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1 Unité Mixte de Recherche (UMR) Centre National de la Recherche Scientifique (CNRS)-UHP 7567, Maturation des ARN et Enzymologie Molé culaire, Faculté des Sciences, Université Henri Poincaré Nancy I, 54506 Vandoeuvre-lès-Nancy, France
2 Laboratoire de Chimie et Toxicologie des métaux, Faculté de Médecine, Université Henri Poincaré Nancy I, 54505 Vandoeuvre-lès-Nancy, France
Reprint requests to: Guy Branlant, UMR CNRS-UHP 7567, Maturation des ARN et Enzymologie Molé culaire, Faculté des Sciences, Université Henri Poincaré Nancy I, Bld. des Aiguillettes, BP 239, 54506 Vandoeuvre-lès-Nancy, France; e-mail: guy.branlant{at}maem.uhp-nancy.fr; fax: +(33) 3-83-68-43-07.
(RECEIVED July 21, 2005; FINAL REVISION August 26, 2005; ACCEPTED August 26, 2005)
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Keywords: methionine sulfoxide reductase B; metal binding site; zinc; iron; apoenzyme; thioredoxin
Abbreviations: MetSO, methionine sulfoxide • ROS, reactive oxygen species • Msr, MsrA, and MsrB, methionine sulfoxide reductase, methionine sulfoxide reductase A, methionine sulfoxide reductase B, respectively • eMsrB and nmMsrB, Escherichia coli and Neisseria meningitidis MsrB, respectively • IPTG, isopropyl-
-D-thiogalactopyranoside • DTT, dithiothreitol • Trx, thioredoxin • TCA, trichloroacetic acid • CD, circular dichroism • ESI, electrospray ionization • DSC, differential scanning calorimetry • ANS, 8-anilino-1-naphtalenesulfonic acid.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051711105.
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). It consists of three steps that include a reductase step resulting in formation of a sulfenic acid intermediate on the catalytic cysteine and release of 1 mol of methionine per mol of Msr followed by a second step in which an intramolecular disulfide bond is formed between the catalytic and the recycling cysteines. Finally, in the third step, called the recycling step, thioredoxin (Trx), which interacts specifically with the oxidized form of the Msr, reduces the Msr disulfide bond leading to regeneration of the reduced form of the Msr (Boschi-Muller et al. 2000, 2005; Olry et al. 2002). The kinetics of the three steps have also been extensively studied. The overall rate-limiting step is associated with the recycling process, but more precisely with dissociation of reduced Msr and oxidized Trx, while the rate-determining step in the process leading to disulfide formation is associated with the sulfenic acid formation (Antoine et al. 2003; Olry et al. 2004; Boschi-Muller et al. 2005).
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). In contrast, Neisseria gonorrhoeae MsrB domain contains no CXXC signatures as revealed by its crystal structure, and represents the only published report of MsrB 3D structure so far (Lowther et al. 2002). It shows a core domain composed of two anti-parallel -sheets surrounded by three 
-helices and five 310-helices. One of the two active sites in the asymmetric unit is occupied by a cacodylate molecule that likely mimicks the binding of the substrate with one of the two methyl groups representing the 
-methyl group of the MetSO and which points to W65. The site resides in a pocket exposed on the surface of the protein. The catalytic C117 is located on a -strand, while the recycling C63 and the fluorescent probe W65 (Olry et al. 2004) are both situated within a loop. The respective position of the two cysteines is compatible with formation of a disulfide bond between C117 and C63. The three-dimensional structure also reveals a putative metal binding site, at a distance of 20 Å from the active site, the fold of which is structurally related to that of the exchange factor Mss4 in which a Zn2+ atom is strongly bound via two CXXC signatures (Yu and Schreiber 1995a, b).
