The haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications.IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols.

Loschmidt Laboratories Research Centre for Toxic Compounds in the Environment Faculty of Science Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Janssen DB, Gerritse J, Brackman J, Kalk C, Jager D, Witholt B. 1988. Purification and characterization of a bacterial dehalogenase with activity toward halogenated alkanes, alcohols and ethers. Eur J Biochem 171:67–72. doi:10.1111/j.1432-1033.1988.tb13759.x. PubMed DOI

Keuning S, Janssen DB, Witholt B. 1985. Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. J Bacteriol 163:635–639. PubMed PMC

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

Koudelakova T, Chovancova E, Brezovsky J, Monincova M, Fortova A, Jarkovsky J, Damborsky J. 2011. Substrate specificity of haloalkane dehalogenases. Biochem J 2410:345–354. doi:10.1042/BJ20101405. 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

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

Nagata Y, Nariya T, Ohtomo R, Fukuda M, Yano K, Takagi M. 1993. Cloning and sequencing of a dehalogenase gene encoding an enzyme with hydrolase activity involved in the degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis. J Bacteriol 175:6403–6410. doi:10.1128/jb.175.20.6403-6410.1993. PubMed DOI PMC

Curragh H, Flynn O, Larkin MJ, Stafford TM, Hamilton JT, Harper DB. 1994. Haloalkane degradation and assimilation by Rhodococcus rhodochrous NCIMB 13064. Microbiology 140:1433–1442. doi:10.1099/00221287-140-6-1433. PubMed DOI

Poelarends GJ, Saunier R, Janssen DB. 2001. trans-3-chloroacrylic acid dehalogenase from Pseudomonas pavonaceae 170 shares structural and mechanistic similarities with 4-oxalocrotonate tautomerase. J Bacteriol 183:4269–4277. doi:10.1128/JB.183.14.4269-4277.2001. PubMed DOI PMC

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

Sato Y, Monincova M, Chaloupkova R, Prokop Z, Ohtsubo Y, Minamisawa K, Tsuda M, Damborsky J, Nagata Y. 2005. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, encode haloalkane dehalogenases with novel structures and substrate specificities. Appl Environ Microbiol 71:4372–4379. doi:10.1128/AEM.71.8.4372-4379.2005. PubMed DOI PMC

Wood DW, Setubal JC, Kaul R, Monks DE, Kitajima JP, Okura VK, Zhou Y, Chen L, Wood GE, Almeida NF Jr, Woo L, Chen Y, Paulsen IT, Eisen JA, Karp PD, Bovee D Sr, Chapman P, Clendenning J, Deatherage G, Gillet W, Grant C, Kutyavin T, Levy R, Li MJ, McClelland E, Palmieri A, Raymond C, Rouse G, Saenphimmachak C, Wu Z, Romero P, Gordon D, Zhang S, Yoo H, Tao Y, Biddle P, Jung M, Krespan W, Perry M, Gordon-Kamm B, Liao L, Kim S, Hendrick C, Zhao ZY, Dolan M, Chumley F, Tingey SV, Tomb JF, Gordon MP, Olson MV, Nester EW. 2001. The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294:2317–2323. doi:10.1126/science.1066804. PubMed DOI

Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE III, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544. doi:10.1038/31159. PubMed DOI

Lee HS, Kwon KK, Kang SG, Cha S-S, Kim S-J, Lee JH. 2010. Approaches for novel enzyme discovery from marine environments. Curr Opin Biotechnol 21:353–357. doi:10.1016/j.copbio.2010.01.015. PubMed DOI

Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D, Paulsen I, Nelson KE, Nelson W, Fouts DE, Levy S, Knap AH, Lomas MW, Nealson K, White O, Peterson J, Hoffman J, Parsons R, Baden-Tillson H, Pfannkoch C, Rogers Y-H, Smith HO. 2004. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304:66–74. doi:10.1126/science.1093857. PubMed DOI

Gehret JJ, Gu L, Geders TW, Brown WC, Gerwick L, Gerwick WH, Sherman DH, Smith JL. 2012. Structure and activity of DmmA, a marine haloalkane dehalogenase. Protein Sci 21:239–248. doi:10.1002/pro.2009. PubMed DOI PMC

Daniel L, Buryska T, Prokop Z, Damborsky J, Brezovsky J. 2015. Mechanism-based discovery of novel substrates of haloalkane dehalogenases using in silico screening. J Chem Inf Model 55:54–62. doi:10.1021/ci500486y. 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

Gschwend PM, Macfarlane JK, Newman KA. 1985. Volatile halogenated organic compounds released to seawater from temperate marine macroalgae. Science 227:1033–1035. doi:10.1126/science.227.4690.1033. PubMed DOI

Chovancova E, Pavelka A, Benes P, Strnad O, Brezovsky J, Kozlikova B, Gora A, Sustr V, Klvana M, Medek P, Biedermannova L, Sochor J, Damborsky J. 2012. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput Biol 8:e1002708. doi:10.1371/journal.pcbi.1002708. PubMed DOI PMC

Stepankova V, Khabiri M, Brezovsky J, Pavelka A, Sykora J, Amaro M, Minofar B, Prokop Z, Hof M, Ettrich R, Chaloupkova R, Damborsky J. 2013. Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents. Chembiochem 14:890–897. doi:10.1002/cbic.201200733. PubMed DOI

Stepankova V, Damborsky J, Chaloupkova R. 2013. Organic co-solvents affect activity, stability and enantioselectivity of haloalkane dehalogenases. Biotechnol J 8:719–729. doi:10.1002/biot.201200378. PubMed DOI

Hesseler M, Bogdanović X, Hidalgo A, Berenguer J, Palm GJ, Hinrichs W, Bornscheuer UT. 2011. Cloning, functional expression, biochemical characterization, and structural analysis of a haloalkane dehalogenase from Plesiocystis pacifica SIR-1. Appl Microbiol Biotechnol 91:1049–1060. doi:10.1007/s00253-011-3328-x. PubMed DOI

Li A, Shao Z. 2014. Biochemical characterization of a haloalkane dehalogenase DadB from Alcanivorax dieselolei B-5. PLoS One 9:e89144. doi:10.1371/journal.pone.0089144. PubMed DOI PMC

Nagata Y, Prokop Z, Sato Y, Jerabek P, Kumar A, Ohtsubo Y, Tsuda M. 2005. Degradation of β-hexachlorocyclohexane by haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26. Appl Environ Microbiol 71:2183–2185. doi:10.1128/AEM.71.4.2183-2185.2005. PubMed DOI PMC

Pavlova M, Klvana M, Prokop Z, Chaloupkova R, Banas P, Otyepka M, Wade RC, Tsuda M, Nagata Y, Damborsky J. 2009. Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate. Nat Chem Biol 5:727–733. doi:10.1038/nchembio.205. PubMed DOI

Micsonai A, Wien F, Kernya L, Lee Y-H, Goto Y, Réfrégiers M, Kardos J. 2015. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci U S A 112:E3095–E3103. doi:10.1073/pnas.1500851112. PubMed DOI PMC

Britton HTS, Robinson RA. 1931. Universal buffer solutions and the dissociation constant of veronal. J Chem Soc 0:1456–1462. doi:10.1039/JR9310001456. DOI

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

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

Rakels JLL, Straathof AJJ, Heijnen JJ. 1993. A simple method to determine the enantiomeric ratio in enantioselective biocatalysis. Enzyme Microb Technol 15:1051–1056. doi:10.1016/0141-0229(93)90053-5. PubMed DOI

Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family

Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique (S)-Enantiopreference, and High Thermostability

Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation