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Characterization of two recombinant 3-hexulose-6-phosphate synthases from the halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z

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Abstract

Two key enzymes of the ribulose monophosphate (RuMP) cycle for formaldehyde fixation, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexulose isomerase (PHI), in the aerobic halotolerant methanotroph Methylomicrobium alcaliphilum 20Z are encoded by the genes hps and phi and the fused gene hps-phi. The recombinant enzymes HPS-His6, PHI-His6, and the two-domain proteinHPS–PHI were obtained by heterologous expression in Escherichia coli and purified by affinity chromatography. PHI-His6, HPS-His6 (2 × 20 kDa), and the fused protein HPS–PHI (2 × 40 kDa) catalyzed formation of fructose 6-phosphate from formaldehyde and ribulose 5-phosphate with activities of 172 and 22 U/mg, respectively. As judged from the k cat/K m ratio, HPS-His6 had higher catalytic efficiency but lower affinity to formaldehyde compared to HPS–PHI. AMP and ADP were powerful inhibitors of both HPS and HPS–PHI activities. The two-domain HPS–PHI did not show isomerase activity, but the sequences corresponding to its HPS and PHI regions, when expressed separately, were found to produce active enzymes. Inactivation of the hps-phi fused gene did not affect the growth rate of the mutant strain. Analysis of annotated genomes revealed the separately located genes hps and phi in all the RuMP pathway methylotrophs, whereas the hps-phi fused gene occurred only in several methanotrophs and was absent in methylotrophs not growing under methane. The significance of these tandems in adaptation and biotechnological potential of methylotrophs is discussed.

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Abbreviations

FA:

formaldehyde

F6P:

fructose 6-phosphate

GPD:

glucose-6-phosphate dehydrogenase

HPS:

3-hexulose-6-phosphate synthase

LB:

Luria–Bertani medium

ORF:

open reading frame

PGI:

phosphoglucoisomerase

PHI:

6-phospho-3-hexulose isomerase

RuMP:

ribulose monophosphate

Ru5P:

ribulose 5-phosphate

References

  1. Ferenci, T., Strom, T., and Quayle, J. R. (1974) Purification and properties of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase from Methylococcus capsulatus, Biochem. J., 144, 477–486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sahm, H., Schutte, H., and Kula, M. R. (1976) Purification and properties of 3-hexulosephosphate synthase from Methylomonas M15, Eur. J. Biochem., 66, 591596.

    Article  Google Scholar 

  3. Kato, N., Ohashi, H., Tani, Y., and Ogata, K. (1978) 3Hexulosephosphate synthase from Methylomonas aminofaciens 77a: purification, properties and kinetics, Biochim. Biophys. Acta, 523, 238–244.

    Google Scholar 

  4. Arfman, N., Bystrykh, L., Govorukhina, N. I., and Dijkhuizen, L. (1990) 3-Hexulose-6-phosphate synthase from thermotolerant methylotroph Bacillus C1, Methods Enzymol., 188, 391–397.

    Article  CAS  PubMed  Google Scholar 

  5. Quayle, J. R., and Ferenci, T. (1978) Evolutionary aspects of autotrophy, Microbiol. Rev., 42, 251–273.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Yasueda, H., Kawahara, Y., and Sugimoto, S. (1999) Bacillus subtilis yckG and yckF encode two key enzymes of the ribulose monophosphate pathway used by methylotrophs, and yckH is required for their expression, J. Bacteriol., 181, 7154–7160.

    CAS  PubMed  Google Scholar 

  7. Yurimoto, H., Hirai, R., Yasueda, H., Mitsui, R., Sakai, Y., and Kato, N. (2002) The ribulose monophosphate pathway operon encoding formaldehyde fixation in a thermotolerant methylotroph, Bacillus brevis S1, FEMS Microbiol. Lett., 214, 189–193.

