Proc. Natl. Acad. Sci. USA Vol. 92, pp. 964-967, February 1995 Plant Biology

Maize branching catalyzes synthesis of glycogen-like polysaccharide in glgB-deficient Escherichia coli HANPING GUAN, TAKASHI KURIKI, MIRTA SIVAK, AND JACK PREISS* Department of Biochemistry, Michigan State University, East Lansing, MI 48824 Communicated by Martin Gibbs, Brandeis University, Waltham, MA, October 24, 1994

ABSTRACT The structure of a-glucan, isolated from branching amylose than branching amylopectin and preferen- wild-type Escherichia coli B, a glycogen branching enzyme tially transferred longer chains. In contrast, maize BEII had a (BE)-deficient E. coli AC71 (glgB-), or from AC71 trans- lower rate of branching amylose than branching amylopectin formed with genes coding for maize BEI and BEII individually and preferentially transferred shorter chains. as well as with both genes, was analyzed by high-performance The genes coding for maize BEI and BEII have been cloned anion-exchange chromatography (HPAEC) with pulsed am- (8, 9) and expressed in Escherichia coli (10, 11). To further perometric detection. Transformation ofthe maize BE gene(s) characterize the specificity of maize BEI and BEII in vivo, we in AC71 (glgB-) showed complementation in branching ac- expressed the genes coding for mature maize BEI and BEII tivity. Analysis by HPAEC revealed different structures be- individually as well as together in AC71 (glgB-) and charac- tween glycogen ofE. coli B and a-glucan ofAC71 transformed terized the a-glucan structure by high-performance anion- with a different maize BE gene(s). The individual chains ofthe exchange chromatography (HPAEC). Here we report the ci-glucan debranched with isoamylase were distributed be- expression of maize BE and structural analysis of the a-glucan tween chain length (CL) 3 and > 30 and the chain with CL 6 synthesized. was the most abundant. In comparison with the glycogen ofE. coli B, the az-glucan of AC71 transformed with the maize BE gene(s) consisted ofa lesser amount ofchains with CL 7-9 and MATERIALS AND METHODS a larger amount ofchains with CL > 14. It also showed a broad Construction of the Maize BE Expression Vectors. The peak with chains of CL 9-12 as in maize amylopectin. This genes coding for mature maize BEI (MBEI) and BEII study provides in vivo evidence that glycogen BE and maize BE (MBEII) were subcloned from plasmids pET-23d-MBEI and isozymes may have different specificities in the length ofchain pET-23d-MBEII (10, 11) into the expression vector pTrc99A transferred. Furthermore, this study suggests that the spec- (Pharmacia) at the Nco I/Xba I and Nco I/Sal I sites, respec- ificity of and synthase and their tively, to form the plasmids pTrc99A-MBEI and pTrc99A- concerted action with BE play an important role in determin- MBEII (Fig. 1). The Xba I/Xho I fragment containing a ing the structure of the polysaccharide synthesized. ribosome of pET-23d (Novagen) and the mature MBEII gene was subcloned to theXba I/Sal I site of pTrc99A- The biosynthesis of starch in plants requires at least three MBEI to form the plasmid pTrc99A-MBEI-MBEII (Fig. 1). (1)-ADPglucose pyrophosphorylase (-i- Expression of Maize BE in E. coli AC71 (glgB-). An phosphate adenylyltransferase; ADP:a-D-glucose-l-phos- overnight culture of the transformed cells with the maize BE phate adenylyltransferase, EC 2.7.7.27), starch synthase (SS; gene(s) was diluted 1:20 (vol/vol) in fresh Kornberg broth ADPglucose: 1,4-a-D-glucan 4-a-D-, EC [1-liter solution containing 11 g of K2HPO4, 