Derek Parsonage

Protein-sulfenic acid stabilization and function in enzyme catalysis and gene regulation.

Sulfenic acids (R‐SOH) result from the stoichiometric oxidations of thiols with mild oxidants such as H2O2; in solution, however, these derivatives accumulate only transiently due to rapid self‐condensation reactions, further oxidations to the sulfinic and/or sulfonic acids, and reactions with nucleophiles such as R‐SH. In contrast, oxidations of cysteinyl side chains in proteins, where disulfide bond formation can be prevented and where the reactivity of the nascent cysteine‐sulfenic acid (Cys‐SOH) can be controlled, have previously been shown to yield stable active‐site Cys‐SOH derivatives of papain and glyceraldehyde‐3‐phosphate dehydrogenase. More recently, however, functional Cys‐SOH residues have been identified in the native oxidized forms of the FAD‐containing NADH peroxidase and NADH oxidase from Streptococcus faecalis; these two proteins constitute a new class within the flavoprotein disulfide reductase family. In addition, Cys‐SOH derivatives have been suggested to play important roles in redox regulation of the DNA‐binding activities of transcription factors such as Fos and Jun, OxyR, and bovine papillomavirus type 1 E2 protein. Structural inferences for the stabilization of protein‐sulfenic acids, drawn from the refined 2.16‐Å structure of the streptococcal NADH peroxidase, provide a molecular basis for understanding the proposed redox functions of these novel cofactors in both enzyme catalysis and transcriptional regulation.— Claiborne, A., Miller, H., Parsonage, D., Ross, R. P. Protein‐sulfenic acid stabilization and function in enzyme catalysis and gene regulation. FASEB J. 7: 1483‐1490; 1993.

Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli

Bacillithiol (BSH), the α-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid, is a major low-molecular-weight thiol in Bacillus subtilis and related bacteria. Here, we identify genes required for BSH biosynthesis and provide evidence that the synthetic pathway has similarities to that established for the related thiol (mycothiol) in the Actinobacteria. Consistent with a key role for BSH in detoxification of electrophiles, the BshA glycosyltransferase and BshB1 deacetylase are encoded in an operon with methylglyoxal synthase. BshB1 is partially redundant in function with BshB2, a deacetylase of the LmbE family. Phylogenomic profiling identified a conserved unknown function protein (COG4365) as a candidate cysteine-adding enzyme (BshC) that co-occurs in genomes also encoding BshA, BshB1, and BshB2. Additional evolutionarily linked proteins include a thioredoxin reductase homolog and two thiol:disulfide oxidoreductases of the DUF1094 (CxC motif) family. Mutants lacking BshA, BshC, or both BshB1 and BshB2 are devoid of BSH. BSH is at least partially redundant in function with other low-molecular-weight thiols: redox proteomics indicates that protein thiols are largely reduced even in the absence of BSH. At the transcriptional level, the induction of genes controlled by two thiol-based regulators (OhrR, Spx) occurs normally. However, BSH null cells are significantly altered in acid and salt resistance, sporulation, and resistance to electrophiles and thiol reactive compounds. Moreover, cells lacking BSH are highly sensitive to fosfomycin, an epoxide-containing antibiotic detoxified by FosB, a prototype for bacillithiol-S-transferase enzymes.

A high throughput drug screen for Entamoeba histolytica identifies a new lead and target

Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to protozoan infections worldwide, resulting in ∼70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole, which has adverse effects, and potential resistance of E. histolytica to the drug is an increasing concern. To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA.

Peroxiredoxins: guardians against oxidative stress and modulators of peroxide signaling

Peroxiredoxins (Prxs) are a ubiquitous family of cysteine-dependent peroxidase enzymes that play dominant roles in regulating peroxide levels within cells. These enzymes, often present at high levels and capable of rapidly clearing peroxides, display a remarkable array of variations in their oligomeric states and susceptibility to regulation by hyperoxidative inactivation and other post-translational modifications. Key conserved residues within the active site promote catalysis by stabilizing the transition state required for transferring the terminal oxygen of hydroperoxides to the active site (peroxidatic) cysteine residue. Extensive investigations continue to expand our understanding of the scope of their importance as well as the structures and forces at play within these critical defense and regulatory enzymes.

