Biomedical subjects

Emma Lloyd Raven

Publications and source records attributed to Emma Lloyd Raven.

14 recordsLinked to original sources

PubMed2006

Indoleamine 2,3-dioxygenase-expressing dendritic cells form suppurative granulomas following Listeria monocytogenes infection.

Control of pathogens by formation of abscesses and granulomas is a major strategy of the innate immune system, especially when effector mechanisms of adaptive immunity are insufficient. We show in human listeriosis that DCs expressing indoleamine 2,3-dioxygenase (IDO), together with macrophages, are major cellular components of suppurative granulomas in vivo. Induction of IDO by DCs is a cell-autonomous response to Listeria monocytogenes infection and was also observed in other granulomatous infections with intracellular bacteria, such as Bartonella henselae. Reporting on our use of the clinically applied anti-TNF-alpha antibody infliximab, we further demonstrate in vitro that IDO induction is TNF-alpha dependent. Repression of IDO therefore might result in exacerbation of granulomatous diseases observed during anti-TNF-alpha therapy. These findings place IDO(+) DCs not only at the intersection of innate and adaptive immunity but also at the forefront of bacterial containment in granulomatous infections.

Antigens, CD↗

PubMed2006

Ascorbate peroxidase-thioredoxin interaction.

Proteomics data have suggested ascorbate peroxidase (APX) to be a potential thioredoxin-interacting protein. Using recombinant enzymes, we observed that incubation of pea cytosolic APX with reduced poplar thioredoxins h drastically inactivated the peroxidase. A similar inactivation is induced by reduced glutathione and dithiothreitol, whereas diamide and oxidized glutathione have no effect. Oxygen consumption measurements, modifications of the APX visible spectrum and protection by hydrogen peroxide scavenging enzymes suggest that APX oxidizes thiols leading to the generation of thiyl radicals. These radicals can in turn react with thiyl anions to produce the disulfide radical anions, which are responsible for oxygen reduction and subsequent hydrogen peroxide production. The APX inactivation is not due solely to hydrogen peroxide since fluorimetry indicates that the environment of the APX tryptophan residues is dramatically modified only in the presence of thiol groups. The physiological implications of this interaction are discussed.

Arabidopsis↗

PubMed2006

Interaction of ascorbate peroxidase with substrates: a mechanistic and structural analysis.

Previous work [Sharp, K. H., et al. (2003) Nat. Struct. Biol. 10, 303-307] has revealed the location of the ascorbate binding site in ascorbate peroxidase and has identified hydrogen-bonding interactions to Arg172, Lys30, and the heme 6-propionate as important in formation of the enzyme-substrate complex. In this work, the individual and collective contributions of these hydrogen bond interactions have been dissected using site-directed mutagenesis, steady-state and pre-steady-state kinetics, X-ray crystallography, and modified substrate analogues. Steady-state and pre-steady-state kinetic data reveal that the hydrogen bonds to Arg172 and the heme 6-propionate play a major part in stabilization of the bound ascorbate but that the interaction with Lys30 plays only a minor role. Binding of aromatic substrates is not affected by substitutions at Arg172/Lys30. Neutralization or removal of electrostatic charge at (Lys30) or adjacent to (Lys31) the ascorbate site does not substantially disrupt the binding interaction. Substrate oxidation and reduction of Compounds I and II is still possible in the absence of Arg172, but at a much reduced level. Crystallographic data (to 1.8 A) for the R172A variant indicate that the molecular structure of the proposed [Sharp, K. H., et al. (2004) Biochemistry 43, 8644-8651] proton transfer pathway from the ascorbate to the heme is conserved, which accounts for the residual activity. The results are discussed in terms of our wider understanding of the structural features that control substrate binding specificity in other peroxidase enzymes.

Amino Acid Sequence↗

PubMed2006

Conformational mobility in the active site of a heme peroxidase.

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.

Catalytic Domain↗

PubMed2005

Redox and spectroscopic properties of human indoleamine 2,3-dioxygenase and a His303Ala variant: implications for catalysis.

