Skip to main content
SpringerLink
Log in

Identification and functional characterization of sorbitol-6-phosphate dehydrogenase protein from rice and structural elucidation by in silico approach

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

Abstract

The sorbitol-6-phosphate dehydrogenase (S6PDH) is a key enzyme for sorbitol synthesis and plays an important role in the alleviation of salinity stress in plants. Despite the huge significance, the structure and the mode of action of this enzyme are still not known. In the present study, sequence analysis, cloning, expression, activity assays and enzyme kinetics using various substrates (glucose-6-phosphate, sorbitol-6-phosphate and mannose-6-phosphate) were performed to establish the functional role of S6PDH protein from rice (Oryza sativa). For the structural analysis of the protein, a comparative homology model was prepared on the basis of percentage sequence identity and substrate similarity using the crystal structure of human aldose reductase in complex with glucose-6-phosphate and NADP+ (PDB ID: 2ACQ) as a template. Molecular docking was performed for studying the structural details of substrate binding and possible enzyme mechanism. The cloned sequence resulted into an active recombinant protein when expressed into a bacterial expression system. The purified recombinant protein was found to be active with glucose-6-phosphate and sorbitol-6-phosphate; however, activity against mannose-6-phosphate was not found. The K m values for glucose-6-phosphate and sorbitol-6-phosphate were found to be 15.9 ± 0.2 and 7.21 ± 0.5 mM, respectively. A molecular-level analysis of the active site of OsS6PDH provides valuable information about the enzyme mechanism and requisite enantioselectivity for its physiological substrates. Thus, the fundamental studies of structure and function of OsS6PDH could serve as the basis for the future studies of bio-catalytic applications of this enzyme.

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

Access this article

Log in via an institution

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
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12

Similar content being viewed by others

Abbreviations

S6PDH:

Sorbitol-6-phosphate dehydrogenase

OsS6PDH:

Sorbitol-6-phosphate dehydrogenase of rice

G-6-P:

Glucose-6-phosphate

S-6-P:

Sorbitol-6-phosphate

M-6-P:

Mannose-6-phosphate

ROAD:

Rice oligonucleotide array database

PDB:

Protein data bank

References

  • Alia, Saradhi PP, Mohanty P (1993) Proline in relation to free radical production in seedlings of Brassica juncea raised under sodium chloride stress. Plant Soil 155(156):497–500

    Article  Google Scholar 

  • Askonas LJ, Ricigliano JW, Penning TM (1991) The kinetic mechanism catalysed by homogeneous rat liver 3α-hydroxysteroid dehydrogenase. Evidence for binary and ternary dead-end complexes containing non-steroidal anti-inflammatory drugs. Biochem J 278:835–841

    CAS  PubMed Central  PubMed  Google Scholar 

  • Bellaloui N, Yadavc RC, Chern MS, Hu H, Gillen AM, Greve C, Dandekar AM, Ronald PC, Brown PH (2003) Transgenically enhanced sorbitol synthesis facilitates phloem-boron mobility in rice. Physiologia Plantarum 117:79–84

    Article  CAS  Google Scholar 

  • Berrisford JM, Hounslow AM, Akerboom J, Hagen WR, Brouns SJ, van der Oost J, Murray IA, Blackburn GM, Waltho JP, Rice DW, Baker PJ (2006) Evidence supporting a cis-enediol-based mechanism for Pyrococcus furiosus phosphoglucose isomerase. J Mol Biol 358:1353–1366

    Article  CAS  PubMed  Google Scholar 

  • Colovos C, Yeates TO (1993) Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci 2:1511–1519

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cooper WC, Jin Y, Penning TM (2007) Elucidation of a complete kinetic mechanism for a mammalian hydroxysteroid dehydrogenase (HSD) and identification of all enzyme forms on the reaction coordinate. The example of rat liver 3 alpha-HSD (AKR1c9). J Biol Chem 282:33484–33493

    Article  CAS  PubMed  Google Scholar 

  • Couture JF, Legrand P, Cantin L, Labrie F, Luu-The V, Breton R (2004) Loop relaxation, a mechanism that explains the reduced specificity of rabbit 20a-hydroxysteroid dehydrogenase, a member of the aldo-ketoreductase super family. J Mol Biol 339:89–102

    Article  CAS  PubMed  Google Scholar 

  • Couture JF, De Jesus-Tran KP, Roy AM, Cantin L, Cote PL, Legrand P, Luu-The V, Labrie F, Breton R (2005) Comparison of crystal structures of human type 3 3α-hydroxysteroid dehydrogenase reveals an “induced-fit” mechanism and a conserved basic motif involved in the binding of androgen. Protein Sci 14:1485–1497

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific, San Carlos

    Google Scholar 

  • Emanuelsson O, Nielsen H, von Heijne G, Chloro P (1999) A neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Figueroa CM, Iglesias AA (2010) Aldose-6-phosphate reductase from apple leaves: importance of the quaternary structure for enzyme activity. Biochimie 92:81–88

