Structural and biochemical characterization of novel haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94

Tomas Mozga¹, Tatyana Prudnikova², Radka Chaloupkova¹, Tana Koudelakova¹, Pavlina Rezacova^4,5, Yukari Sato⁶, Eva Chovancova¹, Ivana Kuta Smatanova^2,3, Yuji Nagata⁶ and Jiri 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; ²Institute of Physical Biology, University of South Bohemia Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic; ³Institute of Systems Biology and Ecology, Academy of Science of Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic; ⁴Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague, Czech Republic; ⁵Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague, Czech Republic; ⁶Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan

Haloalkane dehalogenases (EC 3.8.1.5; HLDs) are bacterial enzymes that catalyze the hydrolytic conversion of halogenated aliphatic compounds to their corresponding alcohols. These enzymes play a key role in aerobic mineralization of many halogenated compounds that occur as environmental pollutants. HLDs are applicable for bioremediation, decontamination and industrial biocatalysis [1]. Isolation of a new family members is important for evolutionary and mechanistic studies as well as for biotechnological applications.

Three subfamilies, denoted HLD-I, HLD-II and HLD-III, can be recognized within this family [2]. A novel enzyme DbeA belonging to the subfamily HLD-II was isolated from Bradyrhizobium elkani USDA94. This new enzyme is closely related to DbjA from Bradyrhizobium japonicum USDA110 [3], which exhibits a unique insertion in the N-terminus of the cap domain absent in other HLDs. DbeA has 71% identity to DbjA [3], 47% identity to DhaA [4], 41% identity to LinB [5] and 39% identity to DmbA [6].

Proper folding of DbeA was assessed by measurement of CD spectra in far-UV and near-UV spectral regions. Thermal stability of DbeA was evaluated by determination of the melting temperature (T_m = 48.82 ± 0.16°C), which is in the same range as observed with other family members. Gel filtration and native polyacrylamide gel electrophoresis were used for determination of oligomerization state of DbeA. Determined molecular weight of DbeA confirmed dimeric state of DbeA under native conditions. Activity data of HLDs were measured with a set of 30 various substrates. The principal component analysis of the specific activities showed that DbeA is less active than DbjA and LinB, while it has similar activity as DhaA. DbeA posses a unique substrate specificity, the enzyme has the highest activity towards brominated and iodinated compounds from all tested HLDs. DbeA showed high enantioselective conversion of 2-bromopentane, 2-bromohexane and brominated ester of propionic and butyric acid into chiral alcohols. The temperature and pH profiles of DbeA were detected by activity measurement with 1-iodohexane as a substrate. The highest activity of the enzyme was detected at the temperature range 45-55 °C, which is in good agreement with the temperature profiles of other HLDs. Surprisingly, DbeA showed more than one pH optimum with the maximal activity detected at pH conditions 6.0 and 8.5-9.5. Similar pH profile was described only for DmbA, while other HLDs exhibited single pH optimum. Crystallographic analysis of DbeA was initiated to understand the structure-function relationships and evolution of this interesting enzyme.

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