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 β-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.

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-, β- 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.

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.

domains, the latter allowing for monitoring VSP activity by means of total internal reflection microscopy (TIRF-M). The whole-cell patch-clamp configuration allowed for control not only over membrane voltage but also intracellular pH by dialysing the cell with solutions with the desired pH. We find that acidification of the cytoplasm results in increased PI(4,5)P 2 depletion, accompanied by a shift of the apparent voltage dependence towards more negative potentials. An increase in intracellular pH has the opposite effect. The voltage dependence of sensing currents was unaffected by the pH changes, suggesting that alterations of the VSD are not causal for the observed changes in voltage dependent activity. Similar effects were observed in all tested VSPs We conclude that the overall activity of the phosphatase is enhanced under acidic and diminished under alkaline conditions. Kinetic modeling predicts a shift in apparent voltage dependence under these circumstances that is in agreement with the observed shift.In conclusion, we suggest that intracellular pH can play a role in the regulation of the activity of VSPs. Our research aims to understand how changes in ultrafast dynamics compare and correlate to thermophilic enzyme activity. We observe fluctuating electric field effects in a promiscuous, hyperthermophilic ene-reductase from Pyrococcus horikoshii (PhENR) to address this. This enzyme catalyzes the reduction of activated alkenes/alkynes to their respective alkanes/alkenes via proton and hydride transfers from a flavin cofactor in the active site. We exploit the promiscuity of PhENR in order to incorporate a variety of substrates and substrate analogs into the active site for these studies. We have synthesized a set of covalently-attached substituted N-phenylmaleimide infrared labels, which mimic the structures of the enzyme's substrates, and contain unique vibrational chromophores to probe the enzyme's active site dynamics. Current studies focus on the vibrational frequencies and lineshapes of nitrile labels such as those of 4-cyano-N-phenylmaleimide, which sits proximal to the catalytic flavin and can be attached in multiple orientations within the active site. When compared to the label in solution, the covalently attached label undergoes significant inhomogeneous broadening in its FTIR spectrum reflecting the distribution of active site microenvironments. Additionally, protein-based non-natural amino acid labels such as methionine to azidohomoalanine substitutions are also being incorporated into the distal side of the flavin cofactor for similar studies in different location within the enzyme's active site. Using 2D IR spectroscopy, we are examining the contributions of femtosecond to picosecond active site dynamics to the lineshapes of both the covalently attached probes as well as the incorporated non-natural amino acid labels. Future research aims to break the thermophilicity of the enzyme via specific mutations in order to compare the active site dynamics to a corresponding mesophilic version of the p...

No abstract