Substrate specificity and redox potential of AhpC, a bacterial peroxiredoxin

Typical 2-Cys peroxiredoxins (Prxs) are ubiquitous peroxidases that are involved in peroxide scavenging and/or the regulation of peroxide signaling in eukaryotes. Despite their prevalence, very few Prxs have been reliably characterized in terms of their substrate specificity profile and redox potential even though these values are important for gaining insight into physiological function. Here, we present such studies focusing on Salmonella typhimurium alkyl hydroperoxide reductase C component (StAhpC), an enzyme that has proven to be an excellent prototype of this largest and most widespread class of Prxs that includes mammalian Prx I–Prx IV. The catalytic efficiencies of StAhpC (kcat/Km) are >107 M−1·s−1 for inorganic and primary hydroperoxide substrates and ≈100-fold less for tertiary hydroperoxides, with the difference being exclusively caused by changes in Km. The oxidative inactivation of AhpC through reaction with a second molecule of peroxide shows parallel substrate specificity. The midpoint reduction potential of StAhpC is determined to be −178 ± 0.4 mV, a value much higher than most other thiol-based redox proteins. The relevance of these results for our understanding of Prx and the physiological role of StAhpC is discussed.

Structural, redox, and mechanistic parameters for cysteine-sulfenic acid function in catalysis and regulation

A primary objective of this review is to facilitate the application of the chemical and structural approaches that are currently being employed in the identification of Cys-SOH, as both transient intermediates and stable redox forms, in biochemical systems where these derivatives are suspected of playing key roles in redox catalysis or regulation. These range from high-resolution crystallographic analyses benefiting from recent technological advances in rapid data collection at cryogenic temperatures to 13C NMR investigations of [3-(13)C]Cys-labeled proteins and chemical modification protocols that can be integrated with both UV-visible and fluorescence spectroscopic as well as mass spectrometric (especially ESI, MALDI-TOF, and even FT ion-cyclotron-resonance) analyses. In summarizing the diversity of biological functions currently identified with Cys-SH reversible Cys-SOH redox cycles (Fig. 17), it should also be [figure: see text] emphasized that in at least one protein (nitrile hydratase) stable Cys-SOH and Cys-SO2H derivatives play important structural roles while also modulating the electronic properties of the iron center; in neither case is the Cys-SOH residue itself involved in reduction and oxidation. The somewhat incomplete structural descriptions of the oxidized Cys forms involved in redox regulation of some transcription factors (e.g., BPV-1 E2 protein and activator protein-1) indicate that there is ample room for the application of the types of investigations employed, for example, with NADH peroxidase and the AhpC peroxiredoxin, with a view toward defining the potential roles of Cys-SOH in these very important contexts of intracellular redox signaling. These advances will also build on the recent progress in defining sulfenic acid stabilization and properties in small molecule model systems, as evidenced in the work of Okazaki, Goto, and others. When viewed in the perspective of Allisons 1976 review on the subject of sulfenic acids in proteins, the reader will hopefully come to appreciate the conclusion that the concept of protein-sulfenic acids has now become a very well-defined and established principle of biochemistry, with current efforts in this and other laboratories being directed to bring about still more detailed understanding of Cys-SOH function in both redox and nonredox modes of enzyme catalysis and regulation of protein function.

Structural Evidence that Peroxiredoxin Catalytic Power Is Based on Transition-State Stabilization

Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cell-wide peroxide reduction, but the origins of their high activity have been mysterious. We present here a novel structure of human PrxV at 1.45 A resolution that has a dithiothreitol bound in the active site with its diol moiety mimicking the two oxygens of a peroxide substrate. This suggests diols and similar di-oxygen compounds as a novel class of competitive inhibitors for the Prxs. Common features of this and other structures containing peroxide, peroxide-mimicking ligands, or peroxide-mimicking water molecules reveal hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen track along which the peroxide oxygens move as the reaction proceeds. Key insights include how the active-site microenvironment activates both the peroxidatic cysteine side chain and the peroxide substrate and how it is exquisitely well suited to stabilize the transition state of the in-line S(N)2 substitution reaction that is peroxidation.

Regulatory effects of ferritin on angiogenesis.

