File S1 - Biochemical Characterization of a Haloalkane Dehalogenase DadB from <i>Alcanivorax dieselolei</i> B-5

Figure S1, Multiple sequence alignment of DadA, DadB and structurally described HLDs. The white letters in red background represent identical residues in all HLDs involved in alignment. The red letters in white background indicate similar residues. The secondary structure elements above the sequences come from LinB (1mj5). Residues of catalytic pentad are labeled at the top(▴ indicates the nucleophile residue D, ▪ indicates the catalytic acid residue E of HLD-II members, □ indicates the catalytic acid residue D of HLD-I, • indicates the catalytic base residue H, ♦ indicates the first halide-binding residue W, ★ indicates the second halide-binding residue of HLD-II members, ☆ indicates the second halide-binding residue of HLD-I members. The multiple sequence alignment was conducted by ClustalX2.1[1] and printed by ESPript 2.2[16]. According to this figure, DadA and DadB has typical catalytic pentad of HLD-II members. In DadB, the catalytic pentad includes the nucleophile residue D108, base residue H271, the acid catalytic residue E132, and two halide-binding residues, N37 and W109. So DadB may have dehalogenation activity as identified HLDs with hydrolysis mechanism. Figure S2, Phylogenetic analyses of DadA, DadB and other 18 identified HLDs. Multiple sequence alignment was conducted by MUSCLE[17] and the tree was constructed with Neighbor-Joining method[18] by MEGA 5.05 [19]. Robustness of output trees were estimated by bootstrapping the data 1000 times. This phylogenetic tree is basically the same with the tree of Chovancova (Chovancova et al. 2007). Figure S3, Substrate specificity profile of DadB toward chlorinated (blue), brominated (red), and iodinated (green) substrates. The activities of 4 chlorinated alkenes and the corresponding chlorinated alkanes are indicated in the black box. Figure S4, Activity comparison of DadB with other HLDs. The values, except for DadB, were obtained from the results published by Koudelakova et al. [20]. Two activities greater than 250 nmol·s−1·mg−1 are cut off and labeled with the values. Figure S5, Effect of temperature and pH on the activity of DadB. Both experiments chose 1,3-dibromopropane as substrate and the data are expressed as relative activities. The data in the left picture are determined in 100 mM glycine buffer, pH 8.6 under different temperatures. The data in the right picture are determined at 37°C in different buffers (▴, 100 mM potassium acetate buffers with pH 4.0, 5.0, 5.5 and 6.0; ▪, 100 mM imidazole buffers with pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0; •, 100 mM MOPS buffers with pH 6.0, 6.5, 7.0, 7.5 and 8.0; ♦, 100 mM potassium phosphate buffers with pH 6.0, 6.5, 7.0, 7.5 and 8.0; ▾, 100 mM glycine buffers with pH 8.0, 8.5, 9.0 and 10.0). Figure S6, Secondary structure elements prediction of HLDs. Sequences of DadB and other 7 HLDs with crystal structures [8], [11], [21]–[25] were submitted to PRIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/). And multiple sequence alignment was conducted by ClustalX2.1[1]. The fragments in blue, magenta and yellow background represent the realistic (7 HLDs with solved structures) or predicted (homology modeling of DadB) β-sheets, α- helices and coiled coils respectively. The blue, magenta and yellow letters means they are belong to β-sheets, α- helices and coiled coils according to the Secondary structure elements prediction. Figure S7, Three-dimensional structure model of DadB. The cyan and green elements constitute the cap domain and the main domain, respectively. The yellow, red, magenta, blue represents the halide-binding residues, the nucleophile residue, the acid residue, and the base residue, respectively. Table S1, Halogenated substrates used in the activity determination. Table S2, Oligonucleotides used for gene cloning and vector construction. Table S3, Conditions used in determination of steady-state kinetic constants by gas chromatography. Table S4, Similarity matrix of DadB and other HLDs identified. (DOCX)