Skip to main content

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum

Abstract

Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5’-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5 times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20 times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Angelaccio S (2013) Extremophilic SHMTs: from structure to biotechnology. BioMed Res Int. 2013:851428. https://doi.org/10.1155/2013/851428

    Article  PubMed  PubMed Central  Google Scholar 

  2. Angelaccio S, Chiaraluce R, Consalvi V, Buchenau B, Giangiacomo L, Bossa F, Contestabile R (2003) Catalytic and thermodynamic properties of tetrahydromethanopterin-dependent serine hydroxymethyltransferase from Methanococcus jannaschii. J Biol Chem 278:41789–41797. https://doi.org/10.1074/jbc.M306747200

    CAS  Article  PubMed  Google Scholar 

  3. Chaves ACSD, Ruas-Madiedo P, Starrenburg M, Hugenholtz J, Lerayer ALS (2003) Impact of engineered Streptococcus thermophilus trains overexpressing glyA gene on folic acid and acetaldehyde production in fermented milk. Braz J Microbiol 34:114–117. https://doi.org/10.1590/S1517-83822003000500039

    Article  Google Scholar 

  4. Chiba Y, Terada T, Kameya M, Shimizu K, Arai H, Ishii M, Igarashi Y (2012) Mechanism for folate-independent aldolase reaction catalyzed by serine hydroxymethyltransferase. FEBS Journal 279:504–514. https://doi.org/10.1111/j.1742-4658.2011.08443.x

    CAS  Article  Google Scholar 

  5. Contestabile R, Paiardini A, Pascarella S, di Salvo ML, D’Aguanno S, Bossa F (2001) l-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase. A subgroup of strictly related enzymes specialized for different functions. Eur J Biochem 268:6508–6525. https://doi.org/10.1046/j.0014-2956.2001.02606.x

    CAS  Article  PubMed  Google Scholar 

  6. Delle Fratte SD, White RH, Maras B, Bossa F, Schirch V (1997) Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus. J Bacteriol 179:7456–7461. https://doi.org/10.1128/jb.179.23.7456-7461.1997

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Dückers N, Baer K, Simon S, Gröger H, Hummel W (2010) Threonine aldolases-screening, properties and applications in the synthesis of non-proteinogenic β-hydroxy-α-amino acids. Appl Microbiol Biotechnol 88:409–424. https://doi.org/10.1007/s00253-010-2751-8

    CAS  Article  PubMed  Google Scholar 

  8. Falb M, Müller K, Königsmaier L, Oberwinkler T, Horn P, von Gronau S, Gonzalez O, Pfeiffer F, Bornberg-Bauer E, Oesterhelt D (2008) Metabolism of halophilic archaea. Extremophiles 12:177–196. https://doi.org/10.1007/s00792-008-0138-x

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Fesko K, Uhl M, Steinreiber J, Gruber K, Griengl H (2010) Biocatalytic access to α, α-dialkyl-α-amino acids by a mechanism-based approach. Angew Chem Int Ed Engl 49:121–124. https://doi.org/10.1002/anie.200904395

    CAS  Article  PubMed  Google Scholar 

  10. Franz SE, Stewart JD (2014) Threonine aldolases. Adv Appl Microbiol 88:57–101. https://doi.org/10.1016/B978-0-12-800260-5.00003-6

    Article  PubMed  Google Scholar 

  11. Fukuda W, Morimoto N, Imanaka T, Fujiwara S (2008) Agmatine is essential for the cell growth of Thermococcus kodakaraensis. FEMS Microbiol Lett 287:113–120. https://doi.org/10.1111/j.1574-6968.2008.01303.x

    CAS  Article  PubMed  Google Scholar 

  12. Gutiérrez-Preciado A, Romero H, Peimbert M (2010) An evolutionary perspective on amino acids. Nature Education 3:29

    Google Scholar 

  13. Hileman TH, Santangelo TJ (2012) Genetic techniques for Thermococcus kodakarensis. Front Microbiol 3:195. https://doi.org/10.3389/fmicb.2012.00195

    Article  PubMed  PubMed Central  Google Scholar 

  14. Hochuli M, Patzelt H, Oesterhelt D, Wüthrich K, Szyperski T (1999) Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica. J Bacteriol 181:3226–3237. https://doi.org/10.1128/JB.181.10.3226-3237.1999

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Honda K, Inoue M, Ono T, Okano K, Dekishima Y, Kawabata H (2017) Improvement of operational stability of Ogataea minuta carbonyl reductase for chiral alcohol production. J Biosci Bioeng 123:673–678. https://doi.org/10.1016/j.jbiosc.2017.01.016

    CAS  Article  PubMed  Google Scholar 

  16. Kameya M, Ikeda T, Nakamura M, Arai H, Ishii M, Igarashi Y (2007) A novel ferredoxin-dependent glutamate synthase from the hydrogen-oxidizing chemoautotrophic bacterium Hydrogenobacter thermophilus TK-6. J Bacteriol 189:2805–2812. https://doi.org/10.1128/JB.01360-06

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Kim K, Chiba Y, Kobayashi A, Arai H, Ishii M (2017) Phosphoserine phosphatase is required for serine and one-carbon unit synthesis in Hydrogenobacter thermophilus. J Bacteriol 199:e00409-17. https://doi.org/10.1128/JB.00409-17

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Kobashi N, Nishiyama M, Tanokura M (1999) Aspartate kinase-independent lysine synthesis in an extremely thermophilic bacterium, Thermus thermophilus: lysine is synthesized via α-aminoadipic acid not via diaminopimelic acid. J Bacteriol 181:1713–1718. https://doi.org/10.1128/JB.181.6.1713-1718.1999

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Liu JQ, Dairi T, Itoh N, Kataoka M, Shimizu S, Yamada H (1998) Gene cloning, biochemical characterization and physiological role of a thermostable low-specificity l-threonine aldolase from Escherichia coli. Eur J Biochem 255:220–226. https://doi.org/10.1046/j.1432-1327.1998.2550220.x

    CAS  Article  PubMed  Google Scholar 

  20. Makino Y, Sato T, Kawamura H, Hachisuka SI, Takeno R, Imanaka T, Atomi H (2016) An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism. Nat Commun 7:13446. https://doi.org/10.1038/ncomms13446

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. McNeil JB, McIntosh EM, Taylor BV, Zhang FR, Tang S, Bognar AL (1994) Cloning and molecular characterization of three genes, including two genes encoding serine hydroxymethyltransferases, whose inactivation is required to render yeast auxotrophic for glycine. J Biol Chem 269:9155–9165. https://doi.org/10.1016/S0021-9258(17)37089-8

    CAS  Article  PubMed  Google Scholar 

  22. Nogués I, Tramonti A, Angelaccio S, Ruszkowski M, Sekula B, Contestabile R (2020) Structural and kinetic properties of serine hydroxymethyltransferase from the halophytic cyanobacterium Aphanothece halophytica provide a rationale for salt tolerance. Int J Biol Macromol 159:517–529. https://doi.org/10.1016/j.ijbiomac.2020.05.081

    CAS  Article  PubMed  Google Scholar 

  23. Ogawa H, Gomi T, Fujioka M (2000) Serine hydroxymethyltransferase and threonine aldolase: are they identical? Int J Biochem Cell Biol 32:289–301. https://doi.org/10.1016/S1357-2725(99)00113-2

    CAS  Article  PubMed  Google Scholar 

  24. Peters-Wendisch P, Stolz M, Etterich H, Kennerknecht N, Sahm H, Eggeling L (2005) Metabolic engineering of Corynebacterium glutamicum for l-serine production. Appl Environ Microbiol 71:7139–7144. https://doi.org/10.1128/AEM.71.11.7139-7144.2005

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Renwick SB, Snell K, Baumann U (1998) The crystal structure of human cytosolic serine hydroxymethyltransferase: a target for cancer chemotherapy. Structure 6:1105–1116. https://doi.org/10.1016/s0969-2126(98)00112-9

    CAS  Article  PubMed  Google Scholar 

  26. di Salvo ML, Remesh SG, Vivoli M, Ghatge MS, Paiardini A, D’Aguanno S, Safo MK, Contestabile R (2014) On the catalytic mechanism and stereospecificity of Escherichia coli l-threonine aldolase. FEBS Journal 281:129–145. https://doi.org/10.1111/febs.12581

    CAS  Article  Google Scholar 

  27. Sambrook J (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  28. Santangelo TJ, Čuboňová L, Reeve JN (2010) Thermococcus kodakarensis genetics: TK1827-encoded β-glycosidase, new positive-selection protocol, and targeted and repetitive deletion technology. Appl Environ Microbiol 76:1044–1052. https://doi.org/10.1128/AEM.02497-09

    CAS  Article  PubMed  Google Scholar 

  29. Sato Y, Okano K, Kimura H, Honda K (2020) TEMPURA: database of growth temperature of usual and rare procaryotes. Microbes Environ. 35:ME20074. https://doi.org/10.1264/jsme2.ME20074

    Article  PubMed  PubMed Central  Google Scholar 

  30. Scarsdale JN, Radaev S, Kazanina G, Schirch V, Wright HT (2000) Crystal structure at 2.4 Å resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate. J Mol Biol 296:155–168. https://doi.org/10.1006/jmbi.1999.3453

    CAS  Article  PubMed  Google Scholar 

  31. Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234. https://doi.org/10.1016/j.pep.2005.01.016

    CAS  Article  PubMed  Google Scholar 

  32. Yoshida A, Tomita T, Fujimura T, Nishiyama C, Kuzuyama T, Nishiyama M (2015) Structural insight into amino group-carrier protein-mediated lysine biosynthesis: crystal structure of the LysZ·LysW complex from Thermus thermophilus. J Biol Chem 290:435–447. https://doi.org/10.1074/jbc.M114.595983

    CAS  Article  PubMed  Google Scholar 

  33. Zheng RC, Hachisuka SI, Tomita H, Imanaka T, Zheng YG, Nishiyama M, Atomi H (2018) An ornithine ω-aminotransferase required for growth in the absence of exogenous proline in the archaeon Thermococcus kodakarensis. J Biol Chem 293:3625–3636. https://doi.org/10.1074/jbc.RA117.001222

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was partly supported by the JSPS KAKENHI program (grant numbers 17K07720 and 20H05586). This work was also supported by a Grant-in-Aid for JSPS Fellows (Grant number 20J00010). We thank Dr. Tamotsu Kanai (Toyama Prefectural University) and Dr. Haruyuki Atomi (Kyoto University) for sample donation and technical instructions for gene-expression experiments with Thermococcus kodakarensis.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Kohsuke Honda.

Ethics declarations

Conflicts of interest

The author declares that they have no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by A. Driessen.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 1961 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fauziah Ma’ruf, I., Sasaki, Y., Kerbs, A. et al. Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum. Extremophiles 25, 393–402 (2021). https://doi.org/10.1007/s00792-021-01238-9

Download citation

Keywords

  • Serine hydroxymethyltransferase
  • Threonine aldolase
  • Glycine
  • Tetrahydrofolate
  • Thermoplasma acidophilum