Glucose-6-Phosphate Dehydrogenase from the Human Pathogen Trypanosoma cruzi Evolved Unique Structural Features to Support Efficient Product Formation.

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer-dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.

HPLC separation of human serum albumin isoforms based on their isoelectric points.

Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA.

Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids.

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ∼threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.

Factors affecting protein thiol reactivity and specificity in peroxide reduction.

Protein thiol reactivity generally involves the nucleophilic attack of the thiolate on an electrophile. A low pK(a) means higher availability of the thiolate at neutral pH but often a lower nucleophilicity. Protein structural factors contribute to increasing the reactivity of the thiol in very specific reactions, but these factors do not provide an indiscriminate augmentation in general reactivity. Notably, reduction of hydroperoxides by the catalytic cysteine of peroxiredoxins can achieve extraordinary reaction rates relative to free cysteine. The discussion of this catalytic efficiency has centered in the stabilization of the thiolate as a way to increase nucleophilicity. Such stabilization originates from electrostatic and polar interactions of the catalytic cysteine with the protein environment. We propose that the set of interactions is better described as a means of stabilizing the anionic transition state of the reaction. The enhanced acidity of the critical cysteine is concurrent but not the cause of catalytic efficiency. Protein stabilization of the transition state is achieved by (a) a relatively static charge distribution around the cysteine that includes a conserved arginine and the N-terminus of an α-helix providing a cationic environment that stabilizes the reacting thiolate, the transition state, and also the anionic leaving group; (b) a dynamic set of polar interactions that stabilize the thiolate in the resting enzyme and contribute to restraining its reactivity in the absence of substrate; but upon peroxide binding these active/binding site groups switch interactions from thiolate to peroxide oxygens, simultaneously increasing the nucleophilicity of the attacking sulfur and facilitating the correct positioning of the substrate. The switching of polar interaction provides further acceleration and, importantly, confers specificity to the thiol reactivity. The extraordinary thiol reactivity and specificity toward H(2)O(2) combined with their ubiquity and abundance place peroxiredoxins, along with glutathione peroxidases, as obligate hydroperoxide cellular sensors.

Distance-dependent diffusion-controlled reaction of •NO and O2•- at chemical equilibrium with ONOO-.

The fast reaction of (•)NO and O(2)(•-) to give ONOO(-) has been extensively studied at irreversible conditions, but the reasons for the wide variations in observed forward rate constants (3.8 ≤ k(f) ≤ 20 × 10(9) M(-1) s(-1)) remain unexplained. We characterized the diffusion-dependent aqueous (pH > 12) chemical equilibrium of the form (•)NO + O(2)(•-) = ONOO(-) with respect to its dependence on temperature, viscosity, and [ONOO(-)](eq) by determining [ONOO(-)](eq) and [(•)NO](eq). The equilibrium forward reaction rate constant (k(f)(eq)) has negative activation energy, in contrast to that found under irreversible conditions. In contradiction to the law of mass action, we demonstrate that the equilibrium constant depends on ONOO(-) concentration. Therefore, a wide range of k(f)(eq) values could be derived (7.5-21 × 10(9) M(-1) s(-1)). Of general interest, the variations in k(f) can thus be explained by its dependence on the distance between ONOO(-) particles (sites of generation of (•)NO and O(2)(•-)).

Structural and enzymatic insights into the ATP binding and autophosphorylation mechanism of a sensor histidine kinase.

DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface.

Thiol and sulfenic acid oxidation of AhpE, the one-cysteine peroxiredoxin from Mycobacterium tuberculosis: kinetics, acidity constants, and conformational dynamics.

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen's antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhpE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Reactivity of sulfenic acid in human serum albumin.

Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA-SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was approximately 20-fold faster than the second phase and first order in HSA-SOH and TNB (105 +/- 11 M-1 s-1, 25 degrees C, pH 7.4), allowing quantitative data on HSA-SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37 degrees C, 4 min) yielded 0.18 +/- 0.02 mol of HSA-SOH per mol of HSA. HSA-SH reacted with hydrogen peroxide at 2.7 +/- 0.7 M-1 s-1 (37 degrees C, pH 7.4), while HSA-SOH reacted at 0.4 +/- 0.2 M-1 s-1, yielding sulfinic acid (HSA-SO2H), as detected by mass spectrometry. The rate constants of HSA-SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA-SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA-SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 +/- 0.2, 2.9 +/- 0.5, 9.3 +/- 0.9, and 55 +/- 3 M-1 s-1 (25 degrees C, pH 7.4), respectively, supporting a role for HSA-SOH in the formation of mixed disulfides.