The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis genes. The properties of the enzymatic activities under pressure conditions indicated that the central region, which contained the active center and the dimer forming domains, was shown to be the most important region for pressure tolerance in the deep-sea enzyme

HAMAJIMA, Yuki

IMAI, Takeo

KATO, Chiaki

KATO-YAMADA, Yasuyuki

NAGAE, Takayuki

WATANABE, Nobuhisa

今井, 竹夫

加藤, 千明

山田, 康之

永江, 峰幸

渡邉, 信久

濱島, 裕輝

JAMSTEC Repository

1 Running title: Pressure Effects on the Shewanella Chimeric IPMDH 1  2 Pressure Effects on the Chimeric 3-Isopropylmalate Dehydrogenases of the Deep-Sea 3 Piezophilic Shewanella benthica and the Atmospheric Pressure Adapted Shewanella 4 oneidensis 5  6 Yuki HAMAJIMA,1,2 Takayuki NAGAE,3 Nobuhisa WATANABE,3,4 Yasuyuki 7 KATO-YAMADA,1 Takeo IMAI,1 and Chiaki KATO2† 8  9 1Department of Life Science, Rikkyo University, 3-34-1 Nishi-ikebukuro, Toshima-ku, 10 Tokyo 171-8501, Japan 11 2Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, 12 2-15 Natsushima-cho, Yokosuka 237-0061, Japan 13 3Department of Biotechnology, Graduate School of Engineering, Nagoya University, 14 Furo-cho, Chikusa-Ku, Nagoya 464-8601, Japan 15 4Synchrotron Radiation Research Center, Nagoya University, Furo-cho, Chikusa-Ku, 16 Nagoya 464-8601, Japan 17  18 Received: October 4, 2013; Accepted: December 2, 2013 19  20 †To whom correspondence should be addressed.  21 Fax: 046-867-9525, E-mail: kato_chi@jamstec.go.jp   22 2   The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the 23 deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis 24 genes. The properties of the enzymatic activities under pressure conditions indicated 25 that the central region, which contained the active center and the dimer forming 26 domains, was shown to be the most important region for pressure tolerance in the 27 deep-sea enzyme.  28  29 Key words: chimeric enzyme; high pressure; isopropylmalate dehydrogenase; 30 piezophilic bacteria; Shewanella  31  32   We have isolated a number of piezophilic bacteria from the deep-sea environment in 33 the past twenty years using the manned and unmanned submersibles, Shinkai 6500 and 34 Kaiko systems operated by the Japan Agency for Marine-Earth Science and Technology 35 (JAMSTEC).1) Shewanella benthica DB21MT-2 is one of the isolates from the deepest 36 Mariana Trench Challenger Deep at a depth of 11,000 m and shown to possess an 37 extremely piezophilic growth profile.2) Enzymatic activities for 3-isopropylmalate 38 dehydrogenase (IPMDH) from S. benthica DB21MT-2 were relatively high at higher 39 pressure conditions compared with the IPMDH from S. oneidensis MR-1,3,4) which was 40 isolated from the Oneida Lake at a depth of 6.4 m.5) Other enzymes, eg. dihydrofolate 41 reductase (DHFR), were also studied relative to the pressure effects on their activities 42 for comparison between the enzymes from deep-sea piezophilic strains and atmospheric 43 adapted strains. Ohmae et al. reported that the DHFR of piezophilic S. violacea showed 44 3 enhanced activities up to 100 MPa, and almost the same level of activity at atmospheric 45 pressure (0.1 MPa) remained under 250 MPa conditions. However, the activities of 46 atmospheric adapted Escherichia coli’s DHFR were remarkably decreased at the 47 elevated pressure conditions.6) These observations indicated that the enzymes from 48 piezophilic strains could be more pressure tolerant than the enzymes from atmospheric 49 adapted strains, and they might have special structures for adapting to such 50 high-pressure environments for survival at the bottom of the deep-sea. Structural 51 analyses of the Shewanella IPMDHs from S. benthica DB21MT-2 (SbMT2_IPMDH) 52 and S. oneidensis MR-1 (SoMR1_IPMDH) were also performed and both enzymes 53 were almost identical. However, the deep-sea enzyme (SbMT2_IPMDH) had a 54 somewhat larger internal cavity volume than the SoMR1_IPMDH at ambient 55 atmospheric pressure.7) To elucidate the molecular mechanisms of pressure adaptation 56 in piezophilic proteins, we selected those Shewanella IPMDHs as model enzymes.  