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In the present study, substitutions of C45, C48, C94, and C97 of the E. coli MsrB (eMsrB) with the corresponding amino acids of the consensus sequence D45, S48, S94, S97, or A97 of the MsrBs devoid of the CXXC signatures have been performed (Fig. 2
). Substitution of cysteines for D45, S48, S94, and A97 of the Neisseria meningitidis MsrB (nmMsrB) has also been carried out in reverse. The results show that (1) the absence of bound metal in the eMsrB provokes drastic structural modifications that explains why the reductase activity is lost; (2) D45C/S48C/S94C/A97C nmMsrB tightly binds the metal that is indicative of a metal binding site preformed in MsrBs which does not possess the two CXXC signatures; and (3) metal binding strongly increases the thermal stability of the D45C/S48C/S94C/A97C nmMsrB, although the eMsrB and nmMsrB, which contain bound metal or not, respectively, display similar thermal stabilization as shown by differential scanning calorimetry (DSC) analysis. These results are discussed in the context of the knowledge of the three-dimensional structure of the Neisseria MsrBs and in terms of evolution.
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). It is a plausible assumption that zinc is the metal relevant from a physiological point of view and the presence of iron is likely due to the shortage of zinc when eMsrB is overexpressed in E. coli. To support this experimentally, it would be necessary to isolate the eMsrB from a nontransformed E. coli strain.
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).
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).
Catalytic properties of the wild type and of the C45D/C48S/C94S/C97S eMsrB
In the presence of either saturating concentration of Trx or 10 mM DTT, a kcat value of 0.18 sec–1 and a kobs value of 1.10–2 sec–1, respectively, were determined for the wild-type eMsrB, similar to those of the nmMsrB (Table 2; Olry et al. 2002). MsrBs with different ratios of zinc and iron displayed similar kinetic parameters, suggesting that the nature of the metal bound has no effect on the conformation of the protein particularly in relation to the active site (data not shown). When C45, C48, C94, and C97 were altogether substituted, no activity was detected for the quadruple-mutated eMsrB in the presence of either Trx or DTT (Table 2).
The reductase step of the wild type was studied under single turnover quenched-flow conditions, at pH 8, in the absence of reductant, at a saturating concentration of Ac-L-Met-R,S-SO-NHMe as already described by Olry et al. (2004). The results showed formation of 1 mol Ac-Met-NHMe per mol of wild-type eMsrB within the aging time of the stopped-flow apparatus adapted for quenched-flow use, which is ~30 msec. This indicated a rate of the reductase step of at least 33 sec–1, in the range of that observed for wild-type nmMsrB (Olry et al. 2004). For the C45D/C48S/C94S/C97S eMsrB, no Ac-Met-NHMe was formed after an overnight incubation with saturating concentration of Ac-L-Met-R,S-SO-NHMe, which confirms the absence of activity with DTT.
Biochemical properties of the MsrBs with no bound metal, and comparison with those of the wild-type eMsrB
As shown in Figure 3A, the CD spectrum of the wild-type eMsrB and of the apo wild-type and C45D/C48S/C94S/C97S eMsrBs were quite different. In particular, the negative band at 208 nm observed for the wild-type eMsrB, which is likely representative of the content of -helices, shifted <204 nm. These results suggested that eMsrB in the nonmetal-bound state has either a significant proportion of random coil structures or regions of high flexibility, which probably reflect a less ordered conformation of the eMsrBs. The fact that the same perturbed spectrum was observed with the apo wild-type and the C45D/C48S/C94S/C97S eMsrBs showed that the structural perturbation observed is likely caused by the absence of the metal and not by the amino acids, which have been introduced in place of the cysteines. This is confirmed by the fact that adding zinc to the apo form led to an active enzyme with kinetic properties and CD spectrum similar to those of the wild type (data and spectrum not shown).
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max of the W65 fluorescence emission was centered at 334 nm under native conditions. The max was shifted to 353 nm in the case of the apo wild-type and C45D/C48S/C94S/C97S eMsrBs (Fig. 4A), similarly to what is observed with the wild type under denaturating conditions (in the presence of urea 4 M; curve not shown).
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). In contrast, no thermogram was observed for the apo wild type and the C45D/C48S/C94S/C97S eMsrB that reflects the absence of a significant 
H contribution in the thermal unfolding.