Indoleamine 2,3-dioxygenase is an important mammalian target that catalyses the oxidative cleavage of l-tryptophan to N-formylkynurenine. In this work, the redox properties of recombinant human indoleamine 2,3-dioxygenase (rhIDO) and its H303A variant have been examined for the first time and the spectroscopic and substrate-binding properties of rhIDO and H303A in the presence and absence of substrate are reported. The Fe(3+)/Fe(2+) reduction potential of H303A was found to be -30 +/- 4 mV; in the presence of l-Trp, this value increases to +16 +/- 3 mV. A variety of spectroscopies indicate that ferric rhIDO at pH 6.6 exists as a mixture of six-coordinate, high-spin, water-bound heme and a low-spin species that contains a second nitrogenous ligand; parallel experiments on H303A are consistent either with His303 as the sixth ligand or with His303 linked to a conformational change that affects this transition. There is an increase in the low-spin component at alkaline pH for rhIDO, but this is not due to hydroxide-bound heme. Substrate binding induces a conformational rearrangement and formation of low-spin, hydroxide-bound heme; analysis of the H303A variant indicates that His303 is not required for this conversion and is not essential for substrate binding. The Fe(3+)/Fe(2+) reduction potential of H303A variant is approximately 70 mV lower than that of rhIDO, leading to a destabilization of the ferrous-oxy complex, which is an obligate intermediate in the catalytic process. In comparison with the properties of other heme enzymes, the data can be used to build a more detailed picture of substrate binding and catalysis in indoleamine 2,3-dioxygenase. The wider implications of these results are discussed in the context of our current understanding of the catalytic mechanism of the enzyme.

Alanine↗

PubMed2004

Autocatalytic formation of green heme: evidence for H2O2-dependent formation of a covalent methionine-heme linkage in ascorbate peroxidase.

The mammalian heme peroxidases are distinguished from their plant and fungal counterparts by the fact that the heme group is covalently bound to the protein through ester links from glutamate and aspartate residues to the heme 1- and 5-methyl groups and, in the case of myeloperoxidase, through an additional sulfonium link from the Cbeta of the 2-vinyl group to a methionine residue. To duplicate the sulfonium link in myeloperoxidase and to obtain information on its mechanism of formation, we have engineered a methionine residue close to the 2-vinyl group in recombinant pea cytosolic ascorbate peroxidase (rpAPX) by replacement of Ser160 by Met (S160M variant). The S160M variant is isolated from Escherichia coli as apo-protein. Reconstitution of apo-S160M with exogenous heme gives a red protein (S160M(R)) which has UV-visible (lambda(max)/nm = 407, 511, 633) and steady-state kinetic (kcat = 156 +/- 7 s(-1), KM = 102 +/- 15 microM) properties that are analogous to those of rpAPX. The reaction of S160M(R) with H2O2 gives a green protein (S160M(G)). Electronic spectroscopy, mass spectrometry, and HPLC analyses are consistent with the formation of a covalent linkage between the methionine residue and the heme vinyl group in S160M(G). Single-wavelength and photodiode array stopped-flow kinetic analyses identify a transient Compound I species as a reaction intermediate. The results provide the first direct evidence that covalent heme linkage formation occurs as an H2O2-dependent process that involves Compound I formation. A mechanism that is consistent with the data is presented.

Amino Acid Sequence↗

PubMed2004

Crystal structure of the ascorbate peroxidase-salicylhydroxamic acid complex.

Ascorbate peroxidase is a bifunctional peroxidase that catalyzes the H(2)O(2)-dependent oxidation of both ascorbate and various aromatic substrates. The ascorbate binding site was recently identified as being close to the gamma-heme edge [Sharp, K. H., Mewies, M., Moody, P. C. E., and Raven, E. L. (2003)Nat. Struct. Biol. 10, 303-307]. In this work, the X-ray crystal structure of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) in complex with salicylhydroxamic acid (SHA) has been determined to 1.46 A. The SHA molecule is bound close to the delta-heme edge in a cavity that connects the distal side of the heme to the surface of the protein. There are hydrogen bonds between the phenolic hydroxide of the SHA and the main chain carbonyl of Pro132, between the carbonyl oxygen of SHA and the side chain guanadinium group of Arg38, and between the hydroxamic acid group and the indole nitrogen of Trp41. The structure provides the first information about the location of the aromatic binding site in ascorbate peroxidase and, together with our previous data [Sharp, K. H., et al. (2003) Nat. Struct. Biol. 10, 303-307], completes the structural description of the binding properties of ascorbate peroxidase. The mechanistic implications of the results are discussed in terms of our current understanding of how APX catalyzes oxidation of different types of substrates bound at different locations.

