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. 2005 Aug 9;44(31):10583-92.
doi: 10.1021/bi050448i.

VSports在线直播 - Analysis of the link between enzymatic activity and oligomeric state in AhpC, a bacterial peroxiredoxin

Derek Parsonage  1 , Derek S Youngblood, Ganapathy N Sarma, Zachary A Wood, P Andrew Karplus, Leslie B Poole
Affiliations

Analysis of the link between enzymatic activity and oligomeric state in AhpC, a bacterial peroxiredoxin

Derek Parsonage et al. Biochemistry. .

Abstract

Peroxiredoxins (Prxs) make up a ubiquitous class (proposed EC 1. 11. 1. 15) of cysteine-dependent peroxidases with roles in oxidant protection and signal transduction. An intriguing biophysical property of typical 2-Cys Prxs is the redox-dependent modulation of their oligomeric state between decamers and dimers at physiological concentrations. The functional consequences of this linkage are unknown, but on the basis of structural considerations, we hypothesized that decamer-building (dimer-dimer) interactions serve to stabilize a loop that forms the peroxidatic active site. Here, we address this important issue by studying mutations of Thr77 at the decamer-building interface of AhpC from Salmonella typhimurium. Ultracentrifugation studies revealed that two of the substitutions (T77I and T77D) successfully disrupted the decamer, while the third (T77V) actually enhanced decamer stability. Crystal structures of the decameric forms of all three mutant proteins provide a rationale for their properties. A new assay allowed the first ever measurement of the true k(cat) and K(m) values of wild-type AhpC with H(2)O(2), placing the catalytic efficiency at 4 x 10(7) M(-)(1) s(-)(1). T77V had slightly higher activity than wild-type enzyme, and both T77I and T77D exhibited ca. 100-fold lower catalytic efficiency, indicating that the decameric structure is quite important for, but not essential to, activity. The interplay between decamer formation and active site loop dynamics is emphasized by a decreased susceptibility of T77I and T77D to peroxide-mediated inactivation, and by an increase in the crystallographic B-factors in the active site loop, rather than at the site of the mutation, in the T77D variant VSports手机版. .

