Purification and molecular genetic characterization of ZPU1, a pullulanase-type starch-debranching enzyme from maize.
This study identified and purified specific isoamylase- and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.
Beatty
M K
MK
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames 50011, USA.
Rahman
A
A
Cao
H
H
Woodman
W
W
Lee
M
M
Myers
A M
AM
James
M G
MG
D000595:Amino Acid Sequence; D002874:Chromosome Mapping; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification*; D007517:Isoamylase / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D006026:Glycoside Hydrolases; C118576:ZPU1 protein, Zea mays; C100181:pullulanase; D007517:Isoamylase