    Article  CAS  PubMed  Google Scholar 

  8. Mitsui, R., Kusano, Y., Yurimoto, H., Sakai, Y., Kato, N., and Tanaka, M. (2003) Formaldehyde fixation contributes to detoxification for growth of a nonmethylotroph, Burkholderia cepacia TM1, on vanillic acid, Appl. Environ. Microbiol., 69, 6128–6132.

    Article  CAS  PubMed  Google Scholar 

  9. Orita, I., Yurimoto, H., Hirai, R., Kawarabayasi, Y., Sakai, Y., and Kato, N. (2005) The archaeon Pyrococcus horikoshii possesses a bifunctional enzyme for formaldehyde fixation via the ribulose monophosphate pathway, J. Bacteriol., 187, 3636–3642.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Orita, I., Sato, T., Yurimoto, H., Kato, N., Atomi, H., Imanaka, T., and Sakai, Y. (2006) The ribulose monophosphate pathway substitutes for the missing pentose phosphate pathway in the archaeon Thermococcus kodakaraensis, J. Bacteriol., 188, 4698–4704.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Khmelenina, V. N., Kalyuzhnaya, M. G., Sakharovsky, V. G., Suzina, N. E., Trotsenko, Y. A., and Gottschalk, G. (1999) Osmoadaptation in halophilic and alkaliphilic methanotrophs, Arch. Microbiol., 172, 321–329.

    Article  CAS  PubMed  Google Scholar 

  12. Kalyuzhnaya, M. G., Yang, S., Rozova, O. N., Smalley, N. E., Clubb, J., Lamb, A., Nagana Gowda, G. A., Raftery, D., Fu, Y., Bringel, F., Vuilleumier, S., Beck, D. A. C., Trotsenko, Y. A., Khmelenina, V. N., and Lidstrom, M. E. (2013) Highly efficient methane biocatalysis revealed in methanotrophic bacterium, Nat. Commun., 4, 2785.

    Article  CAS  PubMed  Google Scholar 

  13. Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: a Laboratory Manual, 3rd Edn., Cold Spring Harbor Laboratory, N.-Y.

    Google Scholar 

  14. Kalyuzhnaya, M., Khmelenina, V. N., Kotelnikova, S., Holmquist, L., Pedersen, K., and Trotsenko, Y. A. (1999) Methylomonas scandinavica sp. nov., a new methanotrophic psychrotrophic bacterium isolated from deep igneous rock ground water of Sweden, Syst. Appl. Microbiol., 22, 565–572.

    CAS  Google Scholar 

  15. Catanzariti, A. M., Soboleva, T. A., Jans, D. A., Board, P. G., and Baker, R. T. (2004) An efficient system for highlevel expression and easy purification of authentic recombinant proteins, Protein Sci., 13, 1331–1339.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Reshetnikov, A. S., Mustakhimov, I. I., Rozova, O. N., Beschastny, A. P., Khmelenina, V. N., Murrell, J. C., and Trotsenko, Y. A. (2008) Characterization of the pyrophosphate-dependent 6-phosphofructokinase from Methylococcus capsulatus Bath, FEMS Microbiol. Lett., 288, 202–210.

    Article  CAS  PubMed  Google Scholar 

  17. Slater, G. G. (1969) Stable pattern formation and determination of molecular size by pore-limit electrophoresis, Anal. Chem., 41, 1039–1041.

    Article  CAS  PubMed  Google Scholar 

  18. Nash, T. (1953) The colorimetric estimation of formaldehyde by means of the Hantsch reaction, Biochem. J., 55, 416–421.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mustakhimov, I. I., Reshetnikov, A. S., Glukhov, A. S., Khmelenina, V. N., Kalyuzhnaya, M. G., and Trotsenko, Y. A. (2010) Identification and characterization of EctR1, a new transcriptional regulator of the ectoine biosynthesis genes in the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z, J. Bacteriol., 192, 410–417.

    CAS  PubMed  Google Scholar 

  20. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0, Mol. Biol. Evol., 24, 15961599.