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Figures

Figure 1
Figure 1
Structurally detailed model of the 2-Cys peroxiredoxin catalytic cycle and redox-sensitive oligomerization. Each panel represents a different structure of the proposed mechanism. Hatched surfaces at the top right represent the interfacial region (residues 73–84) contributed by an adjacent dimer in the decameric enzyme; panels lacking it indicate dissociation of the decamer to dimers. Thr77 in this interfacial region was targeted for mutagenesis in this study to disfavor decamer formation and potentially affect the catalytic properties of the enzyme. Boxed structures represent determined crystal structures identified by name in the top right corner [TryP (1E2Y), HBP-23 (PrxI, 1QQ2), TPx-B (PrxII, 1QMV), and AhpC (1KYG)]. The dashed box represents an alternate conformation present in the TryP structure. Unboxed structures are proposed intermediates. The active site loop (residues 45–49) containing the peroxidatic cysteine thiol(ate) (SPH), cysteine sulfenic acid (SPOH), or cysteine sulfinic acid (SPO2H) is represented in either its fully folded conformation (hashed cylinder) or its locally unfolded conformation (thick line). The dynamic equilibrium between folded and unfolded states is represented by KLU. Loop residues 40–44 leading up to the active site loop are represented as well-ordered (curved line), loosely packed (dotted line), or restructured (distorted curved line with hydrogen bonds represented by small dashed lines, species 5 and 6). The C-terminus containing the resolving cysteine (SRH or SR) is depicted as a thick line. Redox steps are represented by one-way arrows, and equilibrium steps are denoted with two-way arrows, with the length relative to the proposed direction of the reaction. The catalytic cycle of AhpC is identified by a circular arrow. See the original reference for further discussion (6).
Figure 2
Figure 2
Three views emphasizing the location of proximal Thr77 residues at the AhpC decamer-building interface. The dimer–dimer interface (panels a and b) in decameric AhpC (also known as the “alternate” or “A” interface) includes Thr residues in the opposing subunits (Thr77 and Thr77′) which are near one another, suggesting a potentially useful target for mutagenesis to destabilize decamers. In panel c, the close-up of this interface region shows that Oγ of Thr77 is proximal to Cγ of Thr77′ (3.8 Å) and to Oδ of Asp73′ (3.7 Å), across the dimer–dimer interface. Chain C (gray carbons) and chain B (green carbons) are colored according to atom type (red oxygens and blue nitrogens). Dashed lines represent hydrogen bonds (2.4–3.2 Å), and the striped lines represent the van der Waals interaction between Cγ2 of Thr77 and C∊1 of Phe42 (3.8 Å). An approximate 2-fold axis relates the two molecules at this interface and also relates the preferred hydration sites (water 2131/3131, and water 2097/3097, 2xxx and 3xxx designation for subunits b and c, respectively).
Figure 3
Figure 3
Sedimentation velocity studies of oxidized wild-type and Thr77 mutant AhpC proteins. Analytical ultracentrifugation studies of AhpC proteins were carried out with centrifugation at 42 000 rpm, 20 °C, and neutral pH. Eight to 16 consecutive 280 nm data sets for each run, where the boundaries had moved to approximately the middle of the cells, were included in the analyses by DCDT+ software to give the g*(s) distributions that are shown. Only data for the oxidized proteins (40 of at least 240 data points used in the fit) are shown. Curves fit to a single-species model (except for wild-type AhpC, which was fit to a two-species model) represent data for wild-type AhpC at 10 μM (black), T77V at 10 μM (blue), T77D at 100 μM (red), and T77I at 100 μM (green). All plots were normalized by area to ease comparisons. Results show that the T77V mutation actually promotes decamerization of the oxidized enzyme, whereas the T77I and T77D mutations destabilize the decameric forms of both the oxidized and reduced proteins (latter not shown).
Figure 4
Figure 4
Structures at the mutation site are well-defined. Each mutant model (chain C) is displayed along with its 2Fo – Fc electron density. Atom coloring is as in Figure 2c. As a reference, the wild-type structure is shown as a semitransparent model. Because of their relevance, AspB73 and water 131 are also shown. Hydrogen bonds are represented as dashed lines: (a) ValC77 contoured at 1.2ρrms, (b) IleC77 contoured at 2.5ρrms, and (c) AspC77 contoured at 2.1ρrms.
Figure 5
Figure 5
Structural differences between T77D and wild-type AhpC. T77D and wild-type AhpC are superimposed in this stereoview. All colors are as in Figure 2c with the wild-type structure shown as a semitransparent image. The view is as in Figure 2c.
Figure 6
Figure 6
Active site loop becomes much less ordered in T77D. For each residue, the average T77D main chain B-factor values are averaged over all five chains (dashed line). The wild-type main chain B-factor values are also averaged over all five chains (dotted line). The difference between these two (solid line) displays a sharp peak from residue 43 to 55. Values greater than zero are for residues for which T77D is on average more mobile than the wild-type. T77I shows no such difference in B-factors.
Figure 7
Figure 7
Wild-type and T77V AhpC show a susceptibility to peroxide inactivation that T77I and T77D mutants do not. NADPH oxidation was assessed at 25 °C in the presence of 50 mM HEPES-NaOH (pH 7.0) with 1 mM EDTA and 0.1 M ammonium sulfate, and with 80 nM thioredoxin reductase, 2.5 μM thioredoxin, 6 μM AhpC [wild-type (a) or T77D mutant (b)] and H2O2 at 0 (●), 1 (◻), 5 (◯), 10 (▵), and 30 mM (◇). T77V results were very similar to those from wild-type AhpC (a), while T77I results were essentially identical to those with T77D (b).
Chart 1
Chart 1
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References

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