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Characterization of SU1 Isoamylase, a Determinant of Storage Starch Structure in Maize1

Afroza Rahman, Kit-sum Wong, Jay-lin Jane, Alan M. Myers, and Martha G. James*

Department of Biochemistry and Biophysics (A.R., A.M.M., M.G.J.), and Department of Food Sciences and Human Nutrition (K.-s.W., J.-I.J.), Iowa State University, Ames, Iowa 50011

Function of the maize (Zea mays) gene sugary1 (su1) is required for normal starch biosynthesis in endosperm. Homozygous su1- mutant endosperms accumulate a highly branched polysaccharide, phytoglycogen, at the expense of the normal branched component of starch, amylopectin. These data suggest that both branched polysaccharides share a common precursor, and that the product of the su1 gene, designated SU1, participates in kernel starch biosynthesis. SU1 is similar in sequence to alpha -(1right-arrow6) glucan hydrolases (starch-debranching enzymes [DBEs]). Specific antibodies were produced and used to demonstrate that SU1 is a 79-kD protein that accumulates in endosperm coincident with the time of starch biosynthesis. Nearly full-length SU1 was expressed in Escherichia coli and purified to apparent homogeneity. Two biochemical assays confirmed that SU1 hydrolyzes alpha -(1right-arrow6) linkages in branched polysaccharides. Determination of the specific activity of SU1 toward various substrates enabled its classification as an isoamylase. Previous studies had shown, however, that su1- mutant endosperms are deficient in a different type of DBE, a pullulanase (or R enzyme). Immunoblot analyses revealed that both SU1 and a protein detected by antibodies specific for the rice (Oryza sativa) R enzyme are missing from su1- mutant kernels. These data support the hypothesis that DBEs are directly involved in starch biosynthesis.

1   This work was supported by the U.S. Department of Agriculture (grant no. 95-34340-1605, North Central Biotechnical Initiative subgrant agreement no. 593-0220-06 to M.G.J. and A.M.M.). This is journal paper no. J-17677 of project no. 3390 of the Iowa Agriculture and Home Economics Experiment Station, Ames.
*   Corresponding author; e-mail mgjames{at}iastate.edu; fax 1-515-294-0453.

Plant Physiol. (1998) 117: 425-435
Copyright Clearance Center:   0032-0889/98/117/0425/11
© 1998 American Society of Plant Physiologists




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