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NADP+-dependent isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina

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Abstract

The gene encoding NADP+-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) of a psychrophilic bacterium, Psychromonas marina, was cloned and sequenced. The open reading frame of the gene encoding IDH of P. marina (PmIDH) was 2229 bp in length and corresponded to a polypeptide composed of 742 amino acids. The molecular mass of IDH was calculated as 80,426 Da. The deduced amino acid sequence of PmIDH exhibited high degrees of homology with the monomeric IDH from other bacteria such as Colwellia maris (62% identity) and Azotobacter vinelandii (AvIDH) (64%). His-tagged PmIDH overexpressed in Escherichia coli cells was purified and characterized. The optimum temperature of PmIDH activity was about 35 °C; however, the enzyme lost 74% of the activity after incubation for 10 min at 30 °C, indicating that this enzyme is thermolabile. Chimeric enzymes produced through domain swapping between PmIDH and mesophilic AvIDH were constructed and their optimum temperatures and thermostability were determined. The results suggest that regions 2 and 3, especially region 3, of the two IDHs are involved in their catalytic activities and optimum temperature and thermostability for activity.

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References

  • Aghajari N, Feller G, Gerday C, Haser R (1998) Structures of psychrophilic Alteromonas haloplanctis α-amylase give insights into cold adaptation at molecular level. Structure 6:1503–1516

    Article  CAS  PubMed  Google Scholar 

  • Auman AJ, Breezee JL, Gosink JJ, Kämpfer P, Staley JT (2006) Psychromonas ingrahamii sp. nov., a novel gas vacuolate, psychrophilic bacterium isolated from Arctic polar sea ice. Int J Syst Evol Microbiol 56:1001–1007

    Article  CAS  PubMed  Google Scholar 

  • Bruke WF, Johanson RA, Reeves HC (1974) NADP+-specific isocitrate dehydrogenase of Escherichia coli. II: subunit structure. Biochim Biophys Acta 351:333–340

    Article  Google Scholar 

  • Chung AE, Frauzen JS (1969) Oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase from Azotobacter vinelandii. Isolation and characterization. Biochemistry 8:3175–3184

    CAS  PubMed  Google Scholar 

  • Eguchi H, Wakagi T, Oshima T (1989) A highly stable NADP+-dependent isocitrate dehydrogenase from Thermus thermophiles HB8. Biochim Biophys Acta 990:133–137

    Article  CAS  PubMed  Google Scholar 

  • Eikmanns BJ, Rittmann D, Sham H (1995) Cloning sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J Bacteriol 177:774–782

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fukunaga N, Imagawa S, Sahara T, Ishii A, Suzuki M (1992) Purification and characterization of monomeric isocitrate dehydrogenase with NADP+-specificity from Vibrio parahaemolyticus Y4. J Biochem 112:849–855

    Article  CAS  PubMed  Google Scholar 

  • Gerday C, Aittaleb M, Arpigny JL, Baise E, Chessa JP, Garsoux G, Petrescu I, Feller G (1997) Psychrophilic enzyme: a thermodynamic challenge. Biochm Biophys Acta 1342:119–131

    CAS  Google Scholar 

  • Hayashi T, Matsuzaki W, Takada Y (2014) Characterization of chimeric and mutated isocitrate lyases of a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, and a psychrophilic bacterium, Colwellia maris. Biosci Biotechnol Biochem 78:195–201

    Article  CAS  PubMed  Google Scholar 

  • Howard RL, Becker RR (1970) Isolation and some properties of the triphosphopyridine nucleotide isocitrate dehydrogenase from Bacillus stearothermophilus. J Biochem 245:3186–3194

    CAS  Google Scholar 

  • Ishii A, Imagawa S, Fukunaga N, Sasaki S, Minowa O, Mizuno Y, Shiokawa H (1987) Isozymes of isocitrate dehydrogenase from an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1: purification and modulation of activities by growth conditions. J Biochem 102:1489–1498

    Article  CAS  PubMed  Google Scholar 

  • Ishii A, Suzuki S, Sahara T, Takada Y, Sasaki S, Fukunaga N (1993) Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1. J Bacteriol 175:6873–6880

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jaenicke R (1991) Protein stability and molecular adaptation to extreme conditions. Eur J Biochem 202:715–728

