Differential Chain-Length Specificities of Two Isoamylase-Type

70215 (297)

Biosci. Biotechnol. Biochem., 71, 70215-1–5, 2007 Note Differential Chain-Length Specificities of Two Isoamylase-Type Starch-Debranching Enzymes from Developing Seeds of Kidney Bean

Yoshinori TAKASHIMA, Takeshi SENOURA, Takayuki YOSHIZAKI, y Shigeki HAMADA, Hiroyuki ITO, and Hirokazu MATSUI

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received April 16, 2007; Accepted June 1, 2007; Online Publication, September 7, 2007 [doi:10.1271/bbb.70215]

Plant isoamylase-type starch-debranching enzymes mutants deficient in ISA3 show a starch-excess pheno- (ISAs) hydrolyze -1,6-linkages in -1,4/ -1,6-linked type in leaves and an altered amylopectin structure with polyglucans. Two ISAs, designated PvISA1/2 and Pv- increases in very short chains, suggesting that ISA3 ISA3, were purified from developing seeds of kidney plays a role in transitory starch degradation, particularly bean by ammonium sulfate fractionation and several in the breakdown of -limit dextrin produced by - column chromatographic procedures. The enzymes amylase.3,10) We have isolated cDNA clones for ISA1, displayedAdvance different substrate specificities for View polyglu- ISA2, and ISA3 from immature seeds of kidney bean, cans: PvISA1/2 showed broad chain-length specificities, and these were designated PvISA1, PvISA2, and whereas PvISA3 liberated specific chains with a DP of 2 PvISA3 cDNA respectively (the respective nucleotide to 4. sequence data can be accessed from the DDBJ Nucleo- tide Sequence Database under accession nos. AB300052, Key words: kidney bean (Phaseolus vulgaris L.); iso- AB300053, and AB300054). RNA gel blot analysis amylase; starch-debranching enzyme; amy- using each cDNA fragment as a probe revealed that the lopectin three ISA transcripts accumulated both in leaves and in developing seeds (Fig. 1A). In this paper, we describe Starch-debranching enzymes (DBEs) catalyze the the purification ofProofs two ISAs (designated PvISA1/2 and hydrolysis of -1,6 glucosidic linkages of polyglucans. PvISA3) from the developing seeds of kidney bean and In higher plants, two types of DBEs with distinct sub- the distinct substrate specificities of the purified ISAs. strate specificities have been identified: the pullulanase- The plant materials were prepared as described pre- type (EC 3.2.1.41) and the isoamylase-type (EC viously.11) Two ISA proteins were purified from large- 3.2.1.68).1) The isoamylase-type DBEs (ISAs) are sub- sized developing seeds by several successive column divided into three isoforms (ISA1, ISA2, and ISA3) chromatographic procedures. The ISA proteins in the based on the primary sequences.2,3) The sugrary-1 (su1) purification steps were monitored by native-PAGE/ mutants of maize and rice show a common phenotype in active staining, which was performed on polyacrylamide endosperms, which is characterized by reduced starch gels containing 0.3% (w/v) potato amylopectin (Sigma content and accumulation of water-soluble and highly Chemical, St. Louis, MO) according to the method of branched polysaccharides (phytoglycogen).4–6) Since the Fujita et al.12) Approximately 1 kg of large-sized devel- su1 loci encode the genes for ISAs,5,7) it is believed that oping seeds was homogenized with Polytron in 5-liter of the formation of the highly ordered amylopectin in vivo extraction buffer (40 mM MOPS–NaOH, pH 7.0, con- requires the actions not only of starch synthases and taining 4 mM DTT, 5 mM MgCl2, and 1 mM PMSF). The starch-branching enzymes but also of DBEs. Recent homogenate was filtrated through three layers of gauze studies suggest that ISA2 has no catalytic activity due to and the supernatant (crude extract) was obtained by a lack of catalytic residues and that ISA1 and ISA2 form centrifugation at 13;000 g for 10 min at 4 C. Our pilot a heteromultimeric complex.2,8,9) The formation of the study showed that PvISA1/2 and PvISA3 are precipi- ISA1/2 complex is also supported by the fact that tated at ammonium sulfate concentrations of 0 to 25% Arabidopsis mutants in ISA1 and ISA2 display identical and 25 to 40% saturation respectively. phenotypes.8) In contrast to the ISA1/2 complex, ISA3 PvISA1/2 proteins were purified from the crude appears to be a monomeric enzyme.2) Arabidopsis extract by the following procedure: The precipitate