    Article  CAS  PubMed  Google Scholar 

  • Gao M, Tao R, Miura K, Dandekar AM, Sugiura A (2001) Transformation of Japanese persimmon (Diospyros kaki Thunb.) with apple cDNA encoding NADP-dependent Sorbitol-6-phosphate dehydrogenase. Plant Sci 160:837–845

    Article  CAS  PubMed  Google Scholar 

  • Garcia AB, Engler JA, Lyer S, Cerats T, Montagu MV, Caplan AB (1997) Effects of osmoprotectants upon NaCl stress in rice. Plant Physiol 115:159–169

    CAS  PubMed Central  PubMed  Google Scholar 

  • Gouet P, Courcelle E, Stuart DI, Metoz F (1999) ESPript: multiple sequence alignments in PostScript. Bioinformatics 15:305–308

    Article  CAS  PubMed  Google Scholar 

  • Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR (2012) Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminform 4:17

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hirai M (1981) Purification and characteristics of Sorbitol-6-phosphate dehydrogenase from loquat leaves. Plant Physiol 67:221–224

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Huizhong W, Danian H, Ruifang L, Junjun L, Qian Q, Xuexian P (2000) Salt tolerance of transgenic rice (Oryza sativa L.) with mtlD gene and gutD gene. Chin Sci Bull 45:1685–1690

    Article  Google Scholar 

  • Jung KH, Dardick C, Bartley LE, Cao P, Phetsom J, Canlas P, Seo YS, Shultz M, Ouyang S, Yuan Q, Frank BC, Ly E, Zheng L, Jia Y, Hsia AP, An K, Chou HH, Rocke D, Lee GC, Schnable PS, An G, Buell CR, Ronald PC (2008) Refinement of light-responsive transcript lists using rice oligonucleotide arrays: evaluation of gene-redundancy. PLoS One 3:e3337

    Article  PubMed Central  PubMed  Google Scholar 

  • Kanayama Y, Yamaki S (1993) Purification and properties of NADP-dependent sorbitol-6-phosphate dehydrogenase from apple seedling. Plant Cell Physiol 34:819–823

    CAS  Google Scholar 

  • Kanayama Y, Mori H, Imaseki H, Yamaki S (1992) Nucleotide sequence of a cDNA encoding NADP-Sorbitol-6-phosphate dehydrogenase from apple. Plant Physiol 100:1607–1608

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kratzer R, Wilson DK, Nidetzky B (2006) Catalytic mechanism and substrate selectivity of aldo-keto reductases: insights from structure-function studies of Candida tenuisxylose reductase. IUBMB Life 58:499–507

    Article  CAS  PubMed  Google Scholar 

  • Krishnamurthy R, Bhagwat KA (1989) Polyamines as modulators of salt tolerance in rice cultivars. Plant Physiol 91:500–504

    Article  CAS  PubMed Central  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 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948

    Article  CAS  PubMed  Google Scholar 

  • Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291

    Article  CAS  Google Scholar 

  • Li F, Lei HJ, Zhao XJ, Tian RR, Li TH (2012) Characterization of three sorbitol transporter genes in micropropagated apple plants grown under drought stress. Plant Mol Biol Rep 30:123–130

    Article  CAS  Google Scholar 

  • Liang D, Cui M, Wu S, Ma F (2012) Genomic structure, sub-cellular localization and promoter analysis of the gene encoding Sorbitol-6-phosphate dehydrogenase from apple. Plant Mol Biol Rep 30:904–914

    Article  CAS  Google Scholar 

  • Loescher WH, Everard JD (2000) Regulation of sugar alcohol biosynthesis. In: Leegood RC, Sharkey TD, von Caemmerer S (eds) Photosynthesis: physiology and metabolism. Kluwer Academic Pubs, Dordrecht, pp 275–299

    Google Scholar 

  • Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 16:2785–2791

    Article  Google Scholar 

  • Negm FB, Loescher WH (1981) Characterization and partial purification of aldose-6-phosphate reductase (alditol-6- phosphate:NADP 1-oxidoreductase) from apple leaves. Plant Physiol 67:139–142

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Neuhauser W, Haltrich D, Kulbe KD, Nidetzky B (1997) NAD (P) H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme. Biochem J 326:683–692

    CAS  PubMed Central  PubMed  Google Scholar 

  • Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Protein Eng 10:1–6

    Article  CAS  PubMed  Google Scholar 

  • Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815

    Article  CAS  PubMed  Google Scholar 

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

    Google Scholar 

  • Schlegel BP, Jez JM, Penning TM (1998) Mutagenesis of 3α-hydroxysteroid dehydrogenase reveals a “push-pull” mechanism for proton transfer in aldo-keto reductases. Biochemistry 37:3538–3548