    Article  CAS  PubMed  Google Scholar 

  • Kang C, Sun N, Poland BW, Gorrell A, Honzatko RB, Fromm HJ (1997) Residues essential for catalysis and stability of the active site of Escherichia coli adenylosuccinate synthetase as revealed by directed mutation and kinetics. J Biol Chem 272:11881–11885

    Article  CAS  PubMed  Google Scholar 

  • Kawasaki K, Nogi Y, Hishinuma M, Nodasaka Y, Matsuyama H, Yumoto I (2002) Psychromonas marina sp. Nov., a novel halophilic, facultatively psychrophilic bacterium isolated from the coast of the Okhotsk Sea. Int J Syst Evol Microbiol 52:1455–1459

    PubMed  Google Scholar 

  • Kobayashi M, Takada Y (2014) Effects of the combined substitutions of amino acid residues on thermal properties of cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria. Extremophiles 18:755–762

    Article  CAS  PubMed  Google Scholar 

  • Konstantinos M, Iason T, Maria T, Micheal K, Vassillis B (2002) Exploring the role of a glycine cluster in cold adaptation of an alkaline phosphatase. Eur J Biochem 269:2330–2335

    Article  Google Scholar 

  • Kurihara T, Takada Y (2012) Analysis of the amino acid residues involved in the thermal properties of the monomeric isocitrate dehydrogenase of the psychrophilic bacterium Colwellia maris and the mesophilic bacterium Azotobacter vinelandii. Biosci Biotechnol Biochem 76:2242–2248

    Article  CAS  PubMed  Google Scholar 

  • Lauro FM, Stratton TK, Chastain RA, Ferriera S, Johnson J, Goldberg SM, Yayanos AA, Bartlett DH (2013) Complete genome sequence of the deep-sea bacterium Psychromonas strain CNPT3. Genome Announc 1:e00304–e00313

    PubMed  PubMed Central  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:144–148

    Google Scholar 

  • Matsuo S, Shirai H, Takada Y (2010) Isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Psudomonas psychrophila. Arch Microbiol 192:639–650

    Article  CAS  PubMed  Google Scholar 

  • Miyazaki K, Wintrode PL, Grayling RA, Rubingh DN, Arnold FH (2000) Directed evolution study of temperature adaptation in a psychrophilic enzyme. J Mol Biol 297:1015–1026

    Article  CAS  PubMed  Google Scholar 

  • Nogi Y, Kato C, Horikoshi K (2002) Psychromonas kaikoae sp. nov., a novel piezophilic bacterium from the deepest cold-seep sediments in the Japan Trench. Int J Syst Evol Microbiol 52:1527–1532

    CAS  PubMed  Google Scholar 

  • Ochiai T, Fukunaga N, Sasaki S (1979) Purification and some properties of two NADP+-specific isocitrate dehydrogenase from an obligately psychrophilic marine bacterium, Vibrio sp., strain ABE-1. J Biochem 86:377–384

    Article  CAS  PubMed  Google Scholar 

  • Ochiai T, Fukunaga N, Sasaki S (1984) Two structurally different NADP+-specific isocitrate dehydrogenase in an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1. J Gen Appl Microbiol 30:479–487

    Article  CAS  Google Scholar 

  • Riley M, Staley TJ, Danchin A, Wang ZT, Brettin ST, Hauser JL, Land LM, Thompson SL (2008) Genomics of an extreme psychrophile, Psychromonas ingrahamii. BMC Genomics 9:210

    Article  PubMed  PubMed Central  Google Scholar 

  • Russell NJ (2000) Toward a molecular understanding of cold activity of enzyme from psychrophiles. Extremophiles 4:83–90

    Article  CAS  PubMed  Google Scholar 

  • Sahara T, Takada Y, Takeuchi Y, Yamaoka N, Fukunaga N (2002) Cloning, sequencing, and expression of a gene encoding the monomeric isocitrate dehydrogenase of the nitrogen-fixing bacterium, Azotobacter vinelandii. Biosci Biotechnol Biochem 66:489–500

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Shiddiqui K, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433

    Article  Google Scholar 

  • Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Pro Natl Acad Sci USA 71:1342–1346

    Article  CAS  Google Scholar 

  • Takada Y, Ochiai T, Okuyama H, Nishi K, Sasaki S (1979) An obligately psychrophilic bacterium isolated on Hokkaido coast. J Gen Appl Microbiol 25:11–19

