Abstract
The hydrolysis of starch is a key step in plant germination, which also has relevance in the malting and brewing processes for beer and spirit production. Gaps in knowledge about this metabolic process exist that cannot easily be addressed using traditional genetic techniques, due to functional redundancy in many of the enzyme activities required for alpha-glucan metabolism in cereal crop species. Chemical inhibitors provide opportunities to probe the role of carbohydrate-active enzymes and the phenotypes associated with inhibition of specific enzymes. Iminosugars are the largest group of carbohydrate-active enzyme inhibitors and represent an underused resource for the dissection of plant carbohydrate metabolism. Herein we report a method for carrying out a reverse chemical genetic screen on α-glucosidase, the enzyme that catalyzes the final step in starch degradation during plant germination, namely the hydrolysis of maltose to release glucose. This chapter outlines the use of a high-throughput screen of small molecules for inhibition of α-glucosidase using a colorimetric assay which involves the substrate p-nitrophenyl α-d-glucopyranoside. Identified inhibitors can be further utilized in phenotypic screens to probe the roles played by amylolytic enzymes. Furthermore this 96-well plate-based method can be adapted to assay exo-glycosidase activities involved in other aspects of carbohydrate metabolism.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
National Geographic Magazine What the World Eats. http://www.nationalgeographic.com/what-the-world-eats/. Accessed 6 June 2017
BeMiller JN, Whistler RL (2009) Starch: chemistry and technology. Academic Press, San Diego
Borrill P, Adamski N, Uauy C (2015) Genomics as the key to unlocking the polyploid potential of wheat. New Phytol 208(4):1008–1022
Rugen MD, Andriotis VME, Field RA (2017) Small-molecule probes of plant glycopolymer metabolism. In: Reference module in chemistry, molecular sciences and chemical engineering. Elsevier, Oxford
Tóth R, van der Hoorn RAL (2010) Emerging principles in plant chemical genetics. Trends Plant Sci 15(2):81–88
O’Connor CJ, Laraia L, Spring DR (2011) Chemical genetics. Chem Soc Rev 40(8):4332–4345
Robert S, Raikhel NV, Hicks GR (2009) Powerful partners: Arabidopsis and chemical genomics. Arab Book 7:e0109
Dejonghe W, Russinova E (2017) Plant chemical genetics: from phenotype-based screens to synthetic biology. Plant Physiol, vol 174, pp 5–20
Fu H (2012) Chemical genomics. Cambridge University Press, Cambridge
Abdurakhmonov IY (2016) Genomics era for plants and crop species–advances made and needed tasks ahead. In: Plant genomics. InTech, London
Andriotis VM, Rejzek M, Rugen MD, Svensson B, Smith AM, Field RA (2016) Iminosugar inhibitors of carbohydrate-active enzymes that underpin cereal grain germination and endosperm metabolism. Biochem Soc Trans 44(1):159–165
Borges de Melo E, da Silveira Gomes A, Carvalho I (2006) α- and β-glucosidase inhibitors: chemical structure and biological activity. Tetrahedron 62(44):10277–10302
Bras NF, Cerqueira NM, Ramos MJ, Fernandes PA (2014) Glycosidase inhibitors: a patent review (2008-2013). Expert Opin Ther Pat 24(8):857–874
Gloster TM, Vocadlo DJ (2012) Developing inhibitors of glycan processing enzymes as tools for enabling glycobiology. Nat Chem Biol 8(8):683–694
Alonzi DS, Scott KA, Dwek RA, Zitzmann N (2017) Iminosugar antivirals: the therapeutic sweet spot. Biochem Soc Trans 45(2):571–582
Zamoner LOB, Aragão-Leoneti V, Mantoani SP, Rugen MD, Nepogodiev SA, Field RA, Carvalho I (2016) CuAAC click chemistry with N-propargyl 1,5-dideoxy-1,5-imino-D-gulitol and N-propargyl 1,6-dideoxy-1,6-imino-D-mannitol provides access to triazole-linked piperidine and azepane pseudo-disaccharide iminosugars displaying glycosidase inhibitory properties. Carbohydr Res 429:29–37
