Proc. Natl. Acad. Sci. USA Vol. 92, pp. 964-967, February 1995 Plant Biology Maize branching enzyme 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 glycogen synthase and starch 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 binding site 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). enzymes (1)-ADPglucose pyrophosphorylase (glucose-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-glucosyltransferase, 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)-transferase, 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 phosphorylase 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.