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Unexpected property of ectoine synthase and its application for synthesis of the engineered compatible solute ADPC

Applied Microbiology and Biotechnology volume 91, pages 113–122 (2011)Cite this article

Abstract

A new cyclic amino acid was detected in a deletion mutant of the moderately halophilic bacterium Halomonas elongata deficient in ectoine synthesis. Using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) techniques, the substance was identified as 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC). We were able to demonstrate that ADPC is the product of a side reaction of lone ectoine synthase (EC 4.2.1.108), which forms ADPC by cyclic condensation of glutamine. This reaction was shown to be reversible. Subsequently, a number of ectoine derivatives, in particular 4,5-dihydro-2-methylimidazole-4-carboxylate (DHMICA) and homoectoine, were also shown to be cleaved by ectoine synthase, which is classified as a hydro-lyase. This study thus reports for the first time that ectoine synthase accepts more than one substrate and is a reversible enzyme able to catalyze both the intramolecular condensation into and the hydrolytic cleavage of cyclic amino acid derivatives. As ADPC supports growth of bacteria under salt stress conditions and stabilizes enzymes against freeze-thaw denaturation, it displays typical properties of compatible solutes. As ADPC has not yet been described as a natural compound, it is presented here as the first man-made compatible solute created through genetic engineering.

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References

  • Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917

    CAS  Article  Google Scholar 

  • Bursy J, Pierik AJ, Pica N, Bremer E (2007) Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase. J Biol Chem 282:31147–31155. doi:https://doi.org/10.1074/jbc.M704023200

    CAS  Article  Google Scholar 

  • Castellanos L, Duque C, Zea S, Espada A, Rodriguez J, Jimenez C (2006) Isolation and synthesis of (−)-(5S)-2-imino-1-methylpyrrolidine-5-carboxylic acid from Cliona tenuis. Structure revision of pyrostatins. Org Lett 8:4967–4970

    CAS  Article  Google Scholar 

  • Dötsch A, Severin J, Alt W, Galinski EA, Kreft J (2008) A mathematical model for growth and osmoregulation in halophilic bacteria. Microbiology 154:2956–2969

    Article  Google Scholar 

  • Furusho K, Yoshizawa T, Shoji S (2005) Ectoine alters subcellular localization of inclusions and reduces apoptotic cell death induced by the truncated Machado-Joseph disease gene product with an expanded polyglutamine stretch. Neurobiol Dis 20:170–178. doi:https://doi.org/10.1016/j.nbd.2005.02.011

    CAS  Article  Google Scholar 

  • Galinski EA, Pfeiffer HP, Trüper HG (1985) 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur J Biochem 149:135–139

    CAS  Article  Google Scholar 

  • Galinski EA, Herzog RM (1990) The role of trehalose as a substitute for nitrogen-containing compatible solutes (Ectothiorhodospira halochloris). Arch Microbiol 153:607–613

    CAS  Article  Google Scholar 

  • Göller K, Galinski EA (1999) Protection of a model enzyme (lactate dehydrogenase) against heat, urea and freeze-thaw treatment by compatible solute additives. J Mol Catal B Enzym 7:37–45

    Article  Google Scholar 

  • Göller K, Ofer A, Galinski EA (1998) Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis. FEMS Microbiol Lett 161:293–300

    Article  Google Scholar 

  • Graf R, Anzali S, Buenger J, Pfluecker F, Driller H (2008) The multifunctional role of ectoine as a natural cell protectant. Clin Dermatol 26:326–333. doi:https://doi.org/10.1016/j.clindermatol.2008.01.002

    Article  Google Scholar 

  • Grammann K, Volke A, Kunte HJ (2002) New type of osmoregulated solute transporter identified in halophilic members of the bacteria domain: TRAP transporter TeaABC mediates uptake of ectoine and hydroxyectoine in Halomonas elongata DSM 2581(T). J Bacteriol 184:3078–3085

    CAS  Article  Google Scholar 

  • Grammel N (1999) Molekulargenetische und biochemische Analyse der Biosynthese von 2-Methyl-4-carboxy-3,4,5,6-tetrahydropyrimidin und seinem 5-Hydroxyderivat, zwei salzstreßinduzierbaren Osmolyten, in Streptomyces chrysomallus. Dissertation, Technische Universität Berlin

  • Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68

    CAS  Article  Google Scholar 

  • Kanapathipillai M, Lentzen G, Sierks M, Park CB (2005) Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer's β-amyloid. FEBS Lett 579:4775–4780

    CAS  Article  Google Scholar 

  • Kanapathipillai M, Ku SH, Girigoswami K, Park CB (2008) Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106–126. Biochem Biophys Res Commun 365:808–813. doi:https://doi.org/10.1016/j.bbrc.2007.11.074

    CAS  Article  Google Scholar 

  • Koichi M, Mitsuhiko M, Tatsuo N, Yoshio S; Takeda Chem Ind Ltd (Take), assignee (1991) Production of tetrahydropyrimidine derivatives. Jp. Patent JP3031265

  • Kunte HJ, Galinski EA (1995) Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata. FEMS Microbiol Lett 128:293–299

    CAS  Article  Google Scholar 

  • Kunte HJ, Galinski EA, Trueper HG (1993) A modified FMOC-method for the detection of amino acid-type osmolytes and tetrahydropyrimidines (ectoines). J Microbiol Meth 17:129–136

    CAS  Article  Google Scholar 

  • Larsen PI, Sydnes LK, Landfald B, Strøm AR (1987) Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose. Arch Microbiol 147:1–7

    CAS  Article  Google Scholar 

  • Lee M, Coulter DM, Lown JW (1988) Total synthesis and absolute configuration of the antibiotic oligopeptide (4S)-(+)-anthelvencin A and its 4R-(−) enantiomer. J Org Chem 53:1855–1859

    CAS  Article  Google Scholar 

  • Lee M, Lown JW (1987) Synthesis of (4S)- and (4R)-methyl 2-amino-1-pyrroline-5-carboxylate and their application to the preparation of (4S)-(+)- and (4R)-(−)-dihydrokikumycin B. J Org Chem 52:5717–5721

    CAS  Article  Google Scholar 

  • Lippert K, Galinski EA (1992) Enzyme stabilization be ectoine-type compatible solutes: protection against heating, freezing and drying. Appl Microbiol Biotechnol 37:61–65

    CAS  Article  Google Scholar 

  • Louis P (1997) Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology 143:1141–1149

    CAS  Article  Google Scholar 

  • Louis P, Trueper HG, Galinski EA (1994) Survival of Escherichia coli during drying and storage in the presence of compatible solutes. Appl Microbiol Biotechnol 41:684–688

    CAS  Article  Google Scholar 

  • Manzanera M, de García Castro A, Tøndervik A, Rayner-Brandes M, Strøm AR, Tunnacliffe A (2002) Hydroxyectoine is superior to trehalose for anhydrobiotic engineering of Pseudomonas putida KT2440. Appl Environ Microbiol 68:4328–4333

    CAS  Article  Google Scholar 

  • Mitchell RE, Teh KL (2005) Antibacterial iminopyrrolidines from Burkholderia plantarii, a bacterial pathogen of rice. Org Biomol Chem 3:3540–3543. doi:https://doi.org/10.1039/b509319h

    CAS  Article  Google Scholar 

  • Moghaieb REA, Tanaka N, Saneoka H, Murooka Y, Ono H, Morikawa H, Nakamura A, Nguyen NT, Suwa R, Fujita K (2006) Characterization of salt tolerance in ectoine-transformed tobacco plants (Nicotiana tabaccum): photosynthesis, osmotic adjustment, and nitrogen partitioning. Plant Cell Environ 29:173–182

    CAS  Article  Google Scholar 

  • Nakayama H, Yoshida K, Ono H, Murooka Y, Shinmyo A (2000) Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells. Plant Physiol 122:1239–1268

    CAS  Article  Google Scholar 

  • Ono H, Sawada K, Khunajakr N, Tao T, Yamamoto M, Hiramoto M, Shinmyo A, Takano M, Murooka Y (1999) Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata. J Bacteriol 181:91–99

