<p> 1 Supplemental Materials for AMB Express 2 1Supplemental Materials for 2Stepwise enhancement of catalytic performance of haloalkane dehalogenase LinB towards -</p><p>3hexachlorocyclohexane 4</p><p>5Ryota Moriuchi,1$ Hiroki Tanaka,1 Yuki Nikawadori,1 Mayuko Ishitsuka,1 Michihiro Ito, 1# Yoshiyuki </p><p>6Ohtsubo, 1 Masataka Tsuda, 1 Jiri Damborsky, 2 Zbynek Prokop,2 and Yuji Nagata1*</p><p>71 Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku </p><p>8University, Sendai 980-8577, Japan, and 2 Loschmidt Laboratories, Department of Experimental </p><p>9Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, </p><p>10Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic</p><p>11$ Present address: The United Graduate School of Agricultural Science, Gifu University 1-1 </p><p>12Yanagido, Gifu 501-1193, Japan.</p><p>13# Present address: Consolidated Research Institute for Advanced Science and Medical Care, Waseda </p><p>14University, 2-2 Wakamatsu-cho, Shinjuku, Tokyo, 162-8480, Japan 15</p><p>16* Address correspondence to Yuji Nagata, Department of Environmental Life Sciences, Graduate </p><p>17School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan. Tel./fax: +81-</p><p>1822-217-5682; e-mail: [email protected]</p><p>19</p><p>3 1 4 Supplemental Materials for AMB Express 5 20 21 (a) LinB (b) M6-1 (LinB T81A) (c) M6-2 (LinB V112A) 22 MI MI MI 23 ! ! ! ) ) ) M M M m m m ( 24 ( (</p><p> n n n o o o i i i t t t a a a r r r t t t n</p><p>25 n n e e e c c c n n n o o o C 26 C C</p><p>27 T i m e ( m i n ) ! T i m e ( m i n ) ! T i m e ( m i n ) ! 28</p><p>29 (d) M6-3 (LinBMI V134I) (e) M6-4 (LinBMI T135A) (f) M6-5 (LinBMI L138I) 30 ! ) ! ! ) ) M m M M</p><p>31 ( m m</p><p>( ( n</p><p> o n n i t o o i i a t t r t a a r r n 32 t t e n n c e e n c c o n n o o C</p><p>33 C C 34 T i m e ( m i n ) ! T i m e ( m i n ) ! T i m e ( m i n ) ! 35 36 (g) M6-6 (LinBMI H247A) (h) M6-7 (LinBMI I253M) 37 ! ! ) 38 ) M M m m ( (</p><p> n n o o i i t 39 t a a r r t t n n e e c c n n</p><p>40 o o C C 41 T i m e ( m i n ) ! T i m e ( m i n ) ! 42 43 44Fig. S1 Degradation of -HCH (closed circle) and appearance of its metabolites, PCHL (closed</p><p>45triangle) and TCDL (open triangle), in reaction mixtures containing LinBMI wild-type (a), and seven</p><p>46point mutants of LinBMI (b-h). Values given are the mean of triplicates. Kinetic data were fitted to 47the irreversible two-step reaction structure of -HCH conversion to TCDL via PCHL (Scheme 1 in 48Materials and Methods) by using GEPASI 3.2 software (Mendes 1997) and shown in solid lines.</p><p>49The specificity constants and their standard errors for both reaction steps (k1 and k2) were obtained 50from the calculation (Table 1). The same data were used that have already been published by Ito et 51al (2007) (panels d and g) and Okai et alF (2013ig. )S (panels1 M a, orib, c,uc e, f,hi and e th) a. l.! 52</p><p>6 2 7 Supplemental Materials for AMB Express 8 53 54 55 56 57 58 59 60 61 62 63 64 65</p><p>9 3 10 Supplemental Materials for AMB Express 11 66References 67Ito M, Prokop Z, Klvana M, Otsubo Y, Tsuda M, Damborsky J, Nagata Y (2007) Degradation of -</p><p>68 hexachlorocyclohexane by haloalkane dehalogenase LinB from -hexachlorocyclohexane-</p><p>69 utilizing bacterium Sphingobium sp. MI1205. Arch Microbiol 188:313-25 </p><p>70Mendes P (1997) Biochemistry by numbers: simulation of biochemical pathways with Gepasi 3.</p><p>71 Trends Biochem Sci 22:361–363 </p><p>72Okai M, Ohtsuka J, Imai LF, Mase T, Moriuchi R, Tsuda M, Nagata K, Nagata Y, Tanokura M</p><p>73 (2013) Crystal structure and site-directed mutagenesis analyses of haloalkane dehalogenase</p><p>74 LinB from Sphingobium sp. MI1205. J Bacteriol 195:2642-2651 75 76</p><p>77 78 79 80</p><p>12 4</p>