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max of the fluorescence emission was observed in the presence of hydrophobic fluorescent probe such as 8-anilino-1-naphtalenesulfonic acid (ANS) for wild-type, apo wild-type, and C45D/C48S/C94S/C97S eMsrBs under native conditions. On the contrary, an increase and a shift of max from 480 to 520 nm were observed for all the three forms under conditions compatible with the presence of a molten globule intermediate (in the presence of 0.1% SDS) (curves not shown). This suggested that apo and C45D/C48S/C94S/C97S eMsrBs have acquired a fold, although different from that of the wild type, with their hydrophobic residues buried within the protein interior.
Biochemical and catalytic properties of the D45C/S48C/S94C/A97C nmMsrB and comparison with those of the wild-type nmMsrB
nmMsrB belongs to a larger three-domain protein called PilB. PilB is composed of an N-terminal domain, which displays a disulfide reductase activity (Wu et al. 2005), a central domain, which bears an MsrA activity, while the C-terminal domain shows an MsrB activity (Olry et al. 2002). In the present study, the nmMsrB corresponds to the C-terminal domain of PilB, as described by Orly et al. (2002).
When overexpressed D45C/S48C/S94C/A97C nmMsrB was isolated, the concentrated protein solution was also pink colored. Metal analysis by atomic emission spectrometry showed a binding of zinc:iron with a ratio 8:2 and a global stoichiometryof 1atom of metal permol of protein in contrast to the wild type, in which no zinc/iron is bound (Table 2). Moreover, chelating reagents had no effect on the metal content and on the activity, suggesting again a tight binding of the metal (data not shown).
No Trx recycling activity was observed, whereas activity measured with DTT was in the range of that of the wild-type nmMsrB (Table 2). This showed that although the Trx recycling process is not operative, the reductase step was likely as efficient as that of the wild type. To validate this assumption, the rate of the reductase step of the D45C/S48C/S94C/A97C nmMsrB was determined by following the rate associated with a change of the W65 fluorescence intensity upon going from the reduced to the sulfenic acid form as already described by Olry et al. (2004). From the curve of kobs versus substrate concentration, a KS value of 170 ± 30 mM for Ac-L-Met-R,S-SO-NH-Me and a kobs max value of 65 ± 4 sec–1 were determined at pH 8, similar to those described for the wild-type nmMsrB (Fig. 6; Olry et al. 2004). The fact that an efficient Trx recycling activity was observed for the apo D45C/S48C/S94C/A97C nmMsrB with a kcat value of 0.06 sec–1 similar to that of the wild-type nmMsrB showed that the loss of the Trx recycling activity is likely due to the presence of the bound metal.
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) and the emission fluorescence (Fig. 4B) spectra of the wild-type and D45C/S48C/S94C/A97C nmMsrBs were recorded. No significant difference was observed, which indicates the absence of major structural modifications caused by the metal binding.
DSC experiments carried out on the nmMsrB showed that the Tm value is shifted from 39°–42°C for the apo D45C/S48C/S94C/A97C and the wild-type nmMsrBs to 91°C for the D45C/S48C/S94C/A97C nmMsrB in which a metal is bound (Fig. 5).
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H contribution in the thermal unfolding as revealed by DSC analysis in contrast to the wild-type eMsrB, which presents an endotherm with a Tm of 40.2°C.
Therefore, the role of the metal in eMsrB is likely to stabilize the core structure, which renders the conformation of the active site competent for the reductase activity. This is in apparent contradiction with the fact that the nmMsrB, lacking the two CXXC signatures and therefore has no bound metal, is as active as the eMsrB and thus has a tertiary structure competent for revealing activity. Indeed, the kobs max value for the reductase step, the kcat value obtained with the Trx/Trx reductase recycling system, and the KM values for the MetSO substrate and Trx are similar to those of the eMsrB values. Inspection of the three-dimensional structure of nmMsrB (B. Kauffmann, unpubl.), shows a putative metal binding site at a distance of 20 Å from the active site similar to that observed in the structure of the N. gonorrhoeae MsrB (Lowther et al. 2002). A water molecule, hydrogen bonded to the invariant residues D45, S48, and S94, replaces the bound metal. Substituting cysteines for D45, S48, S94, and A97 resulted in a nmMsrB that tightly binds one metal atom per mol of enzyme. Such a result confirms the existence of a preformed metal binding site in the subclass of MsrBs in which C45, C48, C94, and C97 are not present. Moreover, the rate and the affinity constant of the quadruple mutated nmMsrB for the MetSO in the reductase step remained unchanged but the recycling activity with Trx was lost. These results suggest that metal binding induces subtle structural modifications transmitted to the active site that prevents formation of a competent binary complex between oxidized MsrB and reduced Trx but not between the reduced MsrB and MetSO substrate. These hypotheses are supported by the fact that no significant change was observed in the CD spectrum and in the max of the W65 fluorescence emission peak of the reduced quadruple mutated nmMsrB. However, what is particularly striking is the strong thermal stabilization brought by the metal binding as shown by an increase in Tm from 42° to 91°C of the endotherm in DSC. The relationship between the drastic increase of the thermal stability and the fact that a competent complex between oxidized MsrB and reduced Trx is not formed remains to be understood at the structural level.
