Skip to main content

Advertisement

Log in

Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Main conclusion

Recombinant wheat DHDPS was produced for the first time in milligram quantities and shown to be an enzymatically active tetramer in solution using analytical ultracentrifugation and small angle X-ray scattering.

Wheat is an important cereal crop with an extensive role in global food supply. Given our rapidly growing population, strategies to increase the nutritional value and production of bread wheat are of major significance in agricultural science to satisfy our dietary requirements. Lysine is one of the most limiting essential amino acids in wheat, thus, a thorough understanding of lysine biosynthesis is of upmost importance to improve its nutritional value. Dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7) catalyzes the first committed step in the lysine biosynthesis pathway of plants. Here, we report for the first time the expression and purification of recombinant DHDPS from the bread wheat Triticum aestivum (Ta-DHDPS). The optimized protocol yielded 36 mg of > 98% pure recombinant Ta-DHDPS per liter of culture. Enzyme kinetic studies demonstrate that the recombinant Ta-DHDPS has a KM (pyruvate) of 0.45 mM, KM (l-aspartate-4-semialdehyde) of 0.07 mM, kcat of 56 s−1, and is inhibited by lysine (IC LYS50 of 0.033 mM), which agree well with previous studies using labor-intensive purification from wheat suspension cultures. We subsequently employed circular dichroism spectroscopy, analytical ultracentrifugation and small angle X-ray scattering to show that the recombinant enzyme is folded with 60% α/β structure and exists as a 7.5 S tetrameric species with a Rg of 33 Å and Dmax of 118 Å. This study is the first to report the biophysical properties of the recombinant Ta-DHDPS in aqueous solution and offers an excellent platform for future studies aimed at improving nutritional value and primary production of bread wheat.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Atkinson SC, Dogovski C, Downton MT, Pearce FG, Reboul CF, Buckle AM, Gerrard JA, Dobson RC, Wagner J, Perugini MA (2012) Crystal, solution and in silico structural studies of dihydrodipicolinate synthase from the common grapevine. PLoS One 7:e38318

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Atkinson SC, Dogovski C, Downton MT, Czabotar PE, Dobson RC, Gerrard JA, Wagner J, Perugini MA (2013) Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition. Plant Mol Biol 81:431–446

    Article  PubMed  CAS  Google Scholar 

  • Atkinson SC, Hor L, Dogovski C, Dobson RC, Perugini MA (2014) Identification of the bona fide DHDPS from a common plant pathogen. Proteins 82:1869–1883

    Article  PubMed  CAS  Google Scholar 

  • Blagova E, Levdikov V, Milioti N, Fogg MJ, Kalliomaa AK, Brannigan JA, Wilson KS, Wilkinson AJ (2006) Crystal structure of dihydrodipicolinate synthase (BA3935) from Bacillus anthracis at 1.94 Å resolution. Proteins 62:297–301

    Article  PubMed  CAS  Google Scholar 

  • Blickling S, Beisel HG, Bozic D, Knablein J, Laber B, Huber R (1997) Structure of dihydrodipicolinate synthase of Nicotiana sylvestris reveals novel quaternary structure. J Mol Biol 274:608–621

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Burgess BR, Dobson RC, Bailey MF, Atkinson SC, Griffin MD, Jameson GB, Parker MW, Gerrard JA, Perugini MA (2008) Structure and evolution of a novel dimeric enzyme from a clinically important bacterial pathogen. J Biol Chem 283:27598–27603

    Article  PubMed  CAS  Google Scholar 

  • Davis AJ, Perugini MA, Smith BJ, Stewart JD, Ilg T, Hodder AN, Handman E (2004) Properties of GDP-mannose pyrophosphorylase, a critical enzyme and drug target in Leishmania mexicana. J Biol Chem 279:12462–12468

    Article  PubMed  CAS  Google Scholar 

  • Dereppe C, Bold G, Ghisalba O, Ebert E, Schar HP (1992) Purification and characterization of dihydrodipicolinate synthase from pea. Plant Physiol 98:813–821

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Diekmann F (2009) Wheat. J Agric Food Inf 10:289–299

    Article  Google Scholar 

  • Dobson RC, Griffin MD, Jameson GB, Gerrard JA (2005) The crystal structures of native and (S)-lysine-bound dihydrodipicolinate synthase from Escherichia coli with improved resolution show new features of biological significance. Acta Crystallogr D Biol Crystallogr 61:1116–1124

    Article  PubMed  CAS  Google Scholar 

  • Dobson RC, Giron I, Hudson AO (2011) L, L-diaminopimelate aminotransferase from Chlamydomonas reinhardtii: a target for algaecide development. PLoS One 6:e20439

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Dogovski C, Atkinson SC, Dommaraju SR, Dobson RC, Perugini MA, Hor L, Hutton CA, Gerrard JA (2009) Lysine biosynthesis in bacteria: an unchartered pathway for novel antibiotic design. In: Doelle H (ed) Biotechnology part 1, vol 11. Encyclopedia of Life Support Systems (EOLSS), Oxford, pp 116–136

    Google Scholar 

  • Dogovski C, Dommaraju SR, Small LC, Perugini MA (2012a) Comparative structure and function analyses of native and his-tagged forms of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus. Protein Expr Purif 85:66–76

    Article  PubMed  CAS  Google Scholar 

  • Dogovski C, Wubben JM, Paxman JJ, Wagner J, Hor L, Peverelli MG, Perugini MA, Downton M, Reumann M, Taylor NL (2012b) Enzymology of bacterial lysine biosynthesis. In: Ekinci D (ed) Biochemistry. InTech Open Access Publisher, Croatia, pp 225–262. https://doi.org/10.5772/34121

    Chapter  Google Scholar 

  • Dogovski C, Gorman MA, Ketaren NE, Praszkier J, Zammit LM, Mertens HD, Bryant G, Yang J, Griffin MD, Pearce FG, Gerrard JA, Jameson GB, Parker MW, Robins-Browne RM, Perugini MA (2013) From knock-out phenotype to three-dimensional structure of a promising antibiotic target from Streptococcus pneumoniae. PLoS One 8:e83419

    Article  PubMed  PubMed Central  Google Scholar 

  • Dommaraju SR, Dogovski C, Czabotar PE, Hor L, Smith BJ, Perugini MA (2011) Catalytic mechanism and cofactor preference of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus. Arch Biochem Biophys 512:167–174

    Article  PubMed  CAS  Google Scholar 

  • Frisch DA, Gengenbach BG, Tommey AM, Sellner JM, Somers DA, Myers DE (1991) Isolation and characterization of dihydrodipicolinate synthase from maize. Plant Physiol 96:444–452

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Frizzi A, Huang S, Gilbertson LA, Armstrong TA, Luethy MH, Malvar TM (2008) Modifying lysine biosynthesis and catabolism in corn with a single bifunctional expression/silencing transgene cassette. Plant Biotechnol J 6:13–21

    PubMed  CAS  Google Scholar 

  • Georgescauld F, Popova K, Gupta AJ, Bracher A, Engen JR, Hayer-Hartl M, Hartl FU (2014) GroEL/ES chaperonin modulates the mechanism and accelerates the rate of TIM-barrel domain folding. Cell 157:922–934

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Ghislain M, Frankard V, Jacobs M (1990) Dihydrodipicolinate synthase of Nicotiana sylvestris, a chloroplast-localized enzyme of the lysine pathway. Planta 180:480–486

    Article  PubMed  CAS  Google Scholar 

  • Griffin MD, Dobson RC, Pearce FG, Antonio L, Whitten AE, Liew CK, Mackay JP, Trewhella J, Jameson GB, Perugini MA, Gerrard JA (2008) Evolution of quaternary structure in a homotetrameric enzyme. J Mol Biol 380:691–703

    Article  PubMed  CAS  Google Scholar 

  • Griffin MD, Dobson RC, Gerrard JA, Perugini MA (2010) Exploring the dihydrodipicolinate synthase tetramer: how resilient is the dimer–dimer interface? Arch Biochem Biophys 494:58–63

    Article  PubMed  CAS  Google Scholar 

  • Griffin MD, Billakanti JM, Wason A, Keller S, Mertens HD, Atkinson SC, Dobson RC, Perugini MA, Gerrard JA, Pearce FG (2012) Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana. PLoS One 7:e40318

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Gupta R, Soares da Costa TP, Faou P, Dogovski C, Perugini MA (2018) Comparison of untagged and his-tagged dihydrodipicolinate synthase from the enteric pathogen Vibrio cholerae. Protein Expr Purif 145:85–93

    Article  PubMed  CAS  Google Scholar 

  • Hor L, Dobson RC, Downton MT, Wagner J, Hutton CA, Perugini MA (2013) Dimerization of bacterial diaminopimelate epimerase is essential for catalysis. J Biol Chem 288:9238–9248

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Houmard NM, Mainville JL, Bonin CP, Huang S, Luethy MH, Malvar TM (2007) High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi. Plant Biotechnol J 5:605–614

    Article  PubMed  CAS  Google Scholar 

  • Huang S, Kruger DE, Frizzi A, D’Ordine RL, Florida CA, Adams WR, Brown WE, Luethy MH (2005) High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation. Plant Biotechnol J 3:555–569

    Article  PubMed  CAS  Google Scholar 

  • Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Gr 14:33–38

    Article  CAS  Google Scholar 

  • Jacques DA, Trewhella J (2010) Small-angle scattering for structural biology—expanding the frontier while avoiding the pitfalls. Protein Sci 19:642–657

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Kaneko T, Hashimoto T, Kumpaisal R, Yamada Y (1990) Molecular cloning of wheat dihydrodipicolinate synthase. J Biol Chem 265:17451–17455

    PubMed  CAS  Google Scholar 

  • Kefala G, Evans GL, Griffin MD, Devenish SR, Pearce FG, Perugini MA, Gerrard JA, Weiss MS, Dobson RC (2008) Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis. Biochem J 411:351–360

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI (2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Crystallogr 36:1277–1282

    Article  CAS  Google Scholar 

  • Konarev PV, Petoukhov MV, Volkov VV, Svergun DI (2006) ATSAS 2.1, a program package for small-angle scattering data analysis. J Appl Crystallogr 39:277–286

    Article  CAS  Google Scholar 

  • Kozin MB, Svergun DI (2001) Automated matching of high- and low-resolution structural models. J Appl Crystallogr 34:33–41

    Article  CAS  Google Scholar 

  • Kumpaisal R, Hashimoto T, Yamada Y (1987) Purification and characterization of dihydrodipicolinate synthase from wheat suspension cultures. Plant Physiol 85:145–151

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Laue TM, Shah BD, Ridgeway TM, Pelletier SL (1992) Computer-aided interpretation of analytical sedimentation data for proteins. In: Harding SE, Rowe AJ, Horton JC (eds) Analytical ultracentrifugation in biochemistry and polymer science. The Royal Society of Chemistry, Cambridge, pp 90–125

    Google Scholar 

  • Leatherbarrow RJ (1988) Enzfitter: a non-linear regression data analysis program for IBM PC. J Am Chem Soc 110:4098–4100

    Article  Google Scholar 

  • Liu Y, Xie S, Yu J (2016) Genome-wide analysis of the lysine biosynthesis pathway network during maize seed development. PLoS One 11:e0148287

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mank N, Arnette A, Klapper V, Offermann L, Chruszcz M (2015) Structure of dihydrodipicolinate synthase from the commensal bacterium Bacteroides thetaiotaomicron at 2.1 Å resolution. Acta Crystallogr F Struct Biol Commun 71:449–454

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Matthews BF, Widholm JM (1978) Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots. Planta 141:315–321

    Article  PubMed  CAS  Google Scholar 

  • Matthews BF, Widholm JM (1979) Expression of aspartokinase, dihydrodipicolinic acid synthase and homoserine dehydrogenase during growth of carrot cell suspension cultures on lysine- and threonine-supplemented media. Z Naturforsch C 34:1177–1185

    Article  PubMed  CAS  Google Scholar 

  • Mirwaldt C, Korndorfer I, Huber R (1995) The crystal structure of dihydrodipicolinate synthase from Escherichia coli at 2.5 A resolution. J Mol Biol 246:227–239

    Article  PubMed  CAS  Google Scholar 

  • Naqvi KF, Staker BL, Dobson RC, Serbzhinskiy D, Sankaran B, Myler PJ, Hudson AO (2016) Cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the human pathogenic bacterium Bartonella henselae strain Houston-1 at 2.1 Å resolution. Acta Crystallogr F Struct Biol Commun 72:2–9

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Pearce FG, Perugini MA, McKerchar HJ, Gerrard JA (2006) Dihydrodipicolinate synthase from Thermotoga maritima. Biochem J 400:359–366

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Perugini MA, Schuck P, Howlett GJ (2000) Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid. J Biol Chem 275:36758–36765

    Article  PubMed  CAS  Google Scholar 

  • Perugini MA, Schuck P, Howlett GJ (2002) Differences in the binding capacity of human apolipoprotein E3 and E4 to size-fractionated lipid emulsions. Eur J Biochem 269:5939–5949

    Article  PubMed  CAS  Google Scholar 

  • Peverelli MG, Soares da Costa TP, Kirby N, Perugini MA (2016) Dimerization of bacterial diaminopimelate decarboxylase is essential for catalysis. J Biol Chem 291:9785–9795

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sagong HY, Kim KJ (2016) Structural insight into dihydrodipicolinate reductase from Corynebacterium glutamicum for lysine biosynthesis. J Microbiol Biotechnol 26:226–232

    Article  PubMed  CAS  Google Scholar 

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

    Google Scholar 

  • Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J 78:1606–1619

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Schuck P, Perugini MA, Gonzales NR, Howlett GJ, Schubert D (2002) Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. Biophys J 82:1096–1111

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Shaul O, Galili G (1992) Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts. Plant J 2:203–209

    Article  CAS  Google Scholar 

  • Shewry PR (2009) Wheat. J Exp Bot 60:1537–1553

    Article  PubMed  CAS  Google Scholar 

  • Soares da Costa TP, Yap MY, Perugini MA, Wallace JC, Abell AD, Wilce MC, Polyak SW, Booker GW (2014) Dual roles of F123 in protein homodimerization and inhibitor binding to biotin protein ligase from Staphylococcus aureus. Mol Microbiol 91:110–120

    Article  PubMed  CAS  Google Scholar 

  • Soares da Costa TP, Christensen JB, Desbois S, Gordon SE, Gupta R, Hogan CJ, Nelson TG, Downton MT, Gardhi CK, Abbott BM, Wagner J, Panjikar S, Perugini MA (2015) Quaternary structure analyses of an essential oligomeric enzyme. Methods Enzymol 562:205–223

    Article  PubMed  CAS  Google Scholar 

  • Soares da Costa TP, Desbois S, Dogovski C, Gorman MA, Ketaren NE, Paxman JJ, Siddiqui T, Zammit LM, Abbott BM, Robins-Browne RM, Parker MW, Jameson GB, Hall NE, Panjikar S, Perugini MA (2016) Structural determinants defining the allosteric inhibition of an essential antibiotic target. Structure 24:1282–1291

    Article  PubMed  CAS  Google Scholar 

  • Soares da Costa TP, Abbott BM, Gendall AR, Panjikar S, Perugini MA (2018) Molecular evolution of an oligomeric biocatalyst functioning in lysine biosynthesis. Biophys Rev 10:153–162

  • Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287:252–260

    Article  PubMed  CAS  Google Scholar 

  • Sreerama N, Venyaminov SY, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal Biochem 287:243–251

    Article  PubMed  CAS  Google Scholar 

  • Sridharan U, Ebihara A, Kuramitsu S, Yokoyama S, Kumarevel T, Ponnuraj K (2014) Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus. Extremophiles 18:973–985

    Article  PubMed  CAS  Google Scholar 

  • Svergun D (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 25:495–503

    Article  CAS  Google Scholar 

  • Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J 76:2879–2886

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Svergun D, Barberato C, Koch MHJ (1995) CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 28:768–773

    Article  CAS  Google Scholar 

  • Triassi AJ, Wheatley MS, Savka MA, Gan HM, Dobson RC, Hudson AO (2014) L,L-diaminopimelate aminotransferase (DapL): a putative target for the development of narrow-spectrum antibacterial compounds. Front Microbiol 5:509

    Article  PubMed  PubMed Central  Google Scholar 

  • Trigoso YD, Evans RC, Karsten WE, Chooback L (2016) Cloning, expression, and purification of histidine-tagged Escherichia coli dihydrodipicolinate reductase. PLoS One 11:e0146525

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Ufaz S, Galili G (2008) Improving the content of essential amino acids in crop plants: goals and opportunities. Plant Physiol 147:954–961

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • van der Meer IM, Bovy AG, Bosch D (2001) Plant-based raw material: improved food quality for better nutrition via plant genomics. Curr Opin Biotechnol 12:488–492

    Article  PubMed  Google Scholar 

  • Vauterin M, Frankard V, Jacobs M (1999) The Arabidopsis thaliana DHDPS gene encoding dihydrodipicolinate synthase, key enzyme of lysine biosynthesis, is expressed in a cell-specific manner. Plant Mol Biol 39:695–708

    Article  PubMed  CAS  Google Scholar 

  • Voss JE, Scally SW, Taylor NL, Atkinson SC, Griffin MD, Hutton CA, Parker MW, Alderton MR, Gerrard JA, Dobson RC, Dogovski C, Perugini MA (2010) Substrate-mediated stabilization of a tetrameric drug target reveals Achilles heel in anthrax. J Biol Chem 285:5188–5195

    Article  PubMed  CAS  Google Scholar 

  • Wallsgrove RM, Mazelis M (1980) The enzymology of lysine biosynthesis in higher plants: complete localization of the regulatory enzyme dihydrodipicolinate synthase in the chloroplasts of spinach leaves. FEBS Lett 116:189–192

    Article  PubMed  CAS  Google Scholar 

  • Wallsgrove RM, Mazelis M (1981) Spinach leaf dihydrodipicolinate synthase: partial purification and characterization. Phytochemistry 20:2651–2655

    Article  CAS  Google Scholar 

  • Wang W, Galili G (2016) Transgenic high-lysine rice—a realistic solution to malnutrition? J Exp Bot 67:4009–4011

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yang QQ, Zhang CQ, Chan ML, Zhao DS, Chen JZ, Wang Q, Li QF, Yu HX, Gu MH, Sun SS, Liu QQ (2016) Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance. J Exp Bot 67:4285–4296

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

TPSC acknowledges the National Health and Medical Research Council of Australia for fellowship support (APP1091976) and MAP the Australian Research Council for funding support (DP150103313). We would like to acknowledge the La Trobe University-Comprehensive Proteomics Platform, La Trobe University, Melbourne, Australia for providing the infrastructure and expertise. We would also like to acknowledge the support and assistance of the beamline scientists at the Australian Synchrotron, Victoria, Australia. Finally, we thank all members of the Perugini laboratory for helpful discussions during the preparation of this manuscript.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Matthew A. Perugini or Tatiana P. Soares da Costa.

Ethics declarations

Conflict of interest

There are no competing interests to declare.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gupta, R., Hogan, C.J., Perugini, M.A. et al. Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum. Planta 248, 381–391 (2018). https://doi.org/10.1007/s00425-018-2894-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-018-2894-x

Keywords

Navigation