Purification and molecular genetic characterization of ZPU1, a pullulanase-type starch-debranching enzyme from maize

Abstract

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.

Figure 1

A, Overlapping bacteriophage λ clones provide the full-length Zpu1 cDNA. The 3261-bp nucleotide sequence of the Zpu1 cDNA was determined from overlapping sequences of three λ clones. The Zpu1 cDNA sequence is available as accession no. AF080567. B, Primary sequence alignment of ZPU1 with rice RE. Amino acid residues deduced from the Zpu1 cDNA (Zm) and rice RE cDNA (Os; accession no. D50602) are shown in alignment. Boxed residues are the same in both polypeptides. The asterisk designates the transit peptide cleavage site known in RE.

Figure 2

Multiple sequence alignment of pullulanase- and isoamylase-type DBEs from higher plants and prokaryotes. DBEs are grouped based on characterized enzymatic activity and/or sequence similarity; any polypeptide within a class is significantly more similar to others within that group than to those of the other class. Conservative substitutions in the consensus sequences are noted when they fall into the functional groups defined by Dayhoff and Orcutt (1979), which are AGPST, ILMV, HKR, DENQ, RWY, and C. Residues invariant in all 19 sequences are noted by asterisks. Rare exceptions to the consensus sequence are underlined. Numerals refer to amino acid position beginning at the first ATG codon of the open reading frame. The number of nonconserved amino acids adjacent to each conserved motif is indicated. Conserved motifs in boxes are present in both the pullulanase- and isoamylase-type classes, whereas conserved motifs without boxes are specific to one of the classes as indicated. Motifs I to IV are those defined previously that occur in all members of the α-amylase superfamily (Jesperson et al., 1993), and are numbered accordingly. Abbreviations and accession numbers are provided for the following DBES. Pullulanases and pullulanase-type DBEs: Bth (Bacteroides thetaiotaomicron, U67061); Csa (Caldicellulosiruptor saccharolyticus, L39876); Kae (Klebsiella aerogenes, M16187); Hvu (Hordeum vulgare, AF022725); Kpn (Klebsiella pneumoniae, X52181); Osa (Oryza sativa, D50602); Sol (Spinacia oleracea, X83969); Tma (Thermotoga maritima, AJ001087); Tsp (Thermus sp. IM6501, AF060205); Zma (Z. mays, AF080567, this study). Isoamylases and isoamylase-type DBEs: Art (artificial gene, A10906); Ath (Arabidopsis, AF002109); Eco (E. coli, U18997); Fla (Flavobacterium sp., U90120); Psu (Pseudomonas sp., A28109, A37035); Sac (Sulfolobus acidocaldarius, D83245); Sso (Sulfolobus solfataricus, Y08256); Syn1 (Synechocystis sp., U44761); Syn2 (Synechocystis sp., D90908); and Zma (Z. mays, U18908).

Figure 3

The copy number of the zpu1 locus was determined by gel-blot analysis of genomic DNA. DNA from maize inbred W64A was digested with the indicated restriction enzymes (B, BamHI; K, KpnI; N, NotI; P, PstI; S, SstI; X, XbaI; and Xh, XhoI). The gel blot was hybridized at high stringency with probe EE2.3 (left panel), then stripped of probe and hybridized with probe EH.68 (right panel) of the Zpu1 cDNA.

Figure 4

Total RNAs from various sources were hybridized with probe EE2.3. The RNAs as they appeared in the ethidium bromide (EtBr)-stained gel before transfer are shown to indicate RNA integrity and loading differences. A, RNAs from embryo (Em) and endosperm (En) harvested 20 DAP, seedling leaves (L), immature root (R), and immature tassel (T). B, RNAs from maize endosperm harvested at various times after pollination. C, RNAs from nonmutant (Su1) and mutant (su1-Ref ) kernels harvested 20 DAP.

Figure 5

A, Q-Sepharose chromatography. Fractions eluted from the column were assayed for DBE activity using pullulan (○) or amylopectin (▪) as a substrate. Products of the amylopectin reaction were complexed with iodine, and the change in A₅₅₀ value relative to untreated substrate was plotted (▵). Activity units for the amylopectin digestion are microgram maltose equivalents produced after a 2-h incubation of substrate with 100 μL of protein fraction. Activity units for the pullulan digestion are microgram maltotriose equivalents produced after a 2-h incubation of the substrate with 50 μL of the protein fraction. Fractions with DBE activity were subjected to immunoblot analysis with anti-ZPU1 or anti-SU1 antiserum, as indicated (right-hand panels). B, Gel-filtration chromatography. The peak fractions of pullulanase-type activity from Q-Sepharose columns were pooled, concentrated, and applied to a Sephacryl S-200 superfine gel-permeation column. DBE activity in the fractions eluted from this column was assayed using pullulan as the substrate; activity units are as described for A. Fractions were also assayed for the presence of ZPU1 by immunoblot analysis (right-hand panel). C, Mono-Q chromatography. The peak fractions (7–11) of pullulanase-type activity from the Sephacryl S-200 column were pooled, concentrated, and applied to a Pharmacia fast-protein liquid chromatography Mono-Q column. DBE activity in fractions eluted from this column was assayed using pullulan as the substrate; activity units are as described for A. Fractions were assayed for the presence of ZPU1 in immunoblots (right-hand panels). D, Affinity chromatography. The peak fractions of pullulanase-type activity from Q-Sepharose columns were pooled, concentrated, and applied to a column containing epoxy-activated Sepharose conjugated with cyclohexa-amylose. DBE activity in the four fractions eluted from this column was assayed using pullulan as the substrate; activity units are as described for A. Proteins from two of the fractions were separated by SDS-PAGE and the gel was silver stained; a duplicate gel was subjected to immunoblot analysis with anti-ZPU1 (right-hand panels).

Figure 6

Fractionation of DBEs from nonmutant and su1-Ref kernels by gel-permeation chromatography. A, DBEs in nonmutant kernels. Proteins from nonmutant kernels harvested 20 DAP were applied to a Sephacryl S-200 superfine gel-permeation column. Fractions were assayed for pullulanase-type DBE activity by measuring formation of new reducing ends after incubation with pullulan (activity units, ▪), and for isoamylase-type DBE activity by determination of iodine-complex absorbance maxima after incubation with amylopectin (A₅₅₀, □). Equivalent amounts of protein from fractions exhibiting DBE activity were analyzed for the presence of ZPU1 or SU1 on immunoblots with the indicated antisera. B, DBEs in su1-Ref kernels. Proteins from su1-Ref kernels harvested 20 DAP were fractionated and assayed for DBE activity, and immunoblot analysis was performed, as described for A. C, Comparative immunoblot analysis. Nonmutant proteins in fractions 9 to 12 from A (lanes +) and su1-Ref proteins in fractions 9 to 12 from B (lanes m) were subjected to immunoblot analysis with the anti-ZPU1 antibody. Equivalent amounts of protein were loaded, and each lane contained twice as much protein as the immunoblots shown in A and B.