y To whom correspondence should be addressed. Fax: +81-11-706-2508; E-mail: [email protected] Abbreviations: -CD, -cyclodextrin; DBE, starch-debranching enzyme; DP, degree of polymerization; ISA, isoamylase-type DBE; PMSF, phenylmethylsulfonyl fluoride; PvISA, ISA from kidney bean (Phaseolus vulgaris L.) 70215-2 Y. TAKASHIMA et al.

A SMLMaLf B M 12M (kDa) PvISA1 200

PvISA2 116 97

PvISA3 66

rRNA 45

Fig. 1. Accumulation Profiles of PvISA Transcripts (A) and SDS–PAGE of Purified PvISA1/2 and PvISA3 (B). A, Each lane was loaded with 20 mg of total RNA isolated from developing and mature seeds and leaves. Lanes S, M, L, Ma, and Lf indicate small- (4–8 mm), mid- (10–12 mm), and large- (14–16 mm) sized developing seeds, mature seeds, and leaves respectively. Equal loading of RNA was confirmed by methylene blue staining of ribosomal RNA. B, CBB-stained SDS–PAGE (7.5% w/v gel). Lanes 1 and 2 contained 2 mgof purified PvISA1/2 and PvISA3 respectively. Lane M was loaded with the following standard proteins: myosin (200 kDa), -galactosidase Advance(116 kDa), phosphorylase b (97 kDa), BSA (66 kDa), View and ovalbumin (45 kDa). obtained from a fraction of 25% ammonium sulfate was the gel, PvISA1/2 might be a heterotetramer. The final dissolved in a small amount of buffer A (40 mM MOPS– preparation was determined to be 0.15 mg by the method 13) NaOH, pH 7.0, containing 2 mM DTT, 5 mM MgCl2, and of Bradford. 10% v/v glycerol), and then dialyzed against buffer A. PvISA3 proteins were purified from the crude extract The dialyzed sample was passed through a Q-Sepharose by the following procedure: The precipitate from the FF column (GE Healthcare Bio-Sciences, Piscataway, fraction of 25–40% ammonium sulfate was dissolved in NJ) equilibrated with buffer A. The column was washed a small amount of buffer A and dialyzed against with buffer A, followed by a gradient of 0 to 1 M NaCl. buffer A. The dialyzed sample was subjected to Q- The fractions with ISA activity were dialyzed against Sepharose FF columnProofs chromatography as well as the buffer B (5 mM sodium phosphate buffer, pH 7.0, con- case of the PvISA1/2 purification. Ammonium sulfate taining 2 mM DTT and 5 mM MgCl2) and resolved on a was added to the ISA-active fractions to facilitate 30% Gigapite column (Seikagaku, Tokyo) equilibrated with saturation. The solution was loaded on a Toyopearl- buffer B. The enzyme solution was eluted with a 5 to Butyl 650M column (Tosoh Bioscience, Tokyo) equili- 100 mM gradient of sodium phosphate buffer. The active brated with buffer C (40 mM MOPS–NaOH, pH 7.0, fractions were pooled, concentrated, and purified on a containing 4 mM DTT, 5 mM MgCl2, and 10% v/v gly- Superdex 200 HR 10/30 column (GE Healthcare Bio- cerol), containing ammonium sulfate at 30% saturation. Sciences) equilibrated with a buffer (20 mM MOPS– The column was washed with buffer C containing 30% NaOH, pH 7.0, containing 2 mM DTT, 5 mM MgCl2, and ammonium sulfate, followed by a reverse gradient of 0.1 M NaCl). The purified enzyme solution yielded two 30% to 0% ammonium sulfate. The active fractions were polypeptide bands, with molecular masses of approx- dialyzed against buffer A and resolved on a -CD imately 87 and 93 kDa on an SDS–PAGE gel (Fig. 1B). Sepharose 4B column equilibrated with buffer A. The The determined N-terminal sequences of 87 and 93 kDa column was prepared by coupling -CD (Hayashibara polypeptides were AARNG and SIEETEQQVE. They Biochemical Laboratories, Okayama, Japan) to CNBr- were detected in the amino acid sequences deduced from activated Sepharose 4B (GE Healthcare Bio-Sciences) PvISA1 and PvISA2 cDNA respectively. Hence the according to instructions. After the column was washed purified enzyme was a complex of PvISA1 and PvISA2. with buffer A, the proteins were eluted with a linear It was designated PvISA1/2. This indicates that PvISA1 gradient of 0 to 2 M NaCl. The purified enzyme solution and PvISA2 are synthesized as preproteins with signal migrated as a single polypeptide band with a molecular sequences of 50 and 62 residues respectively, and that mass of approximately 75 kDa on an SDS–PAGE gel mature PvISA1 and PvISA2 correspond to 741 and 803 (Fig. 1B). The N-terminal sequence was determined to amino acids with predicted molecular masses of 83.6 be AYGRRAQEGV, which was included in the se- and 89.2 kDa respectively. The molecular mass of quence predicted from PvISA3 cDNA, indicating that PvISA1/2 was estimated to be approximately 370 kDa PvISA3 is synthesized as a preprotein with a signal by gel permeation chromatography (data not shown). sequence of 77 residues and that the mature form Considering the stained intensities of the two bands on corresponds to 706 amino acids with predicted molecu- Characterization of Two Isoamylases from Kidney Bean Seeds 70215-3 Table 1. Activities of Purified PvISA1/2 and PvISA3 toward Several Polyglucans

