Heterologous Expression and Functional Characterization of Catalytic Subunit of Rice Acetohydroxyacid Synthase

Author(s): Ghazaleh Arabzadeh, Azar Shahpiri*.

Journal Name: Protein & Peptide Letters

Volume 26 , Issue 3 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Background: Acetohydroxyacid Synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched chain amino acids. AHAS is the common target site of five herbicide chemical groups: sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl-thiobenzoates, and sulfonyl-aminocarbonyl-triazolinone.

Objective: The purification of protein enabled us to study the physical and biochemical properties of the enzyme. In addition in vitro activity of this enzyme was tested in the presence of four different sulfonylureaherbicides and the feedback regulation of enzyme was analyzed in the presence of branched amino acids.

Methods: The gene encoding catalytic subunit of rice AHAS (cOsAHAS) without part of the chloroplast transit sequence was cloned into the bacterial expression vector pET41a and heterologously expressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST).The soluble protein was purified using affinity chromatography. The measurement of GSTOsAHAS activity was performed under optimized conditions at present of branched-chain amino acids and sulfonylurea herbicides independently.

Results: The optimum pH and temperature for GST-cOsAHAS activity was 8.0 and 37 °C, respectively. The specific activity and Km value of this enzyme toward pyruvate were 0.08 U/mg and 30 mM, respectively.GST-cOsAHAS was inhibited by herbicides tribenuron, sulfosulfuron, nicosulfuron and bensulfuron while the enzyme was insensitivieto end products.

Conclusion: These results suggest that the recombinant form of GST-cOsAHAS is functionally active and carries the binding site for sulfynylurea herbicides. Furthermore, GST-cOsAHAS was insensitive to feedback inhibition by endproducts which indicates the existence of a regulator subunit in rice AHAS as previously has been described in other plant AHASs.

Keywords: Acetohydroxyacid synthase, catalytic subunit, functional characterization, rice, sulfynylurea herbicides, branched chain amino acids.

Umbarger, H.E. Synthesis of Amino Acids and Proteins. In: MTP International Review of Science, Arbstein, H.R.V.; Ed.; University Park Press: Baltimore. 1975, 7, 1-56.
Duggleby, R.G.; Pang, S.S. Acetohydroxyacid synthase. J. Biochem. Mol. Biol., 2000, 33, 1-36.
Duggleby, R.G.; McCourt, J.A.; Guddat, L.W. Structure and mechanism of inhibition of plant acetohydroxyacid synthase. Plant Physiol. Biochem., 2008, 46, 309-324.
Muller, Y.A.; Lindqvist, Y.; Furey, W.; Schulz, G.E.; Jordan, F.; Schneider, G. A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase. Structure, 1993, 1, 95-103.
Chaleff, R.S.; Mauvais, C.J. Acetolactate synthase is the site action of two sulfonylurea herbicides in higher plants. Science, 1984, 224, 1443-1445.
Schloss, J.V.; Ciskanik, L.M.; Van Dyk, D.E. Origin of the herbicide binding site of acetolactate synthase. Nature, 1988, 331, 360-362.
Subramanian, M.V.; Loney-Gallant, V.; Dias, J.M.; Mireles, M.C. Acetolactate synthase inhibiting herbicides bind to the regulatory site. Plant Physiol., 1991, 96, 310-313.
Mourad, G.; King, J. Effect of four classes of herbicides on the growth and acetolactate synthase activity in several variants of Arabidopsis thaliana. Planta, 1992, 188, 491-497.
Hershey, H.P.; Schwartz, L.J.; Gale, J.P.; Abell, L.M. Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia. Plant Mol. Biol., 1999, 40, 795-806.
Eoyang, L.; Silverman, P. Role of small subunit (ilvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity. J. Bacteriol., 1986, 166(3), 901-904.
Falco, C.; Dumas, K.S.; Livak, K.J. Nucleotide sequence of the yeast ILV2 gene which encodes acetolactate synthase. Nucleic Acids Res., 1985, 13, 4011-4027.
Bascomb, N.F.; Gutteridge, J.K.; Smith, J.K.; Leto, K.J. Import of in vitro synthesized acetolactate synthase into isolated pea chloroplast. J. Cell. Biochem. Suppl., 1987, 11, 88-89.
Shaner, D.L.; Anderson, P.C.; Stidham, M.A. Imidazolinones: Potent inhibitors of acetohydroxyacid synthase. Plant Physiol., 1984, 76, 545-546.
Chang, A.K.; Duggleby, R.G. Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase. Biochem. J., 1997, 327, 161-169.
Kim, H.J.; Chang, S.I. Functional expression of Nicotiana tabacum acetolactate synthase gene in Escherichia coli. J. Biochem. Mol. Biol., 1995, 28, 265-270.
Rutledge, R.G.; Ouellet, T.; Hattori, J.; Miki, B.L. Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family. Mol. Gen. Genet., 1991, 229, 31-40.
Singh, B.K.; Stidham, M.A.; Shaner, D.L. Assay of acetohydroxyacid synthase. Anal. Biochem., 1988, 171, 173-179.
Westerfeld, W.W. A colorimetric determination of blood acetoin. J. Biol. Chem., 1945, 161, 495-502.
Muhitch, M.J.; Shaner, D.L.; Stidham, M.A. Imidazolinones and acetohydroxyacid synthase from higher plants. Plant Physiol., 1987, 83, 451-456.
Chang, S.I.; Kang, M.K.; Choi, J.D.; Namgoong, S.K. Soluble overexpression in Escherichia coli, and purification and characterization of wild-type recombinant tobacco acetolactate synthase. Biochem. Biophys. Res. Commun., 1997, 234, 549-553.
Ray, T.B. Sulfonylurea herbicides as inhibitors of amino acid biosynthesis in plants. Trends Biochem. Sci., 1986, 11, 180-183.
Brown, H.M.; Kearney, P.C. Plant biochemistry.In: Environmental Properties, and Global Impact of the Sulfonylurea Herbicides; Synthesis and Chemistry of Agrochemicals II; Baker, D.R.; Fenyes, J.G.; Moberg, W.K., Eds.; American Chemical Society: Washington, DC, 1991, pp. 32-49.
Singh, B.; Szamosi, I.; Hand, J.M.; Misra, R. Arabidopsis acetohydroxy acid synthase expressed in Escherichia coli is insensitive to the feedback inhibitors. Plant Physiol., 1992, 99, 812-816.
Southan, M.D.; Copeland, L. Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves. Physiol. Plant., 1996, 98, 824-832.
Durner, J.; Gailus, V.; Boger, P. New aspects on inhibition of plant acetolactate synthase by chlorsulfuron and imazaquin. Plant Physiol., 1991, 95, 1144-1149.
Lee, Y.T.; Duggleby, R.G. Identification of the regulatory subunit of Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit. Biochemistry, 2001, 40, 6836-6844.
Zhao, Y.; Niu, C.; Wen, X.; Xi, Z. The minimum activation peptide from ilvH can activate the catalytic subunit of AHAS from different species. ChemBioChem, 2013, 14, 746-752.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [176 - 183]
Pages: 8
DOI: 10.2174/0929866525666181114153727
Price: $58

Article Metrics

PDF: 17