Revisiting the Roles of Catalytic Residues in Human Ornithine Transcarbamylase.

Human ornithine transcarbamylase (hOTC) is a mitochondrial transferase protein involved in the urea cycle and is crucial for the conversion of toxic ammonia to urea. Structural analysis coupled with kinetic studies of rat, bovine, and other transferase proteins has identified residues that play key roles in substrate recognition and conformational changes but has not provided direct evidence for all of the active residues involved in OTC function. Here, computational methods were used to predict the likely active residues of hOTC; the function of these residues was then probed with site-directed mutagenesis and biochemical characterization.

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS.

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment.

Biochemical Activity of 17 Cancer-Associated Variants of DNA Polymerase Kappa Predicted by Electrostatic Properties.

DNA damage and repair have been widely studied in relation to cancer and therapeutics. Y-family DNA polymerases can bypass DNA lesions, which may result from external or internal DNA damaging agents, including some chemotherapy agents. Overexpression of the Y-family polymerase human pol kappa can result in tumorigenesis and drug resistance in cancer.

A community effort in SARS-CoV-2 drug discovery.

The COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the "Billion molecules against COVID-19 challenge", to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors.

Functional annotation of haloacid dehalogenase superfamily structural genomics proteins.

Haloacid dehalogenases (HAD) are members of a large superfamily that includes many Structural Genomics proteins with poorly characterized functionality. This superfamily consists of multiple types of enzymes that can act as sugar phosphatases, haloacid dehalogenases, phosphonoacetaldehyde hydrolases, ATPases, or phosphate monoesterases. Here, we report on predicted functional annotations and experimental testing by direct biochemical assay for Structural Genomics proteins from the HAD superfamily.

Virtually the Same? Evaluating the Effectiveness of Remote Undergraduate Research Experiences.

In-person undergraduate research experiences (UREs) promote students' integration into careers in life science research. In 2020, the COVID-19 pandemic prompted institutions hosting summer URE programs to offer them remotely, raising questions about whether undergraduates who participate in remote research can experience scientific integration and whether they might perceive doing research less favorably (i.e.

Identification and characterization of alternative sites and molecular probes for SARS-CoV-2 target proteins.

Three protein targets from SARS-CoV-2, the viral pathogen that causes COVID-19, are studied: the main protease, the 2'-O-RNA methyltransferase, and the nucleocapsid (N) protein. For the main protease, the nucleophilicity of the catalytic cysteine C145 is enabled by coupling to three histidine residues, H163 and H164 and catalytic dyad partner H41. These electrostatic couplings enable significant population of the deprotonated state of C145.

Design, Synthesis, and Characterization of [F]mG2P026 as a High-Contrast PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

An array of triazolopyridines based on JNJ-46356479 () were synthesized as potential positron emission tomography radiotracers for metabotropic glutamate receptor 2 (mGluR2). The selected candidates featured enhanced positive allosteric modulator (PAM) activity (20-fold max.) and mGluR2 agonist activity (25-fold max.

Enzyme active sites: Identification and prediction of function using computational chemistry.

Understanding the biochemically active amino acids in proteins is a key factor to improve the knowledge of how enzymes work, to predict the function of newly discovered protein structures of unknown function, and to establish design principles for enzyme engineering. Here, we explore recently reported computational chemistry-based methods for the prediction of active amino acids in protein 3D structures, including biochemically important distal residues, and their implications for functional genomics, for enzyme design, and for enhancing understanding of the function of enzymes.

Electrostatic fingerprints of catalytically active amino acids in enzymes.

The computed electrostatic and proton transfer properties are studied for 20 enzymes that represent all six major enzyme commission classes and a variety of different folds. The properties of aspartate, glutamate, and lysine residues that have been previously experimentally determined to be catalytically active are reported. The catalytic aspartate and glutamate residues studied here are strongly coupled to at least one other aspartate or glutamate residue and often to multiple other carboxylate residues with intrinsic pK differences less than 1 pH unit.

Stereoselective Synthesis of β-Glycinamide Ribonucleotide.

A diastereoselective synthesis of the β-anomer of glycinamide ribonucleotide (β-GAR) has been developed. The synthesis was accomplished in nine steps from D-ribose and occurred in 5% overall yield. The route provided material on the multi-milligram scale.

Synthesis and Characterization of 5-(2-Fluoro-4-[C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2-pyrano[2,3-]pyridine-7-carboxamide as a PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

Metabotropic glutamate receptor 2 (mGluR2) is a therapeutic target for several neuropsychiatric disorders. An mGluR2 function in etiology could be unveiled by positron emission tomography (PET). In this regard, 5-(2-fluoro-4-[C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2-pyrano[2,3-]pyridine-7-carboxamide ([C], [C]mG2N001), a potent negative allosteric modulator (NAM), was developed to support this endeavor.

Functional Characterization of Structural Genomics Proteins in the Crotonase Superfamily.

Members of the Crotonase superfamily, a mechanistically diverse family of proteins that share a conserved quaternary structure, can often catalyze more than one reaction. However, the spectrum of activity for its members has not been well studied. We report on measured crotonase and hydrolase activity for eight structural genomics (SG) proteins from the Crotonase superfamily plus two previously characterized proteins, intended as controls: human enoyl CoA hydratase (ECH) and β-diketone hydrolase.