Polyglucan ISA Amylopectin Glycogen -Limit dextrin Pullulan PvISA1/2 14:98 0:51 8:95 1:43 6:58 1:34 0:95 0:15 PvISA3 3:51 0:10 17:16 0:38 29:05 1:08 0:77 0:12

ISA activities are represented in terms of maltotriose equivalent reducing power (mmol)/min/mg protein. Values are the means with S.E. of measurements obtained in three independent experiments. lar mass of 80.1 kDa. The molecular mass of PvISA3 from -limit dextrin were limited to DPs of 2 and 3. was estimated to be 50–60 kDa by gel permeation These results suggest that PvISA1/2 has broad chain- chromatography (data not shown), suggesting that length specificity for amylopectin and glycogen, while PvISA3 is a monomeric enzyme. The final preparation PvISA3 has high specificity for a chain-length of DP 2 to was determined to be 0.2 mg from 1 kg of immature 4. Hence the high specific activity of PvISA3 for -limit seeds. dextrin (Table 1) was attributed to the elevated concen- The ISA activities of purified enzymes were assayed tration of chains (DPs 2 and 3) that can be reacted. The using potato amylopectin (Sigma), rabbit liver glycogen chain-length specificity of PvISA3 was identical to those (Sigma), pullulan (Hayashibara), and -limit dextrin as of GlgX proteins from Escherichia coli17) and cyano- substrates. The reaction mixture (0.5 ml) contained bacteria.18) 50 mM MES–NaOH (pH 6.0), 0.1 mg/ml BSA, 10 mg/ Currently, the ISA1/2 complex is assumed to play a ml of each polyglucan, and 50 ml of enzyme solution role in the biosynthesis of starch because a mutation (0.1–0.3 mg), and was incubated at 30 C for 10 min. in the ISA1 or ISA2 gene causes an accumulation of TheAdvance activities were measured based on the View increase in phytoglycogen in the plastids of both photosynthetic reducing power by the methods of Somogyi and and storage organs.4–6) Although the role of the ISA3 Nelson,14,15) using maltotriose as a standard, and were isoform in starch biosynthesis is not yet established, its represented in terms of 1 mmol of maltotriose equiv- occurrence in storage organs such as immature seeds and alent/min/mg protein. We prepared -limit dextrin from tubers2) suggests that the isoform is also involved in potato starch (Sigma) according to the method of starch synthesis. Considering the enzymatic properties Doehlert and Knutson.1) As shown in Table 1, purified of the isoform, its presence in storage organs might PvISA1/2 and PvISA3 displayed distinct reactivities for contribute to the maturation of starch granules by the several polyglucans. PvISA1/2 showed high activity for removal of short chains on the surface of growing amylopectin, whereas PvISA3 preferred -limit dextrin granules.19) Proofs and glycogen to amylopectin. The two enzymes barely Plant ISAs belong to the -amylase family and 20–22) reacted to pullulan. The substrate specificities of contain a catalytic (/)8 barrel structure. In ad- PvISA1/2 and PvISA3 were consistent with those of dition to the barrel domain, ISAs have N-terminal and potato ISAs, Stisa1, and Stisa3 respectively.2) C-terminal domains whose functions remain unclear. To determine the chain-length specificities of Pv- Our preliminary experiments using recombinant PvISA ISA1/2 and PvISA3, glucan chains liberated by enzyme proteins suggest that PvISA1 alone displays ISA activity reactions were analyzed using