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PDBsum entry 2afy
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Hydrolase activator, protein binding
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PDB id
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2afy
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References listed in PDB file
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Key reference
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Title
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A general binding mechanism for all human sulfatases by the formylglycine-Generating enzyme.
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Authors
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D.Roeser,
A.Preusser-Kunze,
B.Schmidt,
K.Gasow,
J.G.Wittmann,
T.Dierks,
K.Von figura,
M.G.Rudolph.
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Ref.
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Proc Natl Acad Sci U S A, 2006,
103,
81-86.
[DOI no: ]
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PubMed id
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Abstract
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The formylglycine (FGly)-generating enzyme (FGE) uses molecular oxygen to
oxidize a conserved cysteine residue in all eukaryotic sulfatases to the
catalytically active FGly. Sulfatases degrade and remodel sulfate esters, and
inactivity of FGE results in multiple sulfatase deficiency, a fatal disease. The
previously determined FGE crystal structure revealed two crucial cysteine
residues in the active site, one of which was thought to be implicated in
substrate binding. The other cysteine residue partakes in a novel oxygenase
mechanism that does not rely on any cofactors. Here, we present crystal
structures of the individual FGE cysteine mutants and employ chemical probing of
wild-type FGE, which defined the cysteines to differ strongly in their
reactivity. This striking difference in reactivity is explained by the distinct
roles of these cysteine residues in the catalytic mechanism. Hitherto, an
enzyme-substrate complex as an essential cornerstone for the structural
evaluation of the FGly formation mechanism has remained elusive. We also present
two FGE-substrate complexes with pentamer and heptamer peptides that mimic
sulfatases. The peptides isolate a small cavity that is a likely binding site
for molecular oxygen and could host reactive oxygen intermediates during
cysteine oxidation. Importantly, these FGE-peptide complexes directly unveil the
molecular bases of FGE substrate binding and specificity. Because of the
conserved nature of FGE sequences in other organisms, this binding mechanism is
of general validity. Furthermore, several disease-causing mutations in both FGE
and sulfatases are explained by this binding mechanism.
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Figure 2.
Substrate binding to FGE. (a) The surface representation of
FGE shows a groove with the redox-active cysteine pair
Cys-336/Cys-341 (red surface) at one end. Pro-182 (green
surface) marks the site of a cross-link with a photoreactive
substrate peptide (7) and hence is also close to the substrate
binding site (8). (b) FGE–peptide complex. The peptide LCTPSRA
binds to Cys-341 via an intermolecular disulfide bond. The FGE
surface is colored according to electrostatic potential
(±10 kT), showing a negative patch close to the C
terminus of the peptide, which is neutralized by Arg-P73. (c)
Close-up of b rotated 45° clockwise showing the exquisite
surface complementarity of the peptide with FGE.
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Figure 3.
Substrate binding and mechanistic details. (a) Hydrogen
bonds are shown as dashed lines, and water molecules are drawn
as red spheres. (b) General binding mechanism of FGE to all
human sulfatases. The schematic drawing generalizes the binding
of unfolded sulfatases to FGE as the first step in FGly
formation. (c) Magnification of the region adjacent to the
intermolecular disulfide bond. The orientation of the Tyr-340
side chain in the apo– and peptide–FGE structures differs by
6.4 Å (compare with Fig. 4). Only residues Cys-P69 and
Thr-P70 of the peptide are drawn. The solvent-inaccessible
volume between the disulfide bond and serine residues 333 and
336 (transparent gray sphere) is occupied by Cl^– (green) in
the complex structure. (d) Possible mechanisms after the
activation of molecular oxygen by FGE. Atoms from O[2] are
indicated in red. A novel hydroperoxide intermediate is
formulated from which two alternative avenues for FGly formation
are conceivable. Currently, no distinction between these two
pathways is possible.
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