David R. Bevan

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1.

In plants, Glycoside Hydrolase (GH) Family 1 β-glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thalianagenome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron–exon organizations. The forty-eighth member of this family (At3g06510; sfr2) is a β-glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44-encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for β-mannosides including 1,4-β-D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm β-mannosidase and barley seed β-glucosidase/β-mannosidase BGQ60.

Assessing the Stability of Alzheimer’s Amyloid Protofibrils Using Molecular Dynamics

Amyloid fibrils represent a stable form of many misfolded proteins associated with numerous diseases. Among these are Parkinsons disease (alpha-synuclein), Type II diabetes (islet amyloid polypeptide), and Alzheimers disease (amyloid beta-peptide, Abeta). The appearance of Abeta fibrils in neural tissue is a hallmark of Alzheimers disease, and many studies have been conducted to determine and analyze the structure of these protein aggregates. The principal toxic species in Alzheimers disease are believed to be soluble, oligomeric aggregates of Abeta, but numerous studies have found that the insoluble fibrillated form of the peptide also contributes to neurotoxicity. Thus, to design therapeutic agents to combat the progression of Alzheimers disease, it is worthwhile to understand the thermodynamics of destabilizing these aggregates and the features that contribute to their stability. In this work, we present a systematic study of several factors that influence the stability of Abeta(42) fibrils following in silico mutation. We have employed standard molecular dynamics, as well as center-of-mass pulling and umbrella sampling, to study the thermodynamics of peptide dissociation from the core of a model protofibril at physiological temperature. Results indicate that a finite level of hydration around the Asp23-Lys28 salt bridge is crucial to protofibril stability, while mutation of Phe19 to glycine has no effect on the binding free energy of the terminal peptide. Packing between Ile32 and the aliphatic portion of the Lys28 side chain serves to regulate the level of hydration in the core of the protofibril and thus rigidify the Asp23-Lys28 salt bridge. These observations are important for designing compounds that target Abeta aggregates; interrupting these native interactions may destabilize these assemblies and ameliorate their toxicity.

GridMAT-MD: a grid-based membrane analysis tool for use with molecular dynamics.

GridMAT‐MD is a new program developed to aid in the analysis of lipid bilayers from molecular dynamics simulations. It reads a GROMACS coordinate file and generates two types of data: a two‐dimensional contour plot depicting membrane thickness, and a polygon‐based tessellation of the individual lipid headgroups. GridMAT‐MD can also account for proteins or small molecules within the headgroups of the lipids, closely approximating their occupied lateral area. The program requires no installation, is fast, and is freely available.

Crystal structure of a monocotyledon (maize ZMGlu1) beta-glucosidase and a model of its complex with p-nitrophenyl beta-D-thioglucoside.

The maize beta-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Angstrom resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl beta-D-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.

Destabilizing Alzheimer's Aβ42 Protofibrils with Morin: Mechanistic Insights from Molecular Dynamics Simulations

Alzheimers disease is a progressive, neurodegenerative disorder that is the leading cause of senile dementia, afflicting millions of individuals worldwide. Since the identification of the amyloid beta-peptide (Abeta) as the principal toxic entity in the progression of Alzheimers disease, numerous attempts have been made to reduce endogenous Abeta production and deposition, including designing inhibitors of the proteases that generate the peptide, generating antibodies against Abeta aggregates, utilizing metal chelators, and identifying small molecules that target the peptide during the aggregation pathway. The last approach is particularly attractive, as Abeta is normally present in vivo, but aggregation is a purely pathological event. Studies conducted in vitro and in vivo have suggested that administration of flavonoids, compounds naturally present in many foods, including wine and tea, can prevent and reverse Abeta aggregation, but mechanistic details are lacking. In this work, we employ atomistic, explicit-solvent molecular dynamics (MD) simulations to identify the mechanism of Abeta fibril destabilization by morin, one of the most effective anti-aggregation flavonoids, using a model of the mature Abeta fibril. Through the course of 24 simulations totaling 4.3 mus, we find that morin can bind to the ends of the fibrils to block the attachment of an incoming peptide and can penetrate into the hydrophobic core to disrupt the Asp23-Lys28 salt bridges and interfere with backbone hydrogen bonding. The combination of hydrophobicity, aromaticity, and hydrogen bonding capacity of morin imparts the observed behavior.

Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis

Terpene volatiles play important roles in plant-organism interactions as attractants of pollinators or as defense compounds against herbivores. Among the most common plant volatiles are homoterpenes, which are often emitted from night-scented flowers and from aerial tissues upon herbivore attack. Homoterpene volatiles released from herbivore-damaged tissue are thought to contribute to indirect plant defense by attracting natural enemies of pests. Moreover, homoterpenes have been demonstrated to induce defensive responses in plant–plant interaction. Although early steps in the biosynthesis of homoterpenes have been elucidated, the identity of the enzyme responsible for the direct formation of these volatiles has remained unknown. Here, we demonstrate that CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family, is responsible for the breakdown of the C20-precursor (E,E)-geranyllinalool to the insect-induced C16-homoterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Recombinant CYP82G1 shows narrow substrate specificity for (E,E)-geranyllinalool and its C15-analog (E)-nerolidol, which is converted to the respective C11-homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Homology-based modeling and substrate docking support an oxidative bond cleavage of the alcohol substrate via syn-elimination of the polar head, together with an allylic C-5 hydrogen atom. CYP82G1 is constitutively expressed in Arabidopsis stems and inflorescences and shows highly coordinated herbivore-induced expression with geranyllinalool synthase in leaves depending on the F-box protein COI-1. CYP82G1 represents a unique characterized enzyme in the plant CYP82 family with a function as a DMNT/TMTT homoterpene synthase.