    Article  CAS  PubMed  Google Scholar 

  • Sato Y, Antonio B, Namiki N, Takehisa H, Minami H, Kamatsuki K, Sugimoto K, Shimizu Y, Hirochika H, Nagamura Y (2011) RiceXPro: a platform for monitoring gene expression in japonica rice grown under natural field conditions. Nucleic Acids Res 39:D1141–D1148

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sheveleva EV, Marquez S, Chmara W, Zegeer A, Jensen RG, Bohnert HJ (1998) Sorbitol-6-phosphate dehydrogenase expression in transgenic tobacco. Plant Physiol 117:831–839

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tao R, Uratsu SL, Dandekar AM (1995) Sorbitol synthesis in transgenic tobacco with apple cDNA encoding NADP- dependent sorbitol-6-phosphate dehydrogenase. Plant Cell Physiol 36:525–532

    CAS  PubMed  Google Scholar 

  • Teo G, Suzuki Y, Uratsu SL, Lampinen B, Ormonde N, Hu WK, DeJong TM, Dandekar AM (2006) Silencing leaf sorbitol synthesis alters long-distance partitioning and apple fruit quality. PNAS 103:18842–18847

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Urzhumtsev A, TeteFavier F, Mitschler A, Barbanton J, Barth P, Urzhumtseva L (1997) A ‘specificity’ pocket inferred from the crystal structures of the complexes of aldose reductase with the pharmaceutically important inhibitors tolrestat and sorbinil. Structure 5:601–612

    Article  CAS  PubMed  Google Scholar 

  • Wiederstein, Sippl (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35:W407–W410

    Article  PubMed Central  PubMed  Google Scholar 

  • Wilson DK, Nakano T, Petrash JM, Quiocho FA (1997) Structure studies ofaldo-keto reductase inhibition. In: Weiner H, Lindahl R, Crabb DW, Flynn TG (eds) Enzymology and Molecular Biology of Carbonyl Metabolism 6. Plenum Press, New York, pp 435–442

    Google Scholar 

  • Yamaki S (1980) Properties and functions of sorbitol-6-phosphate dehydrogenase, sorbitol dehydrogenase and sorbitol oxidase in fruit and cotyledon of apple (Malus pumila Mill. var. domestica Schneid.). J Jpn Soc Hort Sci 49:429–434

    Article  CAS  Google Scholar 

  • Yamaki S (1981) Subcellular localization of NADP-dependent Sorbitol-6-phosphate dehydrogenase in protoplast from apple cotyledons. Plant Cell Physiol 22:359–367

    CAS  Google Scholar 

  • Zhang J, Yao Y, Streeter JG, Ferree DC (2010) Influence of soil drought stress on photosynthesis, carbohydrates and the nitrogen and phosphorus absorb in different section of leaves and stem of Fugi/M.9EML, a young apple seedling. Afr J Biotechnol 9:5320–5325

    CAS  Google Scholar 

  • Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–71

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Department of Biotechnology, IIT Roorkee for providing cloning and computational facilities. This work has been supported financially by Department of Biotechnology (DBT), India. Rajbala thanks Ministry of Human Resource Development (MHRD), India for the financial support. We also thank Dr P. Selvakumar and Ms Sonali Dhindwal for helpful discussions.

Author information

Authors and Affiliations

  1. Molecular Biology and Proteomics Lab, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, Uttarakhand, India

    Rajbala Yadav & Ramasare Prasad

Authors
  1. Rajbala Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ramasare Prasad
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ramasare Prasad.

Electronic supplementary material

Below is the link to the electronic supplementary material.

425_2014_2076_MOESM1_ESM.jpg

Sup fig. 1 HPLC chromatograms showing the increased concentration of sorbitol in salt-stressed rice plants.(a) HPLC chromatogram of control plant (b) HPLC chromatogram of salt-stressed (150 mM) plant.(JPEG 125 kb)

425_2014_2076_MOESM2_ESM.tif

Sup Fig. 2 The comparative stereo view of OsS6PDH (full-length) and OsS6PDH (without N-terminal extension) showing the inhibition of active sites by N-terminal. N-terminal extension has been shown in yellow. Protein (without N-terminal extension) shown in a, c and e is active while in case of the protein (full-length) shown in b, d and f, N-terminal extension inhibits the active sites and substrate-binding sites. (TIFF 1616 kb)

425_2014_2076_MOESM3_ESM.png

Sup fig. 3 phyre 2 model of protein showing comparative folding in case of without N-terminal extension (a and c) and with N-terminal extension (b and d). (PNG 667 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yadav, R., Prasad, R. Identification and functional characterization of sorbitol-6-phosphate dehydrogenase protein from rice and structural elucidation by in silico approach. Planta 240, 223–238 (2014). https://doi.org/10.1007/s00425-014-2076-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-014-2076-4

Keywords

Search

Navigation