Arginine↗

PubMed2004

Crystal structure of Mycobacterium tuberculosis catalase-peroxidase.

The Mycobacterium tuberculosis catalase-peroxidase is a multifunctional heme-dependent enzyme that activates the core anti-tuberculosis drug isoniazid. Numerous studies have been undertaken to elucidate the enzyme-dependent mechanism of isoniazid activation, and it is well documented that mutations that reduce activity or inactivate the catalase-peroxidase lead to increased levels of isoniazid resistance in M. tuberculosis. Interpretation of the catalytic activities and the effects of mutations upon the action of the enzyme to date have been limited due to the lack of a three-dimensional structure for this enzyme. In order to provide a more accurate model of the three-dimensional structure of the M. tuberculosis catalase-peroxidase, we have crystallized the enzyme and now report its crystal structure refined to 2.4-A resolution. The structure reveals new information about dimer assembly and provides information about the location of residues that may play a role in catalysis including candidates for protein-based radical formation. Modeling and computational studies suggest that the binding site for isoniazid is located near the delta-meso heme edge rather than in a surface loop structure as currently proposed. The availability of a crystal structure for the M. tuberculosis catalase-peroxidase also permits structural and functional effects of mutations implicated in causing elevated levels of isoniazid resistance in clinical isolates to be interpreted with improved confidence.

Amino Acid Sequence↗

PubMed2004

Enzyme-catalyzed mechanism of isoniazid activation in class I and class III peroxidases.

There is an urgent need to understand the mechanism of activation of the frontline anti-tuberculosis drug isoniazid by the Mycobacterium tuberculosis catalase-peroxidase. To address this, a combination of NMR spectroscopic, biochemical, and computational methods have been used to obtain a model of the frontline anti-tuberculosis drug isoniazid bound to the active site of the class III peroxidase, horseradish peroxidase C. This information has been used in combination with the new crystal structure of the M. tuberculosis catalase-peroxidase to predict the mode of INH binding across the class I heme peroxidase family. An enzyme-catalyzed mechanism for INH activation is proposed that brings together structural, functional, and spectroscopic data from a variety of sources. Collectively, the information not only provides a molecular basis for understanding INH activation by the M. tuberculosis catalase-peroxidase but also establishes a new conceptual framework for testing hypotheses regarding the enzyme-catalyzed turnover of this compound in a number of heme peroxidases.

Antitubercular Agents↗

PubMed2003

Exploiting the conformational flexibility of leghemoglobin: a framework for examination of heme protein axial ligation.

We have exploited the intrinsic conformational flexibility of leghemoglobin to reengineer the heme active site architecture of the molecule by replacement of the mobile His61 residue with tyrosine (H61Y variant). The electronic absorption spectrum of the ferric derivative of H61Y is similar to that observed for the phenolate derivative of the recombinant wild-type protein (rLb), consistent with coordination of Tyr61 to (high-spin) iron. EXAFS data clearly indicate a 6-coordinate heme geometry and a Fe-O bond length of 185pm. MCD and EPR spectroscopies are consistent with this assignment and support ligation by an anionic (tyrosinate) group. The alteration in heme ligation leads to a 148mV decrease in the reduction potential for H61Y (-127+/-5mV) compared to rLb and destabilisation of the functional oxy-derivative. The results are discussed in terms of our wider understanding of other heme proteins with His-Tyr ligation.

Binding Sites↗

PubMed2003

Crystal structure of the ascorbate peroxidase-ascorbate complex.

Heme peroxidases catalyze the H2O2-dependent oxidation of a variety of substrates, most of which are organic. Mechanistically, these enzymes are well characterized: they share a common catalytic cycle that involves formation of a two-electron, oxidized Compound I intermediate followed by two single-electron reduction steps by substrate. The substrate specificity is more diverse--most peroxidases oxidize small organic substrates, but there are prominent exceptions--and there is a notable absence of structural information for a representative peroxidase-substrate complex. Thus, the features that control substrate specificity remain undefined. We present the structure of the complex of ascorbate peroxidase-ascorbate. The structure defines the ascorbate-binding interaction for the first time and provides new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase catalysis for more than 20 years. A new mechanism for electron transfer is proposed that challenges existing views of substrate oxidation in other peroxidases.