Structural Insights into Rice BGlu1 β-Glucosidase Oligosaccharide Hydrolysis and Transglycosylation

The structures of rice BGlu1 beta-glucosidase, a plant beta-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 A and 1.55 A resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) beta-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long beta-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa beta-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-beta-glucanase/beta-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/beta-II beta-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.

Abscisic Acid Regulates Inflammation via Ligand-binding Domain-independent Activation of Peroxisome Proliferator-activated Receptor γ

Abscisic acid (ABA) has shown efficacy in the treatment of diabetes and inflammation; however, its molecular targets and the mechanisms of action underlying its immunomodulatory effects remain unclear. This study investigates the role of peroxisome proliferator-activated receptor γ (PPAR γ) and lanthionine synthetase C-like 2 (LANCL2) as molecular targets for ABA. We demonstrate that ABA increases PPAR γ reporter activity in RAW 264.7 macrophages and increases ppar γ expression in vivo, although it does not bind to the ligand-binding domain of PPAR γ. LANCL2 knockdown studies provide evidence that ABA-mediated activation of macrophage PPAR γ is dependent on lancl2 expression. Consistent with the association of LANCL2 with G proteins, we provide evidence that ABA increases cAMP accumulation in immune cells. ABA suppresses LPS-induced prostaglandin E2 and MCP-1 production via a PPAR γ-dependent mechanism possibly involving activation of PPAR γ and suppression of NF-κB and nuclear factor of activated T cells. LPS challenge studies in PPAR γ-expressing and immune cell-specific PPAR γ null mice demonstrate that ABA down-regulates toll-like receptor 4 expression in macrophages and T cells in vivo through a PPAR γ-dependent mechanism. Global transcriptomic profiling and confirmatory quantitative RT-PCR suggest novel candidate targets and demonstrate that ABA treatment mitigates the effect of LPS on the expression of genes involved in inflammation, metabolism, and cell signaling, in part, through PPAR γ. In conclusion, ABA decreases LPS-mediated inflammation and regulates innate immune responses through a bifurcating pathway involving LANCL2 and an alternative, ligand-binding domain-independent mechanism of PPAR γ activation.

Fast identification of thermostable beta-glucosidase mutants on cellobiose by a novel combinatorial selection/screening approach

Engineering costly cellulases on natural cellulosic substrates is of importance for emerging biomass‐based biorefineries. Directed enzyme evolution is becoming a popular tool, but identification of desired mutants from a large mutant library remains challenging sometimes. In this work, we demonstrated a novel combinatorial selection/screening strategy for finding thermostable beta‐glucosidase on its natural substrate—cellobiose. First, selection was conducted through complementation of beta‐glucosidase for non‐cellobiose‐utilizing Escherichia coli so that only the cells expressing active beta‐glucosidase can grow on a M9 synthetic medium with cellobiose as the sole carbon source (selection plate). Second, the clones on the selection plates were duplicated by using nylon membranes. After heat treatment, the nylon membranes were overlaid on M9/cellobiose screening plates so that remaining activities of thermostable beta‐glucosidase mutants hydrolyzed cellobiose on the screening plates to glucose. Third, the growth of an indicator E. coli strain that can utilize glucose but not cellobiose on the screening plates helped detect the thermostable beta‐glucosidase mutants on the selection plates. Several thermostable mutants were identified from a random mutant library of the Paenibacillus polymyxa beta‐glucosidase. The most thermostable mutant A17S had an 11‐fold increase in the half‐life of thermoinactivation at 50°C. Biotechnol. Bioeng. 2009;103: 1087–1094.

Lipid composition influences the release of Alzheimer's amyloid β-peptide from membranes

The behavior of the amyloid β‐peptide (Aβ) within a membrane environment is integral to its toxicity and the progression of Alzheimers disease. Ganglioside GM1 has been shown to enhance the aggregation of Aβ, but the underlying mechanism is unknown. Using atomistic molecular dynamics simulations, we explored the interactions between the 40‐residue alloform of Aβ (Aβ40) and several model membranes, including pure palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylserine (POPS), an equimolar mixture of POPC and palmitoyloleoylphosphatidylethanolamine (POPE), and lipid rafts, both with and without GM1, to understand the behavior of Aβ40 in various membrane microenvironments. Aβ40 remained inserted in POPC, POPS, POPC/POPE, and raft membranes, but in several instances exited the raft containing GM1. Aβ40 interacted with GM1 largely through hydrogen bonding, producing configurations containing β‐strands with C‐termini that, in some cases, exited the membrane and became exposed to solvent. These observations provide insight into the release of Aβ from the membrane, a previously uncharacterized process of the Aβ aggregation pathway.