Li C, Li QG, Dunwell JM, Zhang YM (2012) Divergent evolutionary pattern of starch biosynthetic pathway genes in grasses and dicots. Mol Biol Evol 29(10):3227–3236
Stevens KL, Molyneux RJ (1988) Castanospermine—a plant growth regulator. J Chem Ecol 14(6):1467–1473
Bamforth CW (2009) Current perspectives on the role of enzymes in brewing. J Cereal Sci 50(3):353–357
Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408(6814):796
Payen A, Persoz J-F (1833) Mémoire sur la diastase, les principaux produits de ses réactions, et leurs applications aux arts industriels. Annales de Chimie et de Physique 53(2):73–92
Pfister B, Zeeman SC (2016) Formation of starch in plant cells. Cell Mol Life Sci 73(14):2781–2807
Konishi Y, Aitani M, Nakatani N (1998) Effects of Bay m 1099, an alpha-glucosidase inhibitor, on starch degradation in germinating mung beans. Biosci Biotechnol Biochem 62(1):142–144
Naested H, Kramhøft B, Lok F, Bojsen K, Yu S, Svensson B (2006) Production of enzymatically active recombinant full-length barley high pI α-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification. Protein Expr Purif 46(1):56–63
Stanley D, Rejzek M, Naested H, Smedley M, Otero S, Fahy B, Thorpe F, Nash RJ, Harwood W, Svensson B, Denyer K, Field RA, Smith AM (2011) The role of α-glucosidase in germinating barley grains. Plant Physiol 155(2):932–943
Andriotis VME, Saalbach G, Waugh R, Field RA, Smith AM (2016) The maltase involved in starch metabolism in barley endosperm is encoded by a single gene. PLoS One 11(3):e0151642
Andriotis VM, Rejzek M, Barclay E, Rugen MD, Field RA, Smith AM (2016) Cell wall degradation is required for normal starch mobilisation in barley endosperm. Sci Rep 6:33215
Mega T (2004) Conversion of the carbohydrate structures of glycoproteins in roots of Raphanus sativus using several glycosidase inhibitors. The. J Biochem 136(4):525–531
Mega T (2005) Glucose trimming of N-glycan in endoplasmic reticulum is indispensable for the growth of Raphanus sativus seedling (kaiware radish). Biosci Biotechnol Biochem 69(7):1353–1364
Nash RJ, Kato A, Yu CY, Fleet GW (2011) Iminosugars as therapeutic agents: recent advances and promising trends. Future Med Chem 3(12):1513–1521
Asano N, Nash RJ, Molyneux RJ, Fleet GWJ (2000) Sugar-mimic glycosidase inhibitors: natural occurrence, biological activity and prospects for therapeutic application. Tetrahedron Asymmetry 11(8):1645–1680
Frandsen TP, Lok F, Mirgorodskaya E, Roepstorff P, Svensson B (2000) Purification, enzymatic characterization, and nucleotide sequence of a high-isoelectric-point α-glucosidase from barley malt. Plant Physiol 123(1):275–286
Im H, Henson CA (1995) Characterization of high pI α-glucosidase from germinated barley seeds: substrate specificity, subsite affinities and active-site residues. Carbohydr Res 277(1):145–159
Acknowledgments
The authors would like to thank Alison M Smith and Vasilios Andriotis for helpful discussions and advice. This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC, UK) Institute Strategic Programme Grant (MET) [BB/J004561/1] to the John Innes Centre, a BBSRC-Crop Improvement Research Club (CIRC) grant BB/I017291/1 to A.M.S. and R.A.F, and BBSRC PhD studentship BB/J500069/1 to M.D.R.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rugen, M.D., Rejzek, M., Naested, H., Svensson, B., Field, R.A. (2018). High-Throughput In Vitro Screening for Inhibitors of Cereal α-Glucosidase. In: Fauser, F., Jonikas, M. (eds) Plant Chemical Genomics. Methods in Molecular Biology, vol 1795. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7874-8_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7874-8_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7873-1
Online ISBN: 978-1-4939-7874-8
eBook Packages: Springer Protocols