Angiogenesis, the synthesis of new blood vessels from preexisting vessels, plays a critical role in normal wound healing and tumor growth. HKa (cleaved high molecular weight kininogen) is an endogenous inhibitor of angiogenesis formed by the cleavage of kininogen on endothelial cells. Ferritin is a protein principally known for its central role in iron storage. Here, we demonstrate that ferritin binds to HKa with high affinity (Kd 13 nM). Further, ferritin antagonizes the antiangiogenic effects of HKa, enhancing the migration, assembly, and survival of HKa-treated endothelial cells. Effects of ferritin were independent of its iron content. Peptide mapping revealed that ferritin binds to a 22-aa subdomain of HKa that is critical to its antiangiogenic activity. In vivo, ferritin opposed HKas antiangiogenic effects in a human prostate cancer xenograft, restoring tumor-dependent vessel growth. Ferritin-mediated regulation of angiogenesis represents a new angiogenic regulatory pathway, and identifies a new role for ferritin in cell biology.

Cloning and sequencing of two enterococcal glpK genes and regulation of the encoded glycerol kinases by phosphoenolpyruvate-dependent, phosphotransferase system-catalyzed phosphorylation of a single histidyl residue.

The glpK genes of Enterococcus casseliflavus and Enterococcus faecalis, encoding glycerol kinase, the key enzyme of glycerol uptake and metabolism in bacteria, have been cloned and sequenced. The translated amino acid sequences exhibit strong homology to the amino acid sequences of other bacterial glycerol kinases. After expression of the enterococcalglpK genes in Escherichia coli, both glycerol kinases were purified and were found to be phosphorylated by enzyme I and the histidine-containing protein of the phosphoenolpyruvate:glycose phosphotransferase system. Phosphoenolpyruvate-dependent phosphorylation caused a 9-fold increase in enzyme activity. The site of phosphorylation in glycerol kinase of E. casseliflavus was determined as His-232. Site-specific mutagenesis was used to replace His-232 in glycerol kinase of E. casseliflavus with an alanyl, glutamate, or arginyl residue. The mutant proteins could no longer be phosphorylated confirming that His-232 of E. casseliflavusglycerol kinase represents the site of phosphorylation. The His232 → Arg glycerol kinase exhibited an about 3-fold elevated activity compared with wild-type glycerol kinase. Fructose 1,6-bisphosphate was found to inhibit E. casseliflavusglycerol kinase activity. However, neither EIIAGlc fromE. coli nor the EIIAGlc domain ofBacillus subtilis had an inhibitory effect on glycerol kinase of E. casseliflavus.

Cysteine pKa values for the bacterial peroxiredoxin AhpC

Salmonella typhimurium AhpC is a founding member of the peroxiredoxin family, a ubiquitous group of cysteine-based peroxidases with high reactivity toward hydrogen peroxide, organic hydroperoxides, and peroxynitrite. For all of the peroxiredoxins, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), acts as a nucleophile in attacking the peroxide substrate, forming a cysteine sulfenic acid at the active site. Because thiolates are far stronger nucleophiles than thiol groups, it is generally accepted that cysteine-based peroxidases should exhibit pK(a) values lower than an unperturbed value of 8.3-8.5. In this investigation, several independent approaches were used to assess the pK(a) of the two cysteinyl residues of AhpC. Methods using two different iodoacetamide derivatives yielded unperturbed pK(a) values (7.9-8.7) for both cysteines, apparently due to reactivity with the wrong conformation of C(P) (i.e., locally unfolded and flipped out of the active site), as supported by X-ray crystallographic analyses. A functional pK(a) of 5.94 +/- 0.10 presumably reflecting the titration of C(P) within the fully folded active site was obtained by measuring AhpC competition with horseradish peroxidase for hydrogen peroxide; this value is quite similar to that obtained by analyzing the pH dependence of the epsilon(240) of wild-type AhpC (5.84 +/- 0.02) and similar to those obtained for two typical 2-cysteine peroxiredoxins from Saccharomyces cerevisiae (5.4 and 6.0). Thus, the pK(a) value of AhpC balances the need for a deprotonated thiol (at pH 7, approximately 90% of the C(P) would be deprotonated) with the fact that thiolates with higher pK(a) values are stronger nucleophiles.