    Article  Google Scholar 

  21. Martinez-Cruz, L. A., Dreyer, M. K., Boisvert, D. C., Yokota, H., Martinez- Chanter, M. L., Kim, R., and Kim, S. H. (2002) Crystal structure of MJ1247 protein from M. jannaschii at 2.0 Å resolution infers a molecular function of 3-hexulose-6-phosphate isomerase, Structure, 10, 195–204.

    Article  CAS  PubMed  Google Scholar 

  22. Sanishvili, R., Wu, R., Kim, D. E., Watson, J. D., Collart, F., and Joachimiak, A. (2004) Crystal structure of Bacillus subtilis YckF: structural and functional evolution, J. Struct. Biol., 148, 98–109.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Orita, I., Sakamoto, N., Kato, N., Yurimoto, H., and Sakai, Y. (2007) Bifunctional enzyme fusion of 3-hexulose6-phosphate synthase and 6-phospho-3-hexuloisomerase, Appl. Microbiol. Biotechnol., 76, 439–445.

    Article  CAS  PubMed  Google Scholar 

  24. Chen, L. M., Li, K. Z., Orita, I., Yurimoto, H., Sakai, Y., Kato, N., and Izui, K. (2004) Enhancement of plant tolerance to formaldehyde by over-expression of formaldehydeassimilating enzymes from a methylotrophic bacterium, Plant Cell. Physiol., 45, S233.

    Article  Google Scholar 

  25. Jakobsen, O. M., Benichou, A., Flickinger, M. C., Ellingsen, V. S., and Brautaset, T. E. (2006) Upregulated transcription of plasmid and chromosomal ribulose monophosphate pathway genes is critical for methanol assimilation rate and methanol tolerance in the methylotrophic bacterium Bacillus methanolicus, J. Bacteriol., 188, 3063–3072.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Sawada, A., Oyabu, T., Chen, L. M., Li, K. Z., Hirai, N., Yurimoto, H., Orita, I., Sakai, Y., Kato, N., and Izui, K. (2007) Purification capability of tobacco transformed with enzymes from a methylotrophic bacterium for formaldehyde, Int. J. Phytoremediat., 9, 487–496.

    Article  CAS  Google Scholar 

  27. Yurimoto, H., Kato, N., and Sakai, Y. (2009) Genomic organization and biochemistry of the ribulose monophosphate pathway and its application in biotechnology, Appl. Microbiol. Biotechnol., 84, 407–416.

    Article  CAS  PubMed  Google Scholar 

  28. Koopman, F. W., De Winde, J. H., and Ruijssenaars, H. J. (2009) C(1) compounds as auxiliary substrate for engineered Pseudomonas putida S12, Appl. Microbiol. Biotechnol., 83, 705–713.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Laboratory of Methylotrophy, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290, Pushchino, Moscow Region, Russia

    O. N. Rozova, S. Y. But, V. N. Khmelenina, A. S. Reshetnikov & Y. A. Trotsenko

  2. Pushchino State Institute of Natural Sciences, 142290, Pushchino, Moscow Region, Russia

    I. I. Mustakhimov & Y. A. Trotsenko

Authors
  1. O. N. Rozova
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  2. S. Y. But
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  3. V. N. Khmelenina
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  4. A. S. Reshetnikov
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  5. I. I. Mustakhimov
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  6. Y. A. Trotsenko
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Corresponding author

Correspondence to V. N. Khmelenina.

Additional information

Published in Russian in Biokhimiya, 2017, Vol. 82, No. 2, pp. 290-300.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM16-297, December 5, 2016.

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Rozova, O.N., But, S.Y., Khmelenina, V.N. et al. Characterization of two recombinant 3-hexulose-6-phosphate synthases from the halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z. Biochemistry Moscow 82, 176–185 (2017). https://doi.org/10.1134/S0006297917020092

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  • DOI: https://doi.org/10.1134/S0006297917020092

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