Characterization and Enzyme Engineering of a Hyperthermophilic Laccase toward Improving Its Activity in Ionic Liquid

Ionic liquids (ILs) are organic salts molten at room temperature that can be used for a wide variety of applications. Many ILs, such as 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]), have been shown to remove a significant fraction of the complex biopolymer lignin from biomass during pretreatment. Valorizing lignin via biological pathways (e.g., enzymes) holds promise but is limited by the low biocompatibility of many ILs used for pretreatment. The discovery of thermostable enzymes and the application of enzyme engineering techniques have yielded biocatalysts capable of withstanding high concentrations of ILs. Converting lignin from a waste product to value-added chemicals is vital to the success of future cellulosic biorefineries. To that end, we screened the activity of the lignolytic enzyme laccase from a hyperthermophilic bacterium (Thermus thermophilus) in aqueous [C2C1Im][OAc]. Despite the thermophilicity (Topt \u3e 90°C) of this laccase, significant activity loss (\u3e 50%) was observed in only 2% (w/v) [C2C1Im][OAc]. Kinetics studies show that the IL can bind to the free enzyme and the enzyme-substrate complex. Docking simulations suggest that the cation favors binding to a region close to the active site. We then used a rational design strategy to improve the activity of the laccase in [C2C1Im][OAc]. A total of 8 single amino acid mutations were made; however, there were no significant improvements in the activity of the mutants in [C2C1Im][OAc] compared to the wild type. The results of this study shed light on the complex nature of enzyme-IL interactions and the challenges faced when designing a biological lignin valorization strategy

Mutant Insulin Degrading Enzyme and Methods of Use

In one aspect, the present invention provides an isolated mutant insulin degrading enzyme (IDE) having an amino acid sequence that is at least 90% identical to SEQ ID NO:1 over its entire length and comprises at least one amino acid substitution at any of amino acid residues 332, 339, 341, 359, 360, 361, 374, 429, 609, 898, 899 or 901 of the sequence. The mutant IDE has a differential activity relative to that of wild-type IDE. Also provided is a polynucleotide encoding the polypeptide of the invention

Timing of extension in the Pioneer metamorphic core complex with implications for the spatial-temporal pattern of Cenozoic extension and exhumation in the northern U.S. Cordillera

The Pioneer core complex (PCC) in central Idaho lies along a transition between Early Eocene and ca. 40 Ma core complexes to the north and south, respectively. Thus, the age of extensional development of the PCC is important in understanding the spatial-temporal patterns of core-complex development in the North American Cordillera. New results, including structural observations and U-Pb zircon (SHRIMP and ICPMS) geochronology, constrain the early extensional history of the footwall for the first time. High-temperature strain with a top-WNW shear-sense is pervasive throughout metamorphic rocks of the northwestern footwall. An isoclinally folded dike yields a crystallization age of ∼48-47 Ma, whereas a crosscutting dike yielded an age of 46 Ma. Metamorphic rocks are also intruded by the ∼50-48 Ma Pioneer intrusive suite (PIS), a W-dipping granodiorite sheet displaying a magmatic fabric. Northwest-trending lineations are locally visible and also defined by anisotropy of magnetic susceptibility, indicating that during emplacement, the PIS was undergoing similarly oriented extensional strain as the enclosing metamorphic rocks. Therefore, WNW-directed extension spanning this structural section occurred between ∼50 and 46 Ma. Following emplacement of crosscutting 46 Ma dikes, deformation was partitioned into the WNW-directed Wildhorse detachment. Motion on the detachment occurred between ∼38 and 33 Ma, as documented by previous 40Ar/ 39Ar thermochronology. It is not clear, however, whether extension was continuous through the interval between these two time periods. Although Early Eocene extension in the PCC was synchronous with extension in core complexes to the north, rates of footwall exhumation in central Idaho were much lower. This southward slowing is compatible with N-S differences in inferred subduction zone geometry/kinematics and in the internal character of the orogenic wedge

A Monomeric Variant of Insulin Degrading Enzyme (IDE) Loses Its Regulatory Properties

Background: Insulin degrading enzyme (IDE) is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structuture. Methodology/Principal Findings: IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained,25 % of the wild type activity. In contrast with the larger peptide substrates b-endorphin and amyloid b peptide 1–40, monomeric IDE retained only 1 to 0.25 % of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold. Conclusions/Significance: These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since

Crystallization and Structure of a Plant Peptide Deformylase

This invention relates to the crystal structure of a plant peptide deformylase polypeptide and methods of using the structure to design compounds that modulate the activity of the polypeptide

Crystallization and Structure of a Plant Peptide Deformylase

This invention relates to the crystal structure of a plant peptide deformylase polypeptide and methods of using the structure to design compounds that modulate the activity of the polypeptide

Bone density of the femoral neck following Birmingham hip resurfacing: A 2-year prospective study in 27 hips

Background Resurfacing is a popular alternative to a standard hip replacement in young arthritic patients. Despite bone preservation around the femoral component, there is little information regarding the bone quality

Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase

Nitroreductase from Enterobacter cloacae (NR) reduces diverse nitroaromatics including herbicides, explosives and prodrugs, and holds promise for bioremediation, prodrug activation and enzyme-assisted synthesis. We solved crystal structures of NR complexes with bound substrate or analog for each of its two half-reactions. We complemented these with kinetic isotope effect (KIE) measurements elucidating H-transfer steps essential to each half-reaction. KIEs indicate hydride transfer from NADH to the flavin consistent with our structure of NR with the NADH analog nicotinic acid adenine dinucleotide (NAAD). The KIE on reduction of p-nitrobenzoic acid (p-NBA) also indicates hydride transfer, and requires revision of prior computational mechanisms. Our mechanistic information provided a structural restraint for the orientation of bound substrate, placing the nitro group closer to the flavin N5 in the pocket that binds the amide of NADH. KIEs show that solvent provides a proton, enabling accommodation of different nitro group placements, consistent with NR’s broad repertoire

Empirical Model Development for Predicting Shock Response on Composite Materials Subjected to Pyroshock Loading

The NASA Engineering and Safety Center (NESC) received a request to develop an analysis model based on both frequency response and wave propagation analyses for predicting shock response spectrum (SRS) on composite materials subjected to pyroshock loading. The model would account for near-field environment (approx. 9 inches from the source) dominated by direct wave propagation, mid-field environment (approx. 2 feet from the source) characterized by wave propagation and structural resonances, and far-field environment dominated by lower frequency bending waves in the structure. This report documents the outcome of the assessment

Empirical Model Development for Predicting Shock Response on Composite Materials Subjected to Pyroshock Loading: Appendices

The NASA Engineering and Safety Center (NESC) received a request to develop an analysis model based on both frequency response and wave propagation analyses for predicting shock response spectrum (SRS) on composite materials subjected to pyroshock loading. The model would account for near-field environment (approx. 9 inches from the source) dominated by direct wave propagation, mid-field environment (approx. 2 feet from the source) characterized by wave propagation and structural resonances, and far-field environment dominated by lower frequency bending waves in the structure. This document contains appendices to the Volume I report