From an evolutionary point of view, it is probable that, as suggested by Gladyshev et al. (Kumar et al. 2002), the zinc-containing MsrB is the prototypical enzyme that has lost the two CXXC signatures later in evolution. This assumption is supported by the present study which shows that MsrBs, that either possess or lack the two signatures CXXC, exhibit a similar DSC thermal stability. When the two CXXC signatures are present, the metal binding should, however, participate in the thermal stabilization of the MsrB. Thus, the loss of the two CXXC signatures should provoke a destabilization of the MsrB due to the absence of bound metal. Therefore, the destabilizing effect has to be compensated probably by other interactions acquired along the evolutionary cycle that stabilize the core structure of MsrB with no bound metal. Inspection of the X-ray structures of the MsrBs from N. meningitidis and gonorrhoeae does not show the presence of evident stabilizing interactions within the core structure that would be absent in MsrBs with bound metal except the presence of an ion pair between D57 and R70. Positions 57 and 70 are usually occupied by Asp/Glu and Arg/Lys residues, respectively, in MsrB with no bound metal (Fig. 2). Substituting D57 of the nmMsrB by Ala has been recently carried out, while Asp and Lys at positions 57 and 70, respectively, were introduced in the C45D/C48S/C94S/C97S eMsrB. In fact, the biochemical properties of the two mutant MsrBs were shown to be unchanged (A. Olry, unpubl.). Clearly, the stabilizing elements involved in MsrBs with no bound metal remain to be characterized at the structural level. In that context, knowledge of the three-dimensional structure of MsrBs with bound metal and comparison to that of MsrBs with no bound metal could be very informative.
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The E. coli strain used for all eMsrB productions was BL21(DE3) transformed with a pETYeaA plasmid containing the wild-type or mutated eMsrB coding sequence under the control of the T7 promoter. The overexpression of eMsrBs was performed by the addition of 1 mM IPTG in the culture medium at 0.6 A600. After 5 h of induction, cells were harvested by centrifugation. The E. coli strain used for all nmMsrB productions was HB101 transformed with a pSKPILBMsrB plasmid, as previously described by Olry et al. (2002).
All wild-type and mutant MsrBs were produced in a soluble form except for C45D/C48S/C94S/C97S eMsrB. For nmMsrBs, the protocol of purification was carried out as previously described (Olry et al. 2002), except that buffer used was devoid of EDTA. For wild-type eMsrB purification, cells were harvested by centrifugation, resuspended in buffer A (Tris-HCl 50 mM at pH 8) containing 20 mM dithiothreitol (DTT), and sonicated. The MsrB was then precipitated at 50% (NH4)2SO4 saturation. The contaminating proteins were removed by applying the enzymatic solutions onto exclusion size chromatography on ACA 54 resin (IBF) at pH 8 (buffer A). Purified fractions were then pooled and applied onto a Q-Sepharose column equilibrated with buffer A, followed by a linear gradient of KCl (0–0.4 M) using a fast protein liquid chromatography system (Amersham Biosciences). The eMsrB was eluted at 250 mM KCl. Finally, 100 mg/L of culture of the homogeneous eMsrB protein was obtained.