In 57 this study, we describe the construction of non His-tagged chimeric enzymes from those 58 IPMDHs and their activities under elevated pressure conditions in order to determine 59 which regions are important for pressure adaptation.  60   The leuB genes encoding the IPMDH proteins of S. benthica DB21MT-2 and S. 61 oneidensis MR-1 were obtained into the vector pUC19.3) From the sequence of both 62 leuB genes, we identified the common restriction endonuclease sites, Eco52I and HaeII 63 in the structural gene sequences (Supplemental Fig. 1). Using these restriction enzymes, 64 the chimeric leuB genes from SbMT2_leuB and SoMR1_leuB were constructed using 65 various combinations of the N-terminal regions (a.a. 1-65), central regions (a.a. 66-328), 66 4 and C-terminal regions (a.a. 329-364), as shown in Fig. 1. Totally, 8 different chimeric 67 genes including two originals (SbMT2_leuB and SoMR1_leuB) were constructed on the 68 vector pUC19. 69   The constructed plasmids were introduced into E. coli strain HB101 (pro-, leuB-) and 70 the leuB+ transformants were isolated. The transformans were cultivated in LB medium 71 containing ampicillin (50 µg/mL) and IPTG (isopropyl β-D thiogalactopyranoside, 0.5 72 mM) for 18 hours at 30°C with shaking (210 rpm). The chimeric enzymes from those 73 transformants were partially purified by the procedure as described below;  74 The cultivated cells were collected by centrifugation (8,000 rpm for 30 min. at 4°C), 75 and suspended in 10 mM Tris-HCl pH8.0, 1.0 mM EDTA, 1.0 mM β-mercaptoethanol, 76 1.0 mg/mL lysozyme buffer and disrupted by a repeated freeze thawing and sonication 77 procedure. The supernatants were prepared by centrifugation at 15,000 rpm for 30 min., 78 and then ammonium sulfate was added to a final concentration of 50% saturation. The 79 supernatants were collected by centrifugation at 15,000 rpm for 30 min. and ammonium 80 sulfate to be a final concentration of 55~65% saturation was added. The resultant 81 precipitates were collected by centrifugation at 15,000 rpm for 30 min. and solubilized 82 with buffer I (50 mM Tris-HCl pH 8.0, 1.0 mM β-mercaptoethanol) containing 83 ammonium sulfate to be a final concentration of 25%. The crude enzymes were loaded 84 onto a Phenyl Sepharose 6 Fast Flow column (GE Healthcare, Uppsala, Sweden) 85 equilibrated with buffer I containing 25% ammonium sulfate. Proteins absorbed were 86 eluted with a linear gradient of 25% to 0% ammonium sulfate. The active enzyme 87 fractions were pooled, concentrated with an Amicon® Ultra 30K device (Millipore Co., 88 Fig. 1 5 Billerica, MA, USA) and purified using a TSKgel G3000SWXL column (Tosoh Co., 89 Tokyo, Japan) equilibrated with buffer I. The purity of the recombinant enzymes was 90 verified by sodium dodecyl sulfate–12.5% polyacrylamide gel electrophoresis 91 (Supplemental Fig. 2). 92   IPMDH activity was measured in 50 mM Tris-HCl buffer, pH 7.6, containing 300 93 mM KCl, 0.2 mM MnCl2, 0.8 mM NAD+, and 0.4 mM 3- isopropylmalate, by recording 94 the production of NADH at 340 nm at 25℃, modified from the standard method.8) 95 Approximately one unit (NADH µmol/min) of enzyme was used for the assay, and that 96 activity was defined as 100% relative activity. A high-pressure spectrophotometric 97 system (Syn Co., Kyoto, Japan) combined with a Shimadzu spectrophotometer (Model 98 UV1600-PC, Kyoto, Japan) was used to determine the enzyme activity under pressure 99 conditions (0.1 – 300 MPa), following the procedure suggested by the manufacturer. 