Amino Acid Sequence↗

PubMed2003

Understanding functional diversity and substrate specificity in haem peroxidases: what can we learn from ascorbate peroxidase?

This review summarizes the most recent advances in our understanding of the haem enzyme ascorbate peroxidase. The aim is to show how the combined applications of protein engineering, mechanistic and structural studies can be used to provide an overall picture of enzyme catalysis, and how this information can be used to provide new insight into other, more well-characterized peroxidases (in particular cytochrome c peroxidase). It contains 212 references and covers literature up to March 2003.

Amino Acid Sequence↗

PubMed2002

Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.

The catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) and a derivative of rsAPX in which a cysteine residue (Cys32) located close to the substrate (L-ascorbic acid) binding site has been modified to preclude binding of ascorbate [Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617] has been examined using pre-steady-state and steady-state kinetic techniques. Formation (k1 = 3.3 +/- 0.1 x 10(7) M(-1) s(-1)) of Compound I and reduction (k(2) = 5.2 +/- 0.3 x 10(6) M(-1) s(-1)) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (lambda(max) (nm) = 409, 530, 569, 655) are consistent with a porphyrin pi-cation radical. Reduction of Compound II by L-ascorbate is rate-limiting: at low substrate concentration (0-500 microM), kinetic traces were monophasic but above approximately 500 microM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 microM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k(3-slow)) = 3.1 +/- 0.1 x 10(3) M(-1) s(-1)). Kinetic transients for reduction of Compound II by L-ascorbic acid for Cys32-modified rsAPX are monophasic at all substrate concentrations, and the second-order rate constant (k(3) = 0.9 +/- 0.1 x 10(3) M(-1) s(-1)) is similar to that obtained from the slow phase of Compound II reduction for unmodified rsAPX. Steady-state oxidation of L-ascorbate by rsAPX showed a sigmoidal dependence on substrate concentration and data were satisfactorily rationalized using the Hill equation; oxidation of L-ascorbic acid by Cys32-modified rsAPX showed no evidence of sigmoidal behavior. The data are consistent with the presence of two kinetically competent binding sites for ascorbate in APX.

Ascorbate Peroxidases↗

PubMed2002

Redox control in heme proteins: electrostatic substitution in the active site of leghemoglobin.

The effects of electrostatic substitutions on the spectroscopic, ligand binding, and redox properties of the heme in leghemoglobin have been examined by replacement of the proximal leucine 88 residue with an aspartic acid residue (Leu88Asp). Electronic and resonance Raman spectra of the ferric derivative of Leu88Asp indicate a mixture of 6-coordinate, high-spin and 6-coordinate, low-spin hemes, analogous to that observed in the recombinant wild-type protein (rLb). At alkaline pH, formation of hydroxide-bound heme is indicated for Leu88Asp; the pK(a) for this transition (8.7 +/- 0.2, micro = 0.10 M, 25.0 degrees C) is 0.4 pH units higher than for rLb. Equilibrium dissociation constants (sodium phosphate, pH 7.0, micro = 0.10 M, 25.0 +/- 0.1 degrees C) for binding of anionic ligands (N(-)(3), nicotinate) to Leu88Asp are higher (K(d,nicotinate) = 6.8 +/- 0.2 microM; K(d,azide) = 33 +/- 0.6 microM) than the corresponding values for rLb (K(d,nicotinate) = 1.4 +/- 0.3 microM (pH 5.5, micro = 0.10 M, 25.0 +/- 0.1 degrees C); K(d,azide) = 4.8 +/- 0.2 microM). Resonance Raman spectra (sodium phosphate, pH 7.0, micro = 0.10 M) for the ferrous derivatives of Leu88Asp and rLb exhibit a strong nu(Fe-His) stretching frequency at 223 cm(-1) in both cases, indicating that the hydrogen bonding structure on the proximal side is not substantially altered in the variant. The reduction potential of Leu88Asp is -14 +/- 2 mV vs standard hydrogen electrode (SHE) (25.0 degrees C, micro = 0.10 M, pH 7.0), a decrease of 35 mV over the corresponding value for the wild-type protein under the same conditions (21 +/- 3 mV vs SHE). An assessment of these data in terms of electrostatic and hydrogen bonding considerations is presented.

Aspartic Acid↗