The Structure of Maltooctaose-Bound Escherichia coli Branching Enzyme Suggests a Mechanism for Donor Chain Specificity.

Glycogen is the primary storage polysaccharide in bacteria and animals. It is a glucose polymer linked by α-1,4 glucose linkages and branched via α-1,6-linkages, with the latter reaction catalyzed by branching enzymes. Both the length and dispensation of these branches are critical in defining the structure, density, and relative bioavailability of the storage polysaccharide. Key to this is the specificity of branching enzymes because they define branch length. Herein, we report the crystal structure of the maltooctaose-bound branching enzyme from the enterobacteria E. coli. The structure identifies three new malto-oligosaccharide binding sites and confirms oligosaccharide binding in seven others, bringing the total number of oligosaccharide binding sites to twelve. In addition, the structure shows distinctly different binding in previously identified site I, with a substantially longer glucan chain ordered in the binding site. Using the donor oligosaccharide chain-bound Cyanothece branching enzyme structure as a guide, binding site I was identified as the likely binding surface for the extended donor chains that the E. coli branching enzyme is known to transfer. Furthermore, the structure suggests that analogous loops in branching enzymes from a diversity of organisms are responsible for branch chain length specificity. Together, these results suggest a possible mechanism for transfer chain specificity involving some of these surface binding sites.

A structural explanation for the mechanism and specificity of plant branching enzymes I and IIb.

Branching enzymes (BEs) are essential in the biosynthesis of starch and glycogen and play critical roles in determining the fine structure of these polymers. The substrates of these BEs are long carbohydrate chains that interact with these enzymes via multiple binding sites on the enzyme's surface. By controlling the branched-chain length distribution, BEs can mediate the physiological properties of starch and glycogen moieties; however, the mechanism and structural determinants of this specificity remain mysterious. In this study, we identify a large dodecaose binding surface on rice BE I (BEI) that reaches from the outside of the active site to the active site of the enzyme. Mutagenesis activity assays confirm the importance of this binding site in enzyme catalysis, from which we conclude that it is likely the acceptor chain binding site. Comparison of the structures of BE from Cyanothece and BE1 from rice allowed us to model the location of the donor-binding site. We also identified two loops that likely interact with the donor chain and whose sequences diverge between plant BE1, which tends to transfer longer chains, and BEIIb, which transfers exclusively much shorter chains. When the sequences of these loops were swapped with the BEIIb sequence, rice BE1 also became a short-chain transferring enzyme, demonstrating the key role these loops play in specificity. Taken together, these results provide a more complete picture of the structure, selectivity, and activity of BEs.

Crystal structures of Escherichia coli branching enzyme in complex with cyclodextrins.

Branching enzyme (BE) is responsible for the third step in glycogen/starch biosynthesis. It catalyzes the cleavage of α-1,4 glucan linkages and subsequent reattachment to form α-1,6 branch points. These branches are crucial to the final structure of glycogen and starch. The crystal structures of Escherichia coli BE (EcBE) in complex with α-, ß- and γ-cyclodextrin were determined in order to better understand substrate binding. Four cyclodextrin-binding sites were identified in EcBE; they were all located on the surface of the enzyme, with none in the vicinity of the active site. While three of the sites were also identified as linear polysaccharide-binding sites, one of the sites is specific for cyclodextrins. In previous work three additional binding sites were identified as exclusively binding linear malto-oligosaccharides. Comparison of the binding sites shed light on this apparent specificity. Binding site IV is located in the carbohydrate-binding module 48 (CBM48) domain of EcBE and superimposes with the cyclodextrin-binding site found in the CBM48 domain of 5'-AMP-activated protein kinase (AMPK). Comparison of these sites shows the similarities and differences in the two binding modes. While some of the binding sites were found to be conserved between branching enzymes of different organisms, some are quite divergent, indicating both similarities and differences between oligosaccharide binding in branching enzymes from various sources.

Crystal Structures of Escherichia coli Branching Enzyme in Complex with Linear Oligosaccharides.

Branching enzyme is responsible for all branching of glycogen and starch. It is an unusual member of the α-amylase family because it has both α-1,4-amylase activity and α-1,6-transferase activity [Drummond, G. S., et al. (1972) Eur. J. Biochem. 26, 168-176]. It also does not react with shorter glucans, though it will bind much longer substrates and substrate mimics [Binderup, K., et al. (2002) Arch. Biochem. Biophys. 397, 279-285]. In an effort to better understand how branching enzyme interacts with its polymeric substrate, we have determined the structure of Δ112 Escherichia coli branching enzyme bound to maltoheptaose and maltohexaose. Together, these structures define six distinct oligosaccharide binding sites on the surface of E. coli branching enzyme. Most of these binding sites surround the edge of the ß-barrel domain and are quite far from the active site. Surprisingly, there is no evidence of oligosaccharide binding in the active site of the enzyme. The closest bound oligosaccharide resides almost 18 Å from the active site. Mutations to conserved residues in binding sites I and VI had a debilitating effect on the activity of the enzyme.