985. PubMed DOI PMC

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. doi:10.1093/nar/25.17.3389. PubMed DOI PMC

Frickey T, Lupas A. 2004. CLANS: a Java application for visualizing protein families based on pairwise similarity. Bioinformatics 20:3702–3704. doi:10.1093/bioinformatics/bth444. PubMed DOI

Edgar RC. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461. doi:10.1093/bioinformatics/btq461. PubMed DOI

Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD, Higgin DG. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. doi:10.1038/msb.2011.75. PubMed DOI PMC

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser (Oxf) 41:95–98.

Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T. 2018. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46:W296–W303. doi:10.1093/nar/gky427. PubMed DOI PMC

Schrodinger, LLC. 2020. The PyMOL molecular graphics system, version 2.0. Schrodinger, LLC, Portland, OR.

Sanchis J, Fernández L, Carballeira JD, Drone J, Gumulya Y, Höbenreich H, Kahakeaw D, Kille S, Lohmer R, Peyralans JJ, Podtetenieff J, Prasad S, Soni P, Taglieber A, Wu S, Zilly FE, Reetz MT. 2008. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates. Appl Microbiol Biotechnol 81:387–397. doi:10.1007/s00253-008-1678-9. PubMed DOI PMC

Warburton M, Omar Ali H, Liong WC, Othusitse AM, Zubir A, Maddock S, Wong TS. 2015. OneClick: a program for designing focused mutagenesis experiments. AIMS Bioeng 2:126–143. doi:10.3934/bioeng.2015.3.126. DOI

GSL Biotech. 2020. SnapGene Viewer 5.0.2. GSL Biotech, Chicago, IL.

Iwasaki I, Utsumi S, Ozawa T. 1952. New colorimetric determination of chloride using mercuric thiocyanate and ferric ion. BCSJ 25:226–226. doi:10.1246/bcsj.25.226. DOI

Lutje Spelberg JH, Rink R, Kellogg RM, Janssen DB. 1998. Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter. Tetrahedron Asymm 9:459–466. doi:10.1016/S0957-4166(98)00003-2. DOI

O'Boyle NM, Banck M, James CA, Morley C, Vandermeersch T, Hutchison GR. 2011. Open Babel: an open chemical toolbox. J Cheminform 3:33. doi:10.1186/1758-2946-3-33. PubMed DOI PMC

Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ. 2009. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791. doi:10.1002/jcc.21256. PubMed DOI PMC

Trott O, Olson AJ. 2010. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461. doi:10.1002/jcc.21334. PubMed DOI PMC

Anandakrishnan R, Aguilar B, Onufriev AV. 2012. H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res 40:W537–W541. doi:10.1093/nar/gks375. PubMed DOI PMC

Stern O, Volmer M. 1919. Über die Abklingungszeit der Fluoreszenz. Phys Zeitschrift 20:183–188.