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.     

A high yield optimized method for the production of acylated ACPs enabling the analysis of enzymes involved in P. falciparum fatty acid biosynthesis

The natural substrates of the enzymes involved in type-II fatty acid biosynthesis (FAS-II) are acylated acyl carrier proteins (acyl-ACPs). The state of the art method to produce acyl-ACPs involves the transfer of a phosphopantetheine moiety from CoA to apo-ACP by E. coli holo-ACP synthase (EcACPS), yielding holo-ACP which subsequently becomes thioesterified with free fatty acids by the E. coli acyl-ACP synthase (EcAAS). Alternatively, acyl-ACPs can be synthesized by direct transfer of acylated phosphopantetheine moieties from acyl-CoA to apo-ACP by means of EcACPS. The need for native substrates to characterize the FAS-II enzymes of P. falciparum prompted us to investigate the potential and limit of the two methods to efficiently acylate P. falciparum ACP (PfACP) with respect to chain length and β-modification and in preparative amounts. The EcAAS activity is found to be independent from the oxidation state at the β-position and accepts fatty acids as substrates with chain lengths starting from C8 to C20, whereas EcACPS accepts very efficiently acyl-CoAs with chain lengths up to C16, and with decreasing activity also longer chains (C18 to C20). Methods were developed to synthesize and purify preparative amounts of high quality natural substrates that are fully functional for the enzymes of the P. falciparum FAS-II system.     

A class of exact solutions for biomacromolecule diffusion-reaction in live cells

A class of novel explicit analytic solutions for a system of n+1 coupled partial differential equations governing biomolecular mass transfer and reaction in living organisms are proposed, evaluated, and analyzed. The solution process uses Laplace and Hankel transforms and results in a recursive convolution of an exponentially scaled Gaussian with modified Bessel functions. The solution is developed for wide range of biomolecular binding kinetics from pure diffusion to multiple binding reactions. The proposed approach provides solutions for both Dirac and Gaussian laser beam (or fluorescence-labeled biomacromolecule) profiles during the course of a Fluorescence Recovery After Photobleaching (FRAP) experiment. We demonstrate that previous models are simplified forms of our theory for special cases. Model analysis indicates that at the early stages of the transport process, biomolecular dynamics is governed by pure diffusion. At large times, the dominant mass transfer process is effective diffusion. Analysis of the sensitivity equations, derived analytically and verified by finite difference differentiation, indicates that experimental biologists should use full space-time profile (instead of the averaged time series) obtained at the early stages of the fluorescence microscopy experiments to extract meaningful physiological information from the protocol. Such a small time frame requires improved bioinstrumentation relative to that in use today. Our mathematical analysis highlights several limitations of the FRAP protocol and provides strategies to improve it. The proposed model can be used to study biomolecular dynamics in molecular biology, targeted drug delivery in normal and cancerous tissues, motor-driven axonal transport in normal and abnormal nervous systems, kinetics of diffusion-controlled reactions between enzyme and substrate, and to validate numerical simulators of biological mass transport processes in vivo.