Amandeep K. Sangha

Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog

BackgroundDown-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors.ResultsGCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples.ConclusionsDown-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.

Radical Coupling Reactions in Lignin Synthesis: A Density Functional Theory Study

Lignin is a complex, heterogeneous polymer in plant cell walls that provides mechanical strength to the plant stem and confers resistance to degrading microbes, enzymes, and chemicals. Lignin synthesis initiates through oxidative radical-radical coupling of monolignols, the most common of which are p-coumaryl, coniferyl, and sinapyl alcohols. Here, we use density functional theory to characterize radical-radical coupling reactions involved in monolignol dimerization. We compute reaction enthalpies for the initial self- and cross-coupling reactions of these monolignol radicals to form dimeric intermediates via six major linkages observed in natural lignin. The 8-O-4, 8-8, and 8-5 coupling are computed to be the most favorable, whereas the 5-O-4, 5-5, and 8-1 linkages are less favorable. Overall, p-coumaryl self- and cross-coupling reactions are calculated to be the most favorable. For cross-coupling reactions, in which each radical can couple via either of the two sites involved in dimer formation, the more reactive of the two radicals is found to undergo coupling at its site with the highest spin density.

Proteins fold by subdiffusion of the order parameter.

It is shown that the folding of a C(alpha) model of chymotyprsin inhibitor (CI2) protein cannot be described by either diffusion (Smoluchowski equation, SE) or a normal-diffusion continuous time random walk of a single order parameter under the influence of the thermodynamic force. The reason for these failures is that the order parameter follows subdiffusion. A theory is proposed based on the idea that an ordinary SE holds along a contour representative of the folding pathways, and that displacements along the contour obey a fractal relationship to, and are longer than, those along the reaction coordinate defined by the order parameter. With a new, constraint-free method to determine the order-parameter-dependent diffusion constant, and statistical temperature molecular dynamics (STMD) enhanced sampling of the free energy, the fractal SE theory is completely characterized by short-time simulations, and its predictions are in quantitative agreement with simulated long-time folding dynamics. Thus, the fractal SE may serve as an accelerated algorithm to study the folding of proteins too slow to be simulated directly.

Protein folding and confinement: inherent structure analysis of chaperonin action.

A coarse-grained model of the action of a chaperonin cage of tunable hydrophobicity, h, upon a protein with the possibility of misfolding is studied with inherent structure (IS) analysis and statistical temperature molecular dynamics (STMD) simulation. Near the folding temperature, the equilibrium properties of the system may be understood in terms of <10 IS. The known phenomenon of an optimal cage hydrophobicity for productive folding, found at h = 0.25, is seen to arise from a striking suppression of the occupations of IS in the misfolding funnel, which in turn arises from a decrease in translational entropy due to confinement to the region of the cage wall. The kinetics of folding is correspondingly fastest at h = 0.25, where a minimum is found in the h-dependent barrier height. While true kinetics is determined by conventional MD, it is shown that the accelerated dynamics of STMD provide a valuable quantitative perspective.

Relative binding affinities of monolignols to horseradish peroxidase

Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic -OH group and a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic -OH group instead interacting with Pro139. Since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate.

Combining neutron crystallography, high-performance computing for enzyme design

Enzymes continue to expand their role in industry as a “green” option for the synthesis of value-added products. They are targeted for the design of drugs in pharmaceutical applications and also for protein engineering in industry to improve their efficiency, stability, and specificity. Knowledge of the exact mechanisms of enzymatic reactions may provide essential information for more effective drug design and enzyme engineering. For the first time, we are employing a joint X-ray/neutron (XN) protein crystallographic technique in combination with high-performance computing, including QM and QM/MM calculations, MD and Rosetta simulations, to investigate the mechanisms of several enzymes that are important to renewable energy and chemical synthesis. D-xylose isomerase (XI) is an enzyme which can be used to increase the production of biofuels from lignocellulosic biomass and also to synthesize rare sugars for pharmaceutical industry. XI catalyzes the reversible multi-stage sugar inter-conversion reaction facilitated by the presence of two divalent metal cations in its active site. It primarily catalyzes the isomerization of the aldo-sugar D-xylose to the keto-isomer Dxylulose, but can also epimerize L-arabinose into L-ribose, albeit much less efficiently. The reaction involves moving hydrogen atoms between the protein residues, sugar and water molecules, and can only be understood if hydrogen atoms are visualized at each reaction stage. We have obtained a number of joint XN structures of XI complexes representing snapshots along the reaction path with D-glucose, D-xylose and L-arabinose. The suggested reaction mechanism has been verified by QM calculations using the novel O(N) methodology. We are using this structural and mechanistic information to re-design XI to be more efficient on D-xylose and Larabinose for biofuels and biomedical applications by employing QM/MM, MD, and Rosetta methodologies.