8.5 g of KH2PO4, 2.4.1.21), and 1,4-a-glucan branching enzyme [BE; 1,4-a-D- 3 g of yeast extract (pH 7.0)] containing 100 ,ug of ampicillin glucan:1,4-a-D-glucan 6-a-D-(1,4-a-D-glucano)-, per ml and 2% glucose. The cells were grown at 37°C for -2 EC 2.4.1.18]. Similarly, the biosynthesis of glycogen in bacteria h toA600 = 0.8 before the expression of maize BE was induced is also through the ADPglucose pathway catalyzed by ADP- by adding isopropyl ,B-D-thiogalactoside to 0.5 mM. After glucose pyrophosphorylase, glycogen synthase (GS), and gly- growth at room temperature for 20 h, cells were harvested in cogen BE, which are encoded by glgC, glgA, and glgB, respec- a refrigerated centrifuge. tively (2, 3). The rate of glycogen synthesis in bacteria as well BE Activity Assay. BE activity was measured in the sonicate as starch synthesis in plants is allosterically regulated at the supematant by the stimulation assay as de- step of ADPglucose synthesis in response to the intracellular scribed by Guan and Preiss (6). One unit of enzyme activity is metabolite levels (1,-3). However, it is believed that the defined as 1 pimol of glucose incorporated into a-D-glucan per specificities of BE and/or GS and SS-are the main reasons why min at 30°C. the structures ofglycogen and starch are distinct (1). Although Protein Assay. Protein concentration was determined ac- both glycogen and amylopectin are composed of branched cording to the method of Smith et al. (12) using prepared a1->4glucan with al-+6 linkages, glycogen has more branches bicinchoninic acid reagent and bovine serum albumin (Pierce) than amylopectin and a different chain length (CL) distribu- as the standard. tion (4). Glycogen shows a monomodal CL distribution while Isolation of a-Glucan from E. coli. The a-glucan was amylopectin displays a polymodal CL distribution (5). isolated from wild-type E. coli B, AC71, and AC71 trans- Knowledge ofthe relationship between the specificity of BE, formed with genes coding for maize BEI and BEII individually SS, and the structure of the polysaccharide synthesized is as well as with both genes. About 18 g (wet weight) of E. coli crucial for us to understand the mechanism of starch synthesis. cells was suspended and sonicated in 80 ml of 50 mM Tris There are multiple forms of BE and SS in maize endosperm acetate buffer (pH 7.5). The a-glucan was isolated by the (1). Previous studies (6, 7) have indicated that maize BEI and method described by Preiss et al. (13). The final alcohol BEII have different properties. BEI had a higher rate of Abbreviations: BE, branching enzyme; CL, chain length; HPAEC, The publication costs of this article were defrayed in part by page charge high-performance anion-exchange chromatography; GS, glycogen payment. This article must therefore be hereby marked "advertisement" in synthase; SS, starch synthase. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 964 Downloaded by guest on September 30, 2021 Plant Biology: Guan et al. Proc. Natl. Acad Sci. USA 92 (1995) 965 Table 1. Expression of maize BE in E. coli AC71 (glgB-) and 0 _ 0 0 -aX properties of the polysaccharide synthesized z x en Specific activity, Protein, unit(s) per mg Amax, _Ptac rbs Maize BEl Strain mg/ml of protein CL nm E. coli B 3.8 0.11 10 470 AC71 3.3 <0.003 42 554 0 AC71 + MBEI 3.6 2.9 14 476 C)o_cis z CO) AC71 + MBEII 3.4 0.44 16 500 AC71 + MBEI + MBEII* 4.1 1.0 12 471 tac rbsj Maize BE2 Experiments were repeated twice. *BEI/BEII activity ratio was 3:1 determined by chromatography on Mono Q.