amylopectin, glycogen, and has the same chain-length specificity as purified and -limit dextrin as substrates (Fig. 2). The reaction PvISA1/2. PvISA1 and PvISA3 share a significant was done under the same conditions as the assay of ISA similarity (63%), and probably a similar conformation. activity. After the reaction was terminated by boiling for Indeed, no large difference was observed in the overall 3 min, the sample (50 ml) was labeled with 0.2 M 8- structural models of PvISA1 and PvISA3 constructed aminopyrene-1,3,6-trisulfonate, as described by O’Shea from the structure of Pseudomonas isoamylase,23) but et al.16) The labeled products were subjected to capillary both enzymes have distinct differences in substrate electrophoresis (310 Genetic Analyzer; Applied Bio- specificity, indicating that minor structural differences systems, Foster City, CA). The chain-length distribution in the active clefts exert a major effect on enzyme profiles of PvISA1/2 and PvISA3 were compared with properties. Probably, PvISA3 has active clefts which that of isoamylase from Pseudomonas (Nacali Tesque, branch chains with a DP of greater than 5 are unable to Kyoto, Japan), which has broad chain-length specificity access. Identification of the residues or regions deter- and is used routinely in structural analysis of polysac- mining chain-length specificity is current underway. charides. The products obtained by PvISA1/2 from each polyglucan were highly similar to those obtained by Acknowledgments Pseudomonas isoamylase. In contrast, the products obtained by PvISA3 from amylopectin and glycogen This work was supported by grants-in-aid for Scien- predominantly had DPs of 3 and 4, although branch tific Research (C) (13660071, 16580069, and 18580086) chains of various lengths were included in these and the Akiyama Foundation to H.I. from the Japan substrates. The products obtained with these enzymes Society for the Promotion of Science, and a grant-in-aid 70215-4 Y. TAKASHIMA et al.

Amylopectin Glycogen β-Limit dextrin 10 20 80 ABCPseudomonas Pseudomonas Pseudomonas

5 10 40 Relative area (%) 0 0 0 5 1015202530 5 10 15 20 25 30 5 10 15 20 25 30 10 20 80 DEFPvISA1/2 PvISA1/2 PvISA1/2

5 10 40

0 0 0 5 1015202530 51015202530 51015202530 60 80 80 GHPvISA3 PvISA3 IPvISA3

Advance30 40 View 40 Relative area (%) Relative area (%) 0 0 0 5 1015202530 51015202530 51015202530 Degree of polymerization (DP) Degree of polymerization (DP) Degree of polymerization (DP)

Fig. 2. Chain-Length Distributions of Reaction Products Obtained with Pseudomonas Isoamylase, PvISA1/2, and PvISA3. The products obtained with Pseudomonas isoamylase (A–C), PvISA1/2 (D–F), and PvISA3 (G–I) with amylopectin, glycogen, and -limit dextrin were detected by capillary electrophoresis. Chain-length distribution was represented as the percentage area when the total area of the degree of polymerization (DP) of 2 to 30 was 100%. A, D, and G, Products obtained with each enzyme with amylopectin. B, E, and H, Products obtained with each enzyme with glycogen. C, F, and I, Products obtained with each enzymeProofs with -limit dextrin. for Young Scientists (B) (18780067) to S.H. from the Characterization of the maize gene sugary1, a determi- Ministry of Education, Culture, Sports, Science and nant of starch composition in kernels. 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