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Effects of the substituted amino acid residues on the thermal properties of monomeric isocitrate dehydrogenases from a psychrophilic bacterium, Psychromonas marina, and a mesophilic bacterium, Azotobacter vinelandii

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Abstract

A cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina (PmIDH), showed a high degree of amino acid sequential identity (64%) to a mesophilic one from a mesophilic bacterium, Azotobacter vinelandii (AvIDH). In this study, eight corresponding amino acid residues were substituted between them by site-directed mutagenesis, and several thermal properties of the mutated IDHs were examined. In the PmIDH mutants, PmL735F, substituted Leu735 of PmIDH by the corresponding Phe of AvIDH, showed higher specific activity and thermostability of activity than wild-type PmIDH, while the H600Y and N741P mutations of PmIDH resulted in decreased specific activity and thermostability of activity. On the other hand, among the AvIDH mutants, AvP718T showed lower optimum temperature and thermostability of activity than wild-type AvIDH. In PmIDH variously combined the H600Y, L735F and N741P mutations, PmH600YL735F, including the H600Y and L735F mutations, showed higher specific activity than PmH600Y and similar optimum temperature and thermostability of activity to PmH600Y. Furthermore, PmL735FN741P exhibited higher specific activity and thermostability of activity than PmN741P. These results indicated that the effects of the three mutations of PmIDH are additive on the specific activity of both PmH600YL735F and PmL735FN741P and on thermostability of PmL735FN741P.

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References

  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201

    Article  CAS  PubMed  Google Scholar 

  • Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–W258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Burley SK, Petsko GA (1985) Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science 229:23–28

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Eikmanns BJ, Rittmann D, Sahm 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 

  • Fields PA, Somero GN (1998) Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes. Proc Natl Acad Sci USA 95:11476–11481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fields PA, Dong Y, Meng X, Somero GN (2015) Adaptations of protein structure and function to temperature: there is more than one way to ‘skin a cat’. J Exp Biol 218:1801–1811

    Article  PubMed  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 Y-4. J Biochem 112:849–855

    Article  CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  • Guex N, Peitsch MC, Schwede T (2009) Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis 30:S162–S173

    Article  PubMed  Google Scholar 

  • Hirota R, Tsubouchi K, Takada Y (2017) NADP+-dependent isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina. Extremophiles 21:711–721

    Article  CAS  PubMed  Google Scholar 

  • Ishii A, Ochiai T, 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 M, 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 

  • Kannan N, Vishveshwara S (2000) Aromatic clusters: a determinant of thermal stability of thermophilic proteins. Protein Eng 13:753–761

    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 

  • Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T (2009) The SWISS-MODEL repository and associated resources. Nucl Acids Res 37:D387–D392

    Article  CAS  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 

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

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  • MacArthur MW, Thornton JM (1991) Influence of proline residues on protein conformation. J Mol Biol 218:397–412

    Article  CAS  PubMed  Google Scholar 

  • Maki S, Yoneta M, Takada Y (2006) Two isocitrate dehydrogenases from a psychrophilic bacterium, Colwellia psychrerythraea. Extremophiles 10:237–249

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Merlino A, Krauss IR, Castellano I, Vendittis E, Rossi B, Conte M, Vergara A, Sica F (2010) Structure and flexibility in cold-adapted iron superoxide dismutases: the case of the enzyme isolated from Pseudoalteromonas haloplanktis. J Struct Biol 172:343–352

    Article  CAS  PubMed  Google Scholar 

  • Ochiai T, Fukunaga N, Sasaki S (1979) Purification and some properties of two NADP+-specific isocitrate dehydrogenases 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 dehydrogenases in an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1. J Gen Appl Microbiol 30:479–487

    Article  CAS  Google Scholar 

  • Pace CN, Fu H, Fryar KL, Landua J, Trevino SR, Schell D, Thurlkill RL, Imura S, Scholtz JM, Gajiwala K, Sevcik J, Urbanikova L, Myers JK, Takano K, Hebert EJ, Shirley BA, Grimsley GR (2014) Contribution of hydrogen bonds to protein stability. Protein Sci 23:652–661

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Papaleo E, Riccardi L, Villa C, Fantucci P, Gioia LD (2006) Flexibility and enzymatic cold-adaptation: a comparative molecular dynamics investigation of the elastase family. Biochim Biophys Acta 1764:1397–1406

    Article  CAS  PubMed  Google Scholar 

  • Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

    Article  CAS  PubMed  Google Scholar 

  • Pischedda A, Ramasamy KP, Mangiagalli M, Chiappori F, Milanesi L, Miceli C, Pucciarelli S, Lotti M (2018) Antarctic marine ciliates under stress: superoxide dismutases from the psychrophilic Euplotes focardii are cold-active yet heat tolerant enzymes. Sci Rep 8:14721

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucl Acids Res 42:W320–W324

    Article  CAS  PubMed  PubMed Central  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, N.Y.

    Google Scholar 

  • Schimmel PR, Flory PJ (1968) Conformational energies and configurational statistics of copolypeptides containing l-proline. J Mol Biol 34:105–120

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Suzuki Y (1989) A general principle of increasing protein thermostability. Proc Jpn Acad Ser B Phys Biol Sci 65:146–148

    Article  CAS  Google Scholar 

  • Suzuki Y, Oishi K, Nakano H, Nagayama T (1987) A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo-1,6-glucosidases. Appl Microbiol Biotechnol 26:546–551

    Article  CAS  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

    Article  CAS  PubMed  Google Scholar 

  • Vogt G, Argos P (1997) Protein thermal stability: hydrogen bonds or internal packing? Fold Des 2:S40–46

    Article  CAS  PubMed  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 chimeric enzymes. Microbiology 151:1083–1094

    Article  CAS  PubMed  Google Scholar 

  • Yasuda W, Kobayashi M, Takada Y (2013) Analysis of amino acid residues involved in cold activity of monomeric isocitrate dehydrogenase from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea. J Biosci Bioeng 116:567–572

    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 

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

    Article  CAS  PubMed  Google Scholar 

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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

    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. Kango Tsubouchi
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Correspondence to Yasuhiro Takada.

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Communicated by M. Moracci.

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Tsubouchi, K., Takada, Y. Effects of the substituted amino acid residues on the thermal properties of monomeric isocitrate dehydrogenases from a psychrophilic bacterium, Psychromonas marina, and a mesophilic bacterium, Azotobacter vinelandii. Extremophiles 23, 809–820 (2019). https://doi.org/10.1007/s00792-019-01137-0

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