Substrate Recognition and Activation by Two Flavoenzymes Involved in Pyrimidine Metabolism: Flavin-Dependent Thymidylate Synthase and tRNA-Dihydrouridine Synthase.

Pyrimidines are essential components of nucleic acids. In biology, they undergo a number of redox reactions catalyzed by flavin-dependent enzymes. This thesis investigates the mechanism of substrate recognition and activation of two flavin-dependent enzymes with pyrimidine-containing substrates: flavin-dependent thymidylate synthase (FDTS) and tRNA-dihydrouridine synthase (DUS). FDTS catalyzes the reductive methylation of the uracil moiety of 2’-deoxyuridine-5’-monophosphate (dUMP) into thymine, whereas DUS reduces specific uracils in tRNA to dihydrouracil. NMR data indicate that FDTS ionizes N3 of the uracil in dUMP using an active-site arginine, which is proposed to initiate catalysis by enhancing the nucleophilicity of C5 of the uracil. Biochemical data on dUMP analogs suggests that the phosphate of dUMP acts as the base that removes the proton from C5 of dUMP during the FDTS-catalyzed reaction. Notably, both ionization of N3 and acid-base catalysis by the phosphate of dUMP are not implicated in the mechanism used by human thymidylate synthase. Several equilibrium and kinetic methods were used to study the mechanism of deoxynucleotide recognition by Thermotoga maritima FDTS. FDTS binds deoxynucleotides with ~200-fold weaker affinity when the flavin is reduced relative to when it is oxidized, and the differences in affinity are largely due to differences in the dissociation rate constant. There is also a temperature-dependent effect on the mechanism by which FDTS binds deoxynucleotides – below 45°C the FDTS homotetramer behaves as a dimer-of-dimers with deoxynucleotide binding while at temperatures above 45°C the four subunits of FDTS bind deoxynucleotides identically. tRNAs contain a number of nucleobase and ribose modifications – including dihydrouracil – at different positions of the tRNA. Previous work has shown that yeast tRNALeu-CAA reacts poorly with yeast DUS2 unless it contains other modifications, suggesting that tRNA modifications are ordered. The reactivity of yeast DUS2 with other unmodified tRNAs was investigated; unmodified yeast tRNAAsp, tRNAAla, and tRNAHis2 all reacted rapidly with yeast DUS2, indicating that not all tRNAs require prior modifications to react rapidly with yeast DUS2. Native gel electrophoresis showed that unmodified tRNALeu-CAA misfolds, explaining its poor reactivity with yeast DUS2.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/110420/1/fstull_1.pd

Substrate protein folds while it is bound to the ATP-independent chaperone Spy

Chaperones assist the folding of many proteins in the cell. While the most well studied chaperones use cycles of ATP binding and hydrolysis to assist protein folding, a number of chaperones have been identified that promote protein folding in the absence of highenergy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we have characterized the kinetic mechanism of substrate folding by the small, ATP-independent chaperone, Spy. Spy rapidly associates with its substrate, Immunity protein 7 (Im7), eliminating its potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while remaining bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones can assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while continuously bound to a chaperone

Periplasmic Chaperones and Prolyl Isomerases

International audienceThe biogenesis of periplasmic and outer membrane proteins (OMPs) in Escherichia coli is assisted by a variety of processes that help with their folding and transport to their final destination in the cellular envelope. Chaperones are macromolecules, usually proteins, that facilitate the folding of proteins or prevent their aggregation without becoming part of the protein's final structure. Because chaperones often bind to folding intermediates, they often (but not always) act to slow protein folding. Protein folding catalysts, on the other hand, act to accelerate specific steps in the protein folding pathway, including disulfide bond formation and peptidyl prolyl isomerization. This review is primarily concerned with E. coli and Salmonella periplasmic and cellular envelope chaperones; it also discusses periplasmic proline isomerization

Folding while Bound to Chaperones

Chaperones are important in preventing protein aggregation and aiding protein folding. How chaperones aid protein folding remains a key question in understanding their mechanism. The possibility of proteins folding while bound to chaperones was reintroduced recently with the chaperone Spy, many years after the phenomenon was first reported with the chaperones GroEL and SecB. In this review, we discuss the salient features of folding while bound in the cases for which it has been observed and speculate about its biological importance and possible occurrence in other chaperones