0 to 0 a0 a In the wild-type E. coli B, a specific activity of 0.11 unit per mg z x z of protein was detected. The AC71 transformants of the different maize BE gene(s) (Fig. 1) showed specific activities of 2.9 for BEI, 0.44 for BEII, and 1 for both BEI and BEII. -[Ptac- rbs I Maize BEl rbs Maize BE2 Chromatography of the sonicate supernatant of the transfor- FIG. 1. Construction of expression vectors for maize BE in plasmid mant with the maize BEI and BEII genes on Mono Q H/R 5/5 pTrc99A (Pharmacia). Genes coding for mature maize BEI and BEII showed two activity peaks. Western blotting analysis with were cloned at the Nco I/Xba I site and Nco I/Sal I site of plasmid anti-BE antiserum showed that the expressed maize BEI with pTrc99A, respectively. a molecular mass of 86 kDa was in the flow-through fractions containing branching activity and the expressed maize BEII precipitate was washed by acetone and then dried over silica with a molecular mass of 83 kDa was in the fractions of the salt gel. gradient containing branching activity (data not shown). This Preparation of Debranched a-Glucan. The isolated a-glu- is similar to the enzyme isolated from developing maize can (1 mg) was dissolved in 1 ml of water by heating at 100°C. endosperm (6). The BEI/BEII activity ratio was 3:1 in the The solution was cooled and 0.1 ml of 1 M sodium-acetate transformant containing the BEI and BEII genes as deter- buffer (pH 3.5) was added. The a-glucan was debranched with mined by chromatography on Mono Q. Although it is not 1 unit of isoamylase at 45°C for 16 h. The reaction was stopped known whether BEI and BEII have differences in catalytic by adding 150 mM sodium hydroxide, and the samples were turnover capabilities, the activity ratio could be mainly due to stored at 45°C. The average CL was determined as described the overall expression of the two genes. by Takeda et al. (7) using the modified Park-Johnson method Since the maize BEI and BEII are active in the transformed and the phenol/H2SO4 method to measure the reducing AC71 cells, it is of interest to determine the structure of the terminal glucosyl residues and total carbohydrate content, a-glucan synthesized. The amount of a-glucan isolated was 1.3 respectively. mg per g wet cell for AC71, and 1.9, 1.7, and 2.3, respectively, Analysis of CL Distribution of the Debranched a-Glucan by for AC71 transformed with the BEI gene, the BEII gene, and HPAEC. HPAEC was performed with a Dionex BioLC system both BEI and BEII genes. The a-glucan isolated from AC71 with a pulsed amperometric detector, an amperometric flow- had an average CL of 42. The a-glucan isolated from wild-type through cell with a gold working electrode. The column used E. coli B and the mutant AC71 transformed with the maize BE was a CarboPac PA-1 (250 x 4 mm) with an AG6 guard gene(s) showed much shorter average CL and lower Amax of column (50 x 4 mm). EluentAwas 150 mM sodium hydroxide. glucan-iodine complex (Table 1). The average CL of the Eluent B was 150 mM sodium hydroxide containing 500 mM a-glucan in the maize BE gene transformants (CL 14 for BEI, sodium acetate. The gradient program was as follows: 40% 16 for BEII, and 12 for BEI + BEII) was longer than that of eluent B at time 0, 50% at 2 min, 60% at 12 min, and 80% at E. coli B glycogen (CL 10) and much shorter than that of maize 50 min. The debranched a-glucan was filtered through a amylopectin (CL 22-24). Similar to glycogen from E. coli B, 0.22-,um membrane filter and an aliquot of 25 ,ul was analyzed the a-glucan isolated from AC71 transformed with the BEI by HPAEC. All samples were run at room temperature with gene and the BEI and BEII genes showed lower Am.x of the a flow rate of 1 ml/min. A 0.1 mM mixture of maltotriose, glucan-iodine complex than that in AC 71 transformed with maltotetraose, maltopentaose, maltohexaose, and maltohep- the BEII gene. These results indicate that E. coli glycogen BE taose was used as standard. and maize BEI and BEII may have differences in the degree ofbranching and branching patterns. Based on the average CL, the a-glucan synthesized in AC71 transformed with the maize RESULTS AND DISCUSSION BE gene(s) is similar to glycogen. We have previously reported (10, 11) the purification and The a-glucan structure was further analyzed by HPAEC. characterization of maize BE expressed in E. coli BL21(DE3) The individual chains of the debranched a-glucan were dis- and shown that the recombinant enzyme had properties similar tributed between CL 3 and > 30 (Fig. 2). The chain with CL to those of the enzyme purified from developing endosperm. 6 was most abundant except in AC71, which had branched To further characterize the properties of maize BE in vivo, the chains at CL 11, 18, and 26 due to the deficiency of glycogen genes coding for mature maize BEI and BEII were expressed BE activity (Fig. 2). The analysis of a-glucan structure by individually as well as together in E. coli mutant AC71 (glgB-). HPAEC not only showed the complementation of branching Transformation of the maize BE gene(s) into AC71 showed activity by maize BE in vivo but also revealed different complementation in branching activity (Table 1). Very little structures ofthe a-glucan inE. coli B and in AC71 transformed activity (specific activity, <0.003 unit per mg of protein) was with a different maize BE gene(s) (Figs. 2 and 3). Based on the detected in AC71 in a long incubation period (120 min) with relative mole responses in pulsed amperometric detection of undiluted enzyme, and no activity was detected at 60 min. This the maltosaccharides with CL 6-17 (14), the relative mole indicates that mutant AC71 is essentially free of glycogen BE. distributions for CL 6-17 were calculated (Fig. 4). E. coli B Downloaded by guest on September 30, 2021 966 Plant Biology: Guan et aL Proc. Natl. Acadc Sci USA 92 (1995)