For C45D/C48S/C94S/C97S eMsrB, the protocol was different due to its production under a nonsoluble form. Pellets obtained after sonication were resuspended in buffer A containing 6 M urea. The contaminating proteins and nucleic acids were removed by applying the enzymatic solution diluted to 2 M urea final concentration onto a Q-Sepharose column equilibrated with buffer A containing 2 M urea and 1 mM DTT using a fast protein liquid chromatography system (Amersham Biosciences). Elution was performed in two steps: first by a linear inverse gradient of urea (2–0 M), and then by a linear gradient of KCl (0–0.5 M). The C45D/C48S/C94S/C97S eMsrB was eluted at 250 mM KCl. Finally, 80 mg/L of culture of the homogeneous eMsrB protein was obtained.
Purity of wild-type and mutated MsrBs was checked by electrophoresis on 15% SDS-polyacrylamide gel and by electrospray ionization (ESI) mass spectrometry analyses. Purified enzymes were stored at –20°C in the presence of 50 mM DTT and 50% (NH4)2SO4. Under these conditions, the enzymes were stable for several weeks. Their molar concentrations were determined spectrophotometrically, using theoretical extinction coefficients at 280 nm deduced from the method of Scopes (1974), i.e., 13,730 M–1 • cm–1 for wild-type and C45D/C48S/C94S/C97S eMsrBs, and 17,330 M–1 • cm–1 for wild-type and D45C/S48C/S94C/S97C nmMsrBs.
Determination of metal content
The zinc and iron contents of wild-type and mutant MsrBs from E. coli and N. meningitidis were analyzed using inductively coupled plasma-atomic emission spectrometry (ICP-AES). In parallel, analyses of control buffer samples showed that zinc and iron concentrations were <50 and 10 µg • L–1, respectively.
The metal content of wild-type eMsrB was also determined by ESI mass spectrometry analyses. Before molecular mass analyses, all samples were desalted by applying the enzymatic solution onto an exclusion size column (HIPREP 26/10 Desalting, Amersham Biosciences) equilibrated in 50 mM ammonium bicarbonate (pH 7.6) buffer. ESI mass spectrometry measurements were performed on an ESI time-of-flight mass spectrometer (LCT, Micromass) fitted with a standard Z-spray source. Measurements were performed in nondenaturing conditions by diluting enzymes to 10 pmol/µL in ammonium acetate 50 mM (pH 6.8) buffer. For mass analysis in denaturing conditions, MsrB samples were diluted to 10 µM in a 1:1 water–acetonitrile mixture (v/v) containing 1% formic acid. Samples were continuously infused into the ion source at a flow rate of 5 µL/min. The accelerating voltage applied on the sample was set to 80 V. Mass spectra were recorded in the positive ion mode on the mass range 1000–4000 m/z, after calibration with a separate injection of horse heart myoglobin diluted to 2 pmol/µL in a 1:1 water–acetonitrile mixture (v/v) acidified with 1% formic acid.
Preparation of metal-free MsrBs
To prepare metal-free wild-type eMsrB and D45C/S48C/S94C/A97C nmMsrB, enzymes were precipitated at room temperature with trichloroacetic acid (TCA) (10% [w/v]). The precipitate was centrifuged and the pellet was dissolved in Tris-HCl buffer 0.5 M (pH 8) containing 0.1 M DTT. The solution was then incubated for 3 h and the MsrB was then again precipitated with TCA (10% [w/v]). The pellet was dissolved in Tris-HCl buffer 0.5 M (pH 8) containing 8 M urea, and diluted with Tris-HCl buffer 0.5 M (pH 8) to a final urea concentration of 1 M. Urea and DTT were finally eliminated with a small Econopac EP 10DG column (Biorad) equilibrated with Tris-HCl buffer 50 mM (pH 8). The absence of bound metal was checked by plasma atomic emission spectrometry.
Determination of MsrBs activity
Kinetic studies were carried out with D,L-Met-R,S-SO as a substrate and E. coli Trx1 as a reductant in the presence of a Trx-regenerating system (1.28 µM E. coli Trx reductase and 0.3 mM NADPH) as previously described by Olry et al. (2002). The initial rate data were fit to the Michaelis-Menten relationship using least-squares analysis to determine kcat and KM with the program Sigmaplot (Jandel Scientific Software). All KM values were determined at saturating concentrations of the other substrate.