    CAS  Article  Google Scholar 

  • Peters P, Galinski EA, Trüper HG (1990) The biosynthesis of ectoine. FEMS Microbiol Lett 71:157–162

    CAS  Article  Google Scholar 

  • Rai M, Pal M, Sumesh KV, Jain V, Sankaranarayanan A (2006) Engineering for biosynthesis of ectoine (2-methyl 4-carboxy tetrahydro pyrimidine) in tobacco chloroplasts leads to accumulation of ectoine and enhanced salinity tolerance. Plant Sci 170:291–306

    CAS  Article  Google Scholar 

  • Reuter K, Pittelkow M, Bursy J, Heine A, Craan T, Bremer E (2010) Synthesis of 5-hydroxyectoine from ectoine: crystal structure of the non-heme iron(II) and 2-oxoglutarate-dependent dioxygenase EctD. PLoS ONE 5:e10647. doi:https://doi.org/10.1371/journal.pone.0010647

    Article  Google Scholar 

  • Sauer T, Galinski EA (1998) Bacterial milking: a novel bioprocess for production of compatible solutes. Biotechnol Bioeng 57:306–313

    CAS  Article  Google Scholar 

  • Schaefer A, Tauch A, Jaeger W, Kalinowski J (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19 selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73

    CAS  Article  Google Scholar 

  • Schwibbert K, Marin-Sanguino A, Bagyan I, Heidrich G, Lentzen G, Seitz H, Rampp M, Schuster SC, Klenk H, Pfeiffer F, Oesterhelt D, Kunte HJ (2010) A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581(T). Environ Microbiol. doi:https://doi.org/10.1111/j.1462-2920.2010.02336.x

  • Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat Biotech 1:784–791

    CAS  Article  Google Scholar 

  • Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130

    CAS  Article  Google Scholar 

  • Takizawa M, Tsubotani S, Tanida S, Harada S, Hasegawa T (1987) A new pyrrole-amidine antibiotic TAN-868 A. J Antibiot 40:1220–1230

    CAS  Article  Google Scholar 

  • Voß P (2002) Synthese von kompatiblen Soluten mit ectoinanaloger Struktur und Charakterisierung des protektiven Effektes auf biochemische Modellsysteme und Escherichia coli. Dissertation, Westfälische Wilhelms-Universität Münster

  • Vreeland RH, Litchfield CD, Martin EL, Elliot E (1980) Halomonas elongata, a new genus and species of extremely salt-tolerant bacteria. Int J Syst Bacteriol 30:485–495

    CAS  Article  Google Scholar 

  • Wei L, Wedeking A, Buttner R, Kalff JC, Tolba RH, van Echten-Deckert G (2009) A natural tetrahydropyrimidine protects small bowel from cold ischemia and subsequent warm in vitro reperfusion injury. Pathobiology 76:212–220. doi:https://doi.org/10.1159/000218338

    CAS  Article  Google Scholar 

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Acknowledgement

We would like to express our gratitude to Katrin Grammann for the construction of mutant KB1 during her diploma thesis at the Institute of Microbiology & Biotechnology, which turned our attention to and eventually led to the disclosure of the other facets of ectoine synthase.

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Affiliations

  1. Institut für Mikrobiologie & Biotechnologie, Rheinische Friedrich-Wilhelms-Universität, 53115, Bonn, Germany

    Elisabeth M. H. J. Witt & Erwin A. Galinski

  2. Central Science Laboratory, University of Tasmania, Hobart, 7001, Australia

    Noel W. Davies

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  1. Elisabeth M. H. J. Witt
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  2. Noel W. Davies
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  3. Erwin A. Galinski
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Correspondence to Erwin A. Galinski.

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Witt, E.M.H.J., Davies, N.W. & Galinski, E.A. Unexpected property of ectoine synthase and its application for synthesis of the engineered compatible solute ADPC. Appl Microbiol Biotechnol 91, 113–122 (2011). https://doi.org/10.1007/s00253-011-3211-9

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  • DOI: https://doi.org/10.1007/s00253-011-3211-9

Keywords

  • Halomonas elongata
  • Ectoine synthase
  • ADPC
  • Genetic engineering
  • Cyclic amino acid derivatives
  • Compatible solute