F col B

17

AC71 +MBEI LkCalQ 0 CU 20 -- 25 I) 4)

AC71+MBEI

20 25 30

18 CL AC71+MEB+MEII FIG. 3. Relative peak area (%) of the chains of the debranched a-glucan isolated from E. coli B (0), AC71 (glgB-) transformed with genes coding for maize BEI (-) and BEII (K) individually as well as with both BEI + BEII (m). transferred. Previous studies of the fine structures of glycogen (15) and amylopectin (16) have indicated that a Ba chain of glycogen carries fewer A chains than that of amylopectin, AC71 which is less branched than glycogen, and the average CL of

18

0 10 20 30 40 20 10 Time Retention (min) a FIG. 2. HPAEC analysis of the debranched a-glucan isolated from 5 E. coli B, AC71 (glgB-), and AC71 transformed with genes coding for r, maize BEI and BEII individually as well as with both genes. Number 15 on each peak indicates the CL of the corresponding component. .2 AL6 9 12 15 Analysis was repeated twice. lao DP '0 glycogen displayed a structure similar to that of oyster glyco- 4- gen (15). The chain with CL 6 was the most abundant and chains with CL > 7 gradually decreased. This distribution 10 profile was different from that of maize amylopectin, which 4) consisted of a small amount of chains with CL 6-8, and had the most abundant chains with CL 9-12 (Fig. 4 Inset). Al- though the chain with CL 6 was most abundant in the a-glucan of AC71 transformed with the maize BE gene(s) (Fig. 4), it 5 displayed different mole distributions. In comparison to E. coli B glycogen, the a-glucan of AC71 transformed with the maize BE gene(s) consisted of a lesser amount of chains with CL 7-9 and a larger amount of chains with CL > 14. Furthermore, it showed a broad peak with chains of CL 9-12 as in maize 4 6 8 10 12 14 16 18 amylopectin (Fig. 4). There were also significant proportions CL of chains with CL > 25 in the a-glucan of the AC71 transfor- FIG. 4. Relative mole distribution of chains with CL 6-17, which mants, while there were only negligible proportions of chains was calibrated on the basis of the mole responses. (Inset) CL distri- with CL > 25 in E. coli B glycogen (Figs. 2 and 3). These bution of debranched maize amylopectin (14). Sum of relative mole structural differences provide in vivo evidence that maize BE distribution of chains with CL 6-17 was taken as 100%. 0, E. coli B; and glycogen BE have different specificities in the size ofchain A, BEI; o, BEII; *, BEI + BEII. Downloaded by guest on September 30, 2021 Plant Biology: Guan et al. Proc. Natt Acad Sci. USA 92 (1995) 967 the Ba fragments of glycogen (CL 7.9) is much shorter than starch synthesis. This study has established the basis for us to that (CL 15.3) ofwheat amylopectin. From a biosynthesis point study the concerted actions of BE and SS in a bacterial model of view, the fine structure is compatible with the irregular system. branching model (4, 15) and the specificities of BE play an important role in determining the structure of the polysac- This work was supported in part by the U.S. Department of charide synthesized. Therefore, it is crucial to understand the Agriculture Grant USDA-CSRS-93-37306-9148 and by Michigan State specificities of BE in the chain transferred and chain spacing University Research Excellence Funds. (internal CL). It is of interest to note that maize BEI and BEII had different effects on the structure of a-glucan synthesized. 1. Preiss, J. (1991) Oxford Surv. Plant Mol. Cell Bio. 7, 59-114. The a-glucan of the transformant with the maize BEI gene 2. Preiss, J. & Romeo, T. (1994) Prog. Nucleic Acid Res. Mol. Bio. showed a lesser amount of shorter chains with CL 6-9 and a 47, 301-327. > 3. Preiss, J. & Romeo, T. (1989) Adv. Microb. Physiol. 