"How Do We Do This at a Distance?!" A Descriptive Study of Remote Undergraduate Research Programs during COVID-19.

The COVID-19 pandemic shut down undergraduate research programs across the United States. A group of 23 colleges, universities, and research institutes hosted remote undergraduate research programs in the life sciences during Summer 2020. Given the unprecedented offering of remote programs, we carried out a study to describe and evaluate them.

Adapting Undergraduate Research to Remote Work to Increase Engagement.

Demand for undergraduate research experiences typically outstrips the available laboratory positions, which could have been exacerbated during the remote work conditions imposed by the SARS-CoV-2/COVID-19 pandemic. This report presents a collection of examples of how undergraduates have been engaged in research under pandemic work restrictions. Examples include a range of projects related to fluid dynamics, cancer biology, nanomedicine, circadian clocks, metabolic disease, catalysis, and environmental remediation.

Reintegrating Biology Through the Nexus of Energy, Information, and Matter.

Information, energy, and matter are fundamental properties of all levels of biological organization, and life emerges from the continuous flux of matter, energy, and information. This perspective piece defines and explains each of the three pillars of this nexus. We propose that a quantitative characterization of the complex interconversions between matter, energy, and information that comprise this nexus will help us derive biological insights that connect phenomena across different levels of biological organization.

Amino acid interactions that facilitate enzyme catalysis.

Interactions in enzymes between catalytic and neighboring amino acids and how these interactions facilitate catalysis are examined. In examples from both natural and designed enzymes, it is shown that increases in catalytic rates may be achieved through elongation of the buffer range of the catalytic residues; such perturbations in the protonation equilibria are, in turn, achieved through enhanced coupling of the protonation equilibria of the active ionizable residues with those of other ionizable residues. The strongest coupling between protonation states for a pair of residues that deprotonate to form an anion (or a pair that accept a proton to form a cation) is achieved when the difference in the intrinsic pKs of the two residues is approximately within 1 pH unit.

Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase.

The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.

Design, Synthesis, and Characterization of Benzimidazole Derivatives as Positron Emission Tomography Imaging Ligands for Metabotropic Glutamate Receptor 2.

Three benzimidazole derivatives (-) have been synthetized as potential positron emission tomography (PET) imaging ligands for mGluR2 in the brain. Of these compounds, exhibits potent binding affinity (IC = 7.6 ± 0.

Probing remote residues important for catalysis in Escherichia coli ornithine transcarbamoylase.

Understanding how enzymes achieve their tremendous catalytic power is a major question in biochemistry. Greater understanding is also needed for enzyme engineering applications. In many cases, enzyme efficiency and specificity depend on residues not in direct contact with the substrate, termed remote residues.

Synthesis of benzoylbenzamide derivatives of 17α-E-vinyl estradiol and evaluation as ligands for the estrogen receptor-α ligand binding domain.

A series consisting of substituted benzoylbenzamide derivatives of 17α-E-vinyl estradiol 6a-i and 7a-d was prepared in good overall yields from the corresponding novel iodinated benzoylbenzamide precursors using Pd(0)-catalyzed Stille coupling. Biological evaluation using competitive binding assays indicated that all compounds were effective ligands for the ERα- and ERβ-LBD (RBA = 0.5-10.

Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis.

Caspases are cysteine-aspartic proteases involved in the regulation of programmed cell death (apoptosis) and a number of other biological processes. Despite overall similarities in structure and active-site composition, caspases show striking selectivity for particular protein substrates. Exosites are emerging as one of the mechanisms by which caspases can recruit, engage, and orient these substrates for proper hydrolysis.

Functional classification of protein structures by local structure matching in graph representation.

As a result of high-throughput protein structure initiatives, over 14,400 protein structures have been solved by Structural Genomics (SG) centers and participating research groups. While the totality of SG data represents a tremendous contribution to genomics and structural biology, reliable functional information for these proteins is generally lacking. Better functional predictions for SG proteins will add substantial value to the structural information already obtained.

In Vitro and in Vivo Evaluation of C-Labeled Azetidinecarboxylates for Imaging Monoacylglycerol Lipase by PET Imaging Studies.

Monoacylglycerol lipase (MAGL) is the principle enzyme for metabolizing endogenous cannabinoid ligand 2-arachidonoyglycerol (2-AG). Blockade of MAGL increases 2-AG levels, resulting in subsequent activation of the endocannabinoid system, and has emerged as a novel therapeutic strategy to treat drug addiction, inflammation, and neurodegenerative diseases. Herein we report a new series of MAGL inhibitors, which were radiolabeled by site-specific labeling technologies, including C-carbonylation and spirocyclic iodonium ylide (SCIDY) radiofluorination.

Prediction of Active Site and Distal Residues in E. coli DNA Polymerase III alpha Polymerase Activity.

The process of DNA replication is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA Pol III, which is a complex of 10 different subunits that coordinates simultaneous replication on the leading and lagging strands.