Guanine to Inosine Substitution Leads to Large Increases in the Population of a Transient G·C Hoogsteen Base Pair

We recently showed that Watson–Crick base pairs in canonical duplex DNA exist in dynamic equilibrium with G­(<i>syn</i>)·C⁺ and A­(<i>syn</i>)·T Hoogsteen base pairs that have minute populations of ∼1%. Here, using nuclear magnetic resonance <i>R</i>_1ρ relaxation dispersion, we show that substitution of guanine with the naturally occurring base inosine results in an ∼17-fold increase in the population of transient Hoogsteen base pairs, which can be rationalized by the loss of a Watson–Crick hydrogen bond. These results provide further support for transient Hoogsteen base pairs and demonstrate that their population can increase significantly upon damage or chemical modification of the base

A R T I C L E S Substrate protein folds while it is bound to the ATP-independent chaperone Spy

Chaperones are essential for maintaining protein-folding homeostasis, and they have important roles in the cellular stress response. By binding to aggregation-sensitive folding intermediates, chaperones inhibit aberrant interactions between proteins. The most intensively studied folding chaperones, such as the GroEL-GroES and DnaK systems, facilitate substrate protein folding through ATP-and cofactor-driven conformational changes In contrast to ATP-dependent chaperones, a number of chaperones have been identified that can assist in protein folding in the absence of ATP The mechanism by which Spy and the growing class of ATPindependent chaperones function to refold proteins in the absence of energy cofactors is an unresolved question. We addressed this by determining how Spy affects the folding pathway of Im7, the protein that was used to discover Spy. Im7 has a number of attributes that facilitate its study. It is small and monomeric, and it folds via a wellcharacterized mechanism that involves transition through a partially folded on-pathway intermediate before the native state is reache

Enzymatic control of dioxygen binding and functionalization of the flavin cofactor

The reactions of enzymes and cofactors with gaseous molecules such as dioxygen (O2) are challenging to study and remain among the most contentious subjects in biochemistry. To date, it is largely enigmatic how enzymes control and fine-tune their reactions with O2, as exemplified by the ubiquitous flavin-dependent enzymes that commonly facilitate redox chemistry such as the oxygenation of organic substrates. Here we employ O2-pressurized X-ray crystallography and quantum mechanical calculations to reveal how the precise positioning of O2 within a flavoenzyme's active site enables the regiospecific formation of a covalent flavin-oxygen adduct and oxygenating species (i.e., the flavin-N5-oxide) by mimicking a critical transition state. This study unambiguously demonstrates how enzymes may control the O2 functionalization of an organic cofactor as prerequisite for oxidative catalysis. Our work thus illustrates how O2 reactivity can be harnessed in an enzymatic environment and provides crucial knowledge for future rational design of O2-reactive enzymes

An enzymatic activation of formaldehyde for nucleotide methylation

International audienceFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH 2 O-shunt may be relevant in several other important folate-dependent reactions

Binding Interface and Electron Transfer Between Nicotine Oxidoreductase and Its Cytochrome c Electron Acceptor

The enzyme nicotine oxidoreductase (NicA2) is a member of the flavoprotein amine oxidase family that uses a cytochrome c protein (CycN) as its oxidant instead of dioxygen, which is the oxidant used by most other members of this enzyme family. We recently identified a potential binding site for CycN on the surface of NicA2 through rigid body docking [J. Biol. Chem. 2022, 298 (8), 102251]. However, this potential binding interface has not been experimentally validated. In this paper, we used unnatural amino acid incorporation to probe the binding interface between NicA2 and CycN. Our results are consistent with a structural model of the NicA2-CycN complex predicted by protein–protein docking and AlphaFold, suggesting that this is the binding site for CycN on NicA2’s surface. Based on additional mutagenesis of potentially redox active residues in NicA2, we propose that electron transfer from NicA2’s flavin to CycN’s heme occurs without the assistance of a protein-derived wire