30, 183-233. larger amount of longer chains with CL 14 than that with the 4. Gunja-Smith, Z., Marshall, J. J., Mercier, C., Smith, E. E. & maize BEII gene (Figs. 3 and 4). In agreement with our in vitro Whelan, W. J. (1970) FEBS Lett. 12, 101-104. biochemical studies (6, 7), the in vivo complementation study 5. Hizukuri, S. (1986) Carbohydr. Res. 147, 342-347. suggests that maize BEII could play an important role in the 6. Guan, H. P. & Preiss, J. (1993) Plant Physiol. 102, 1269-1273. synthesis of shorter chains (CL 6-9; A chains of amylopectin), 7. Takeda, Y., Guan, H. P. & Preiss, J. (1993) Carbohydr. Res. 240, while BEI could be involved mainly in the synthesis of longer 253-263. chains (CL > 14; B chains of amylopectin). 8. Baba, T., Kimura, K., Mizuno, K, Etoh, H., Ishida, Y., Shida, 0. Another interesting observation is that the chain with CL 6 & Arai, Y. (1991) Biochem. Biophys. Res. Commun. 181, 87-94. in the AC71 transformant is more abundant than that in E. coli 9. Fisher, D. K., Boyer, C. D. & Hannah, L. C. (1993) Plant Physiol. B. This is also different from amylopectin, which has a small 102, 1045-1046. amount of the chain with CL 6 (Fig. 4). The chain with CL 6 10. Guan, H. P., Baba, T. & Preiss, J. (1994) Plant Physiol. 104, was also found to be most abundant in the amylopectin of a 1449-1453. 11. Guan, H. P., Baba, T. & Preiss, J. (1994) Cell. Mol. Biol. 40, SSII-deficient Chlamydomonas reinhardtii and its abundance 981-985. was significantly lower in the wild type (17). These observa- 12. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., tions suggest that maize BE could transfer short chains as Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, glycogen BE, which may not be able to transfer chains shorter N. M., Olson, B. J. & Klenk, D. C. (1985) Anal. Biochem. 150, than maltohexaose, and the elongation of a1-4glucan could 76-85. start with the 6-mer oligomaltosaccharide. The fact that the 13. Preiss, J., Greenberg, E. & Sabraw, A. (1975) J. Bio. Chem. 250, concerted action between GS and maize BE did not produce 7631-7638. amylopectin-like polysaccharide suggests that the specificities 14. Koizumi, K., Fukuda, M. & Hizukuri, S. (1991) J. Chromatogr. of GS and SS are different in primers required and in their 585, 233-238. capacities in a1-->4glucan chain elongation. The concerted 15. Rani, M. R. S., Shibanuma, K. & Hizukuri, S. (1992) Carbohydr. action of GS and SS with different BE could play an important Res. 227, 183-194. role in determining the final structure of the polysaccharide 16. Hizukuri, S. & Maehara, Y. (1990) Carbohydr. Res. 206, 145-159. 17. Fontaine, T., D'Hulst, C., Maddelein, M.-L., Routier, F., Pepin, synthesized in addition to the specific branching patterns of T. M., Decq, A., Wieruszeski, J.-M., Delrue, B., Van den Koorn- BE. This is further supported by the recent report that huyse, N., Bossu, J.-P., Fournet, B. & Ball, S. (1993)J. Bio. Chem. expression of E. coli GS in the tubers of transgenic potatoes 268, 16223-16230. results in a highly branched starch (18). Therefore, it is 18. Shewmaker, C. K., Boyer, C. D., Wiesenborn, D. P., Thompson, necessary to study the interactions between different isozymes D. B., Boersig, M. R., Oakes, J. V. & Stalker, D. (1994) Plant of SS and BE in order to fully understand the mechanism of Physiol. 104, 1159-1166. Downloaded by guest on September 30, 2021