Enzymatic properties and regulation of ZPU1, the maize pullulanase-type starch debranching enzyme.
Starch debranching enzymes (DBE) are required for mobilization of carbohydrate reserves and for the normal structural organization of storage glucan polymers. Two isoforms, the pullulanase-type DBEs and the isoamylase-type DBEs, are both highly conserved in plants. To address DBE functions in starch assembly and breakdown, this study characterized the biochemical activity of ZPU1, a pullulanase-type DBE that is the product of the maize Zpu1 gene. Assays showed directly that recombinant ZPU1 (ZPU1r) expressed in Escherichia coli functions as a pullulanase-type enzyme, and 1H-NMR spectroscopy demonstrated that ZPU1r specifically hydrolyzes alpha(1-->6) branch linkages. Preferred substrates for ZPU1r hydrolytic activity were determined, as were pH, temperature, and thermal stability optima. Kinetic properties of ZPU1r with respect to two substrates, beta-limit dextrin and pullulan, were determined. ZPU1 activity was increased by incubation with thioredoxin h, and native activity was decreased in mutants that accumulate soluble sugars, suggesting potential regulatory mechanisms.
Wu
Chunyuan
C
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 2152 Molecular Biology Building, Ames, Iowa 50011, USA.
Colleoni
Christophe
C
Myers
Alan M
AM
James
Martha G
MG
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D003313:Zea mays / Q000201:enzymology*
D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C118576:ZPU1 protein, Zea mays