With the DTT regenerating system, the reaction mixture contained 10 mM DTT and 20–100 µM wild-type or mutated MsrBs in buffer A. Inital rate measurements were carried out at 25°C by following the appearance of free Met measured by high pressure liquid chromatography as previously described by Boschi-Muller et al. (2000).
Kinetics of the reductase activity by single turnover experiments
Kinetics of the emission fluorescence intensity decrease associated with formation of the sulfenic acid intermediate in the D45C/S48C/S94C/A97C nmMsrB were measured with Ac-L-Met-R,S-SO-NHMe as a substrate at 25°C on a SX18MV-R stopped-flow apparatus (Applied PhotoPhysics) adapted for fluorescence measurements as previously described (Olry et al. 2004). Data were fit to Equation 1 using least-squares analysis to determine kobs max and KS for Ac-L-Met-R,S-SO-NHMe. S represents the Ac-L-Met-R,S-SO-NHMe concentration, and K represents the KS value for the substrate.
 | (1) |
For eMsrB, the formation of Ac-L-Met-NHMe was followed by single turnover quenched-flow experiments at 25°C on a SX18MV-R stopped-flow apparatus (Applied PhotoPhysics) fitted for the double-mixing mode and adapted to recover the quenched samples as previously described (Olry et al. 2004). Briefly, the mixture containing 300 µM MsrB and 350 mM Ac-L-Met-R,S-SO-NHMe in buffer A was allowed to react for 30 msec before being mixed with an equal volume of a quenching aqueous solution containing 2% of trifluoroacetic acid. Ac-L-Met-NHMe quantification in the quenched samples was carried out by reverse-phase chromatography as previously described (Olry et al. 2004).
Fluorescence properties of wild-type and mutated MsrBs
The fluorescence emission spectra of the wild-type and mutated MsrBs (10 µM) in buffer A were recorded on a spectrofluorometer (flx SAFAS) thermostated at 25°C at 291-nm and 310-nm excitation wavelength for eMsrBs and nmMsrBs, respectively. For ANS binding experiments, fluorescence emission was measured by excitation at 380-nm and emission was recorded from 400 to 600 nm in buffer A in the presence or the absence of 0.1% SDS. ANS concentration was 1 mM and protein concentration was 10 µM.
Differential scanning calorimetry
Heat denaturation measurements were carried out on a Micro-Cal VP-DSC instrument in 0.51-mL cells at a scan rate of 60°K • h–1. Samples were extensively dialyzed against potassium phosphate buffer 10 mM (pH 7.1) at 4°C, degassed for 7 min twice before the experiment. Protein concentration was 1 mg • mL–1. The final dialysis buffer was used for the reference DSC cell and to record the buffer baseline. Thermograms were analyzed using Microcal ORIGIN software.
Circular dichroism
Circular dichroism (CD) spectra of the enzymes (5 µM in phosphate buffer 10 mM at pH 7.1) were obtained using a Jobin-Yvon CD6 spectrometer. The spectra were scanned at 25°C with 1-nm steps from 260 to 190 nm and averaged over six scans.
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References |
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Boschi-Muller, S., Azza, S., Sanglier-Cianferani, S., Talfournier, F., Van Dorsselear, A., and Branlant, G. 2000. A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli. J. Biol. Chem. 275: 35908–35913.
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Kumar, R.A., Koc, A., Cerny, R.L., and Gladyshev, V.N. 2002. Reaction mechanism, evolutionary analysis, and role of zinc in Drosophila methionine-R-sulfoxide reductase. J. Biol. Chem. 277: 37527–37535.
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90% are reported). Black points indicate the positions of the two catalytic residues of E. coli and N. meningitidis MsrBs, i.e., C63 (at least 50% invariant in all putative Msr sequences [72]) and C117 (at least 95% invariant), and black stars indicate the positions of the cysteines residues involved in binding of zinc/iron (C45, C48, C94, and C97). The numbering of amino acid residues indicated is based on the numbering of the E. coli MsrB sequence. 
