Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2′-deoxythymidine-5′-monophosphate (dTMP) from 2′-deoxyuridine-5′-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans, and a very different mechanism. Here we report NMR evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogs also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.
The renewed use of arsenicals as chemotherapeutics has rekindled interest in the biochemistry of As(III) species. In this work, simple bis- and tris-arsenical derivatives were synthesized with the aim of exploiting the chelate effect in the inhibition of thiol-disulfide oxidoreductases (here, Quiescin sulfhydryl oxidase, QSOX, and protein disulfide isomerase, PDI) that utilize two or more CxxC motifs in the catalysis of oxidative protein folding. Coupling 4-aminophenylarsenoxide (APAO) to acid chloride or anhydride derivatives yielded two bis-arsenical prototypes, BA-1 and BA-2, and a tris-arsenical, TA-1. Unlike the monoarsenical, APAO, these new reagents proved to be strong inhibitors of oxidative protein folding in the presence of a realistic intracellular concentration of competing monothiol (here, 5 mM reduced glutathione, GSH). However, this inhibition does not reflect direct inactivation of QSOX or PDI, but avid binding of MVAs to the reduced unfolded protein substrates themselves. Titrations of reduced riboflavin-binding protein with MVAs show that all 18 protein −SH groups can be captured by these arsenicals. With reduced RNase, addition of substoichiometric levels of MVAs is accompanied by the formation of Congo Red- and Thioflavin T-positive fibrillar aggregates. Even with Kd values of ∼50 nM, MVAs are ineffective inhibitors of PDI in the presence of millimolar levels of competing GSH. These results underscore the difficulties of designing effective and specific arsenical inhibitors for folded enzymes and proteins. Some of the cellular effects of arsenicals likely reflect their propensity to associate very tightly and nonspecifically to conformationally mobile cysteine-rich regions of proteins, thereby interfering with folding and/or function.
The finding that arsenic trioxide is an effective treatment for acute promyelocytic leukemia has renewed interest in the pharmacological uses of inorganic and organic arsenicals. Here we synthesize and characterize the reactivity of an arsenical-maleimide (As-Mal) that can be efficiently conjugated to exposed cysteine residues in peptides and proteins with the ultimate goal of directing these As(III) species to vicinal thiols in susceptible targets within cells and tissues. As-Mal conjugated to a surface cysteine in thioredoxin provides a more potent inhibitor for Escherichia coli thioredoxin reductase than comparable simple inorganic or organic arsenicals. As-Mal can be coupled to all of the eight cysteine residues of reduced unfolded ribonuclease A, or to site-specific locations using appropriate cysteine mutations. We demonstrate particularly strong binding to the two CxxC motifs of protein disulfide isomerase using a mutant RNase in which As-Mal is specifically incorporated at residues 26 and 110. As-Mal will provide a facile reagent for the incorporation of As(III) species into a wide range of thiol-containing proteins, biomaterials and surfaces.
Thymidylate, an essential DNA nucleotide, is synthesized de novo by all organisms. The enzyme thymidylate synthase catalyses the last committed step of this de novo pathway, which is the reductive methylation of 2′‐deoxyuridine‐5′‐monophosphate (dUMP) to produce 2′‐deoxythymidine‐5′‐monophosphate (dTMP). Most organism including humans, rely on classical thymidylate synthase. Recently the identification of many bacteria and human pathogens lacking this enzyme led to the discovery of flavin‐dependent thymidylate synthase (FDTS) encoded by the thyX gene. The two classes of enzyme share no structural or sequence homology and while the mechanism of classical enzyme has been studied over decades and is well established, the mechanism of thyX is still not clear. Overall, thyX receives reducing equivalents from NADPH and uses its flavin prosthetic group for redox chemistry and methylenetetrahydrofolate (CH2H4folate) as the methylene donor to produce dTMP with the concomitant release of tetrahydrofolate. Many different mechanisms have been proposed for the oxidative half‐reaction of this enzyme. One of the major questions involving its catalytic mechanisms is the activation of the pyrimidine substrate. Here we tested the feasibility of a recently published mechanism by synthesizing one of the proposed intermediates. We also present kinetic and spectroscopic data that provides more details into the kinetic mechanism of this flavoenzyme.
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