Structural Studies of Transition-State Stabilization by a Small Catalytic RNA, Metabolite-Binding by a Regulatory RNA Sequence and the Mechanism of Action of Activation Induced Deaminase.

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2008.Structural knowledge of an enzyme’s architecture can help elucidate the details of how it performs its biological role at the molecular level. In a case study of the hairpin ribozyme, structures were solved to high resolution in complex with reaction-intermediate analogs revealing key chemical groups in catalytically-relevant configurations. Importantly, the non-bridging oxygens of the scissile phosphate were tightly restrained in a conformation apparently promoting an inductive effect to elevate the pKa of a crucial active-site nucleobase while also ameliorating the unfavorable buildup of negative charge at the leaving group during the phosphoryl-transfer reaction. Active-site water molecules were ascribed a new role in both geometric and electrostatic stabilization of the reaction intermediate. By comparison to proteinaceous ribonuclease structures harboring the same reaction-intermediate analog, the catalytic strategies employed by these two evolutionarily-disparate enzyme classes appear to be a case of convergent evolution. In a second investigation, the aptamer domain of a metabolite-sensing non-coding regulatory RNA sequence was analyzed. Construct design, initial crystallization and attempts to solve the structure are described followed by improved methods to prepare a homogeneous RNA-metabolite complex. The resulting complex was evaluated by dynamic light scattering as well as small angle X-ray scattering. Subsequent optimization of crystallization conditions and diffraction analyses are also reported. The final area of focus encompasses biochemical and computational efforts to probe the mechanism of action of activation induced deaminase (AID). AID is a member of the cytidine deaminase superfamily and functions in the immunoglobulin maturation processes of somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Despite considerable research, questions remain regarding how AID is targeted to its substrate(s), what other cellular cofactors are necessary for function and how its activity is regulated since dysfunction or misregulation of AID has the potential to cause cancer. Efforts to crystallize AID were complemented by computational modeling, which has provided the basis for targeted biochemical studies aimed at answering outstanding questions. Perspectives on the success and pitfalls of continued modeling are discussed in light of recent empirical structural information reported for related family members

A comparison of vanadate to a 2′–5′ linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization

The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 Å resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 2′,5′-phosphodiester linkage at the site of cleavage was also solved at 2.35 Å resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 2′,5′ structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 2′-O nucleophile with the scissile G+1 phosphorus. The 2′,5′-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 2′,5′ linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds

Structure of the E. coli agmatinase, SPEB.

Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E. coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P31 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/Rfree values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion

Invisibility Cloaks and Hot Reactions: Applying Infrared Thermography in the Chemistry Education Laboratory

Copyright © 2020 American Chemical Society and Division of Chemical Education, Inc. Infrared (IR) thermography renders invisible infrared radiation with intuitive coloration in images and videos taken of objects, reactions, and processes. Educators can take advantage of this technology to extend students\u27 sensory perception of chemical reactions or processes that absorb or release heat in rich detail. In theory, IR thermography can be applied essentially universally for such analysis given that any change in thermal energy must result in, or from, the change of potential energy due to the interactions among atoms, molecules, and photons. Through the use of IR thermography, students can visualize otherwise invisible evidence of what is occurring on the molecular level in a variety of chemical process such as evaporative cooling, phase change, dissolution, titration, and enzymatic reactions. While not new, IR cameras are rapidly becoming affordable with models that connect easily with smartphones and tablets. The price decrease has opened the door for large-scale implementation in the chemistry education laboratory. We report here several laboratory activities and best practices that will facilitate the exploration of specific chemistry concepts through the use of infrared thermography, as well as integration of this technique into existing general chemistry laboratory courses

Comparison of the Response of Bacterial IscU and SufU to Zn²⁺ and Select Transition-Metal Ions

IscU, the central scaffold protein in the bacterial ISC iron–sulfur (Fe–S) cluster biosynthesis system, has long been recognized to bind a Zn²⁺ ion at its active site. While initially regarded as an artifact, Zn²⁺ binding has been shown to induce stabilization of the IscU structure that may mimic a state biologically relevant to IscU’s role in Fe–S cluster biosynthesis. More recent studies have revealed that SufU, a homologous protein involved in Fe–S cluster biosynthesis in Gram-positive bacteria, also binds a Zn²⁺ ion with structural implications. Given the widespread occurrence of the “IscU-like” protein fold, particularly among Fe–S cluster biosynthesis systems, an interesting question arises as to whether Zn²⁺ ion binding and the resulting structural alterations are common properties in IscU-like proteins. Interactions between IscU and specific metal ions are investigated and compared side-by-side with those of SufU from a representative Gram-positive bacterium in the phylum <i>Firmicutes</i>. These studies were extended with additional transition metal ions chosen to investigate the influence of coordination geometry on selectivity for binding at the active sites of IscU and SufU. Monitoring and comparing the conformational behavior and stabilization afforded by different transition metal ions upon IscU and SufU revealed similarities between the two proteins and suggest that metal-dependent conformational transitions may be characteristic of U-type proteins involved in Fe–S cluster biosynthesis

Fe(III)-polyuronic acid photochemistry: radical chemistry in natural polysaccharide

© 2021, The Author(s), under exclusive licence to European Photochemistry Association,European Society for Photobiology. The photochemistry of Fe(III) coordinated to natural uronate-containing polysaccharides has been investigated quantitatively in aqueous solution. It is demonstrated that the photoreduction of the coordinated Fe(III) to Fe(II) and oxidative decarboxylation occurs in a variety of uronate-containing polysaccharides. The photochemistry of the Fe(III)-polyuronic acid system generated a radical species during the reaction which was studied using the spin trapping technique. The identity of the radical species from this reaction was confirmed as CO2•− indicating that both bond cleavage of the carboxylate and oxidative decarboxylation after ligand to metal charge transfer radical reactions may be taking place upon irradiation. Degradation of the polyuronic acid chain was investigated with dynamic light scattering, showing a decrease in the hydrodynamic radius of the polymer assemblies in solution after light irradiation that correlates with the Fe(II) generation. A decrease in viscosity of Fe(IIII)-alginate after light irradiation was also observed. Additionally, the photochemical reaction was investigated in plant root tissue (parsnip) demonstrating that Fe(III) coordination in these natural materials leads to photoreactivity that degrades the pectin component. These results highlight that this Fe(III)-polyuronic acid can occur in many natural systems and may play a role in biogeochemical cycling of iron and ferrous iron generation in plants with significant polyuronic acid content. Graphic abstract: [Figure not available: see fulltext.