    Article  Google Scholar 

  • Thorsness PE, Koshland DE Jr (1987) Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. J Biol Chem 262:10422–10425

    CAS  PubMed  Google Scholar 

  • Wang ZX, Brämer C, Steinbüchel A (2003) Two phenotypically compensating isocitrate dehydrogenases in Ralstonia eutropha. FEMS Microbiol Lett 227:9–16

    Article  CAS  PubMed  Google Scholar 

  • Warb A, Schweiger A, Schultes V, Jaenicke R, Zavodsky P (1990) Extremely thermostable D-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium, Thermotaga maritime. Biochemistry 29:7584–7592

    Article  Google Scholar 

  • Watanabe S, Yasutake Y, Tanaka I, Takada Y (2005) Elucidation of stability determinants of cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Colwellia maris, by construction of the chimeric enzymes. Microbiology 151:1083–1094

    Article  CAS  PubMed  Google Scholar 

  • Yasutake Y, Watanabe S, Yao M, Takada Y, Fukunaga N, Tanaka I (2002) Structure of the monomeric isocitrate dehydrogenase: evidence of a protein monomerization by a domain duplication. Structure 10:1637–1648

    Article  CAS  PubMed  Google Scholar 

  • Yasutake Y, Watanabe S, Yao M, Takada Y, Fukunaga N, Tanaka I (2003) Crystal structure of the monomeric isocitrate dehydrogenase in the presence of NADP+. J Biol Chem 278:36897–36904

    Article  CAS  PubMed  Google Scholar 

  • Yayanos AA, Dietz AS, Boxtel RV (1979) Isolation of a deep-sea barophilic bacterium and some of its growth characteristics. Science 205:808–810

    Article  CAS  PubMed  Google Scholar 

  • Yoneta M, Sahara T, Nitta K, Takada Y (2004) Characterization of chimeric isocitrate dehydrogenase of mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, and a psychrophilic bacterium, Colwellia maris. Curr Microbiol 48:383–388

    Article  CAS  PubMed  Google Scholar 

  • Yumoto I, Kawasaki K, Iwata H, Matsuyama H, Okuyama H (1998) Assignment of Vibrio sp. strain ABE-1 to Colwellia maris sp. Nov., a new psychrophilic bacterium. Int J Syst Bacteriol 48:1357–1362

    Article  CAS  PubMed  Google Scholar 

  • Zavodszky P, Kardos J, Petsko GA (1998) Adjustment of conformational flexibility is key event in the thermal adaptation of proteins. Proc Natl Acad Sci USA 95:7406–7411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We sincerely thank Dr. Isao Yumoto of the National Institute of Advanced Industrial Science and Technology of kind donation of P. marina.

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Authors and Affiliations

  1. Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku, Sapporo, 060-0810, Japan

    Ryo Hirota & Kango Tsubouchi

  2. Department of Biological Sciences, Faculty of Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku, Sapporo, 060-0810, Japan

    Yasuhiro Takada

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  1. Ryo Hirota
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  2. Kango Tsubouchi
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  3. Yasuhiro Takada
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Correspondence to Yasuhiro Takada.

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Communicated by F. Robb.

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792_2017_936_MOESM1_ESM.pdf

Fig. S1 Nucleotide and deduced amino acid sequences of the PmIDH gene. The underlined sequence is the putative ribosomal binding site. The probable stem-loop structure located downstream of the translational stop codon is shown by two opposite arrows. The position of the nucleotide sequence indicated in this figure corresponds to the white bar in Fig. 1 (PDF 1850 kb)

792_2017_936_MOESM2_ESM.pdf

Fig. S2 SDS-PAGE of purified PmIDH, AvIDH and chimeric IDHs. Three µg of protein was applied to each lane. Lane 1, PmIDH; lane 2, AvIDH; lane 3, PAA; lane 4, APA; lane 5, AAP; lane 6, APP; lane 7, PPA; lane M, marker proteins (PDF 1576 kb)

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Hirota, R., Tsubouchi, K. & Takada, Y. NADP+-dependent isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina . Extremophiles 21, 711–721 (2017). https://doi.org/10.1007/s00792-017-0936-0

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  • DOI: https://doi.org/10.1007/s00792-017-0936-0

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