100   The partially purified chimeric enzymes were assayed under the elevated pressure 101 conditions, and it was clearly shown that the enzymes including the central region of 102 SbMT2_IPMDH were more active under higher-pressure conditions (dotted lines in Fig. 103 2 indicated as the chimeric enzymes, BBB, OBB, BBO and OBO) compared with the 104 enzymes containing the central region of SoMR1_IPMDH (solid lines in Fig. 2 105 indicated as the chimeric enzymes, OOO, BOO, OOB, BOB). However, the chimeric 106 enzymes, OBO and BBO, were shown higher activities than the original deep-sea 107 enzyme, BBB, at up to 200 MPa conditions, and the chimeric enzyme, BOB, was 108 shown more pressure sensitive than the original shallow water enzyme, OOO. Those 109 results indicated that the central region of SbMT2_IPMDH was particularly important 110 Fig. 2 6 for activity under high-pressure conditions, compared with N-terminus and C-terminus 111 regions. The central region (a.a. 66-328) included the active site and the dimer forming 112 interfaces,7,9) thus the structure around the active site or the dimer forming interfaces 113 played an important role in high-pressure habitats. Temperature effects were also 114 measured for those chimeric enzymes. The results indicated that the temperature 115 conditions were not significantly affected in the heat stability and optimal conditions of 116 those enzymes (Supplemental Fig. 3).  117   Our observations concluded that the chimeric enzymes including the central region of 118 the deep-sea SbMT2_IPMDH were particularly affected under high-pressure conditions 119 and more pressure tolerant, but the other regions, N-terminal and C-terminal did not 120 appear to be correlated with their pressure habitats, however C-terminal was less 121 identical than other amino acids sequences between both enzymes (Supplemental Fig. 122 1). The C-terminus regions were expected to be some of the pressure effects according 123 to the predicted 3D structures by the 3D-JIGSAW program (ver. 2.0),10) but the X-ray 124 crystallography of those enzymes indicated that no significant structural changing was 125 identified7). The central regions contained the active sites, Arg97, 107 and 1369) 126 commonly, and there were identified some difference amino acids around the active 127 sites in the 3D structures, Leu106, Ser266 and Ala268 in SoMR1_IPMDH, and Met106, 128 Ala266 and Ile268 in SbMT2_IPMDH, respectively.7) We also found some differences 129 around the dimer forming regions, Ara127, Arg128, Val132 and Ser156 in 130 SoMR1_IPMDH, and Glu127, Lys128, Ile132 and Gln156 in SbMT2_IPMDH, 131 respectively (Supplemental Fig. 1). In the case of thermophilic IPMDH, the 132 7 hydrophobic interactions at the subunit-subunit interface played important roles in 133 protein stability.11,12) Therefore, it is possible that the small structural differences in the 134 central regions between SoMR1_ and SbMT2_IPMDHs, caused by the some amino 135 acids differences indicated before, also play significant adaptation roles in extreme 136 pressure habitats. The structural analysis of both enzymes indicated that 137 SbMT2_IPMDH was in a more open form and has a larger cavity volume than 138 SoMR1_IPMDH under atmospheric pressure conditions.7) This loosely packed structure 139 of the SbMT2_IPMDH might help it to avoid pressure-induced distortion of the native 140 structure and maintain activity at higher pressures. To our knowledge, this is the first 141 observation of the chimeric enzymes from mesophilic and piezophilic enzymes at the 142 elevated pressure conditions. The central regions of this study contain the active site and 143 the dimer forming interfaces, and it is still not clear which is more important for the 144 pressure tolerance. Further experimental studies on the IPMDH pressure adaptation 145 mechanisms involving the 3D-structural analyses under high pressure are now in 146 progress.  147  148 Acknowledgments 149   We thank Prof. H. K. Kuramitsu for assistance in editing the manuscript. This work 150 was partially supported by Grant-in-aid for Exploratory Research (25450121) and the 151 Strategic Research Foundation Grant-aided Project for Private Universities (S1201003) 152 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.  153  154 8 References 155  156 1) Kato C, “Extremophiles Handbook,” eds. Horikoshi K, Antranikian G, Bull A, 157 Robb F and Stetter K, Springer-Verlag, Tokyo, pp. 643-655 (2011). 158 2) Kato C, Li L, Nogi Y, Nakamura Y, Tamaoka J, and Horikoshi K, Appl. Environ. 159 Microbiol., 64, 1510-1513 (1998). 160 3) De Poorter LMI, Suzaki Y, Sato T, Tamegai H, and Kato C, Mar. Biotechnol., 6, 161 S190-194 (2004). 162 4) Kasahara R, Sato T, Tamegai H, and Kato C, Biosci. Biotechnol. Biochem., 73, 163 2541-2543 (2009). 164 5) Venkateswaran K, Moser DP, Dollhopf ME, Lies DP, Saffarini DA, MacGregor BJ, 165 Ringelberg DB, White DC, Nishijima M, Sano H, Burghardt J, Stackebrandt E, and 166 Nealson KH, Int. J. Syst. Bacteriol., 49, 705–724 (1999). 167 6) Ohmae E, Kubota K, Nakasone K, Kato C, and Gekko K, Chem. Lett., 33, 798-799 168 (2004). 169 7) Nagae T, Kato C, and Watanabe N, Acta Cryst., F68, 265-268 (2012). 170 8) Tamakoshi M, Nakano Y, Kakizawa S, Yamagishi A, and Oshima T, Extremophiles, 171 5, 17–22 (2001). 172 9) Nango E, Yamamoto T, Kumasaka T, and Eguchi T, Bioorganic Med. Chem., 17, 173 7789-7794 (2009). 174 10) Kato C, Sato T, Abe F, Ohmae E, Tamegai H, Nakasone K, Siddiqui KS, and 175 Thomas T, “Protein Adaptation in Extremophiles”, eds. Siddiqui KS, and Thomas T, 176 9 Nova Science Publishers, pp. 167-191 (2008). 177 11) Kirino H, Aoki M, Aoshima M, Hayashi Y, Ohba M, Yamagishi A, Wakagi T, and 178 Oshima T, Eur. J. Biochem., 220, 275-281 (1994). 179 12) Akanuma S, Yamagishi A, Tanaka N, and Oshima T, Eur. J. Biochem., 260, 180 499-504 (1999). 181  182  183 Figure legends: 184 Fig. 1. Structures of the Chimeric leuB Genes and their Expressed Protein’s ID. 185  186 Fig. 2. Effects of Pressure on the Chimeric IPMDH Activities.  187 Relative activities were measured in the pressurized cells 2 min after the application of 188 pressure. Symbols; □: BBB, ○: BBO, △: OBB, ▽: OBO, ■: OOO, ●: OOB, ▲: 189 BOO, ▼: BOB. Dotted lines: central region is from SbMT2_IPMDH, solid lines: 190 central region is from SoMR1_IPMDH. 191  192  193 Fig.	1	Eco52I	 HaeII	0.1	 100	 150	 200	 250	 300	50	100	80	60	40	20	0	Pressure (MPa)	Relative activity (%)	Fig.	2	Hamajima et al., supplementary data 1    Supplemental Fig. 1. Amino Acid Sequences of the IPMDH Proteins of S. oneidensis (SoMR1_IPMDH, AE014299) and S. benthica DB21MT-2 (SbMT2_IPMDH, AB506073).  “|” indicates the restriction sites for EcoT52I and HaeII and “○”, “△”, and “◇” indicates the active sites, the different amino acids around the active site in 3D structure, and the different amino acids in dimer forming domain, in both enzymes, respectively.    Hamajima et al., supplementary data 2    Supplemental Fig. 2. SDS-PAGE Profiles of the Purified Chimeric Enzymes.  Lanes, M: Molecular weight marker, 1: BBB, 2: BBO, 3: OBB, 4: OBO, 5: OOO, 6: OOB, 7: BOO, 8: BOB.    1 2 43 65 87M7750342921(KD)Hamajima et al., supplementary data 3                 Supplemental Fig. 3. Temperature Effects on the Chimeric Enzymes’ Activity. (A) Temperature stability of the chimeric IPMDHs. (B) Optimal temperatures on the chimeric enzymes’ activity. Symbols and lines are the same as in Fig. 2.  Heat stabilities of the purified enzymes were measured by incubating the enzymes with buffer I under various temperature conditions (10 – 70°C) for 30 min. and immediately chilling the enzymes in an ice water bath. The aggregated protein was then removed by centrifugation (15,000 rpm for 5 min), and IPMDH activity of the supernatant was measured. Optimal temperatures for the enzyme activities were also measured under 10 to 70°C conditions, particularly each 2°C increment from 50 to 66°C.   Temperature (°C)Relative activity (%)BTemperature (°C)Relative activity (%)A

Pressure Effects on the Chimeric 3-Isopropylmalate Dehydrogenases of the Deep-Sea Piezophilic Shewanella benthica and the Atmospheric Pressure-Adapted Shewanella oneidensis

Pressure Effects on the Chimeric 3-Isopropylmalate Dehydrogenases of the Deep-Sea Piezophilic Shewanella benthica and the Atmospheric Pressure-Adapted Shewanella oneidensis

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