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PDBsum entry 2c7x
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Oxidoreductase
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PDB id
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2c7x
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References listed in PDB file
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Key reference
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Title
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The structural basis for substrate anchoring, Active site selectivity, And product formation by p450 pikc from streptomyces venezuelae.
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Authors
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D.H.Sherman,
S.Li,
L.V.Yermalitskaya,
Y.Kim,
J.A.Smith,
M.R.Waterman,
L.M.Podust.
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Ref.
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J Biol Chem, 2006,
281,
26289-26297.
[DOI no: ]
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PubMed id
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Abstract
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The pikromycin (Pik)/methymycin biosynthetic pathway of Streptomyces venezuelae
represents a valuable system for dissecting the fundamental mechanisms of
modular polyketide biosynthesis, aminodeoxysugar assembly, glycosyltransfer, and
hydroxylation leading to the production of a series of macrolide antibiotics,
including the natural ketolides narbomycin and pikromycin. In this study, we
describe four x-ray crystal structures and allied functional studies for PikC,
the remarkable P450 monooxygenase responsible for production of a number of
related macrolide products from the Pik pathway. The results provide important
new insights into the structural basis for the C10/C12 and C12/C14 hydroxylation
patterns for the 12-(YC-17) and 14-membered ring (narbomycin) macrolides,
respectively. This includes two different ligand-free structures in an
asymmetric unit (resolution 2.1 A) and two co-crystal structures with bound
endogenous substrates YC-17 (resolution 2.35 A)or narbomycin (resolution 1.7 A).
A central feature of the enzyme-substrate interaction involves anchoring of the
desosamine residue in two alternative binding pockets based on a series of
distinct amino acid residues that form a salt bridge and a hydrogen-bonding
network with the deoxysugar C3' dimethylamino group. Functional significance of
the salt bridge was corroborated by site-directed mutagenesis that revealed a
key role for Glu-94 in YC-17 binding and Glu-85 for narbomycin binding. Taken
together, the x-ray structure analysis, site-directed mutagenesis, and
corresponding product distribution studies reveal that PikC substrate tolerance
and product diversity result from a combination of alternative anchoring modes
rather than an induced fit mechanism.
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Figure 2.
FIGURE 2. Ribbon representation of ligand-free PikC. A,
open, and B, closed conformations of ligand-free PikC (2BVJ). C,
overlay of both conformations, open (cyan) and closed (gray),
demonstrating that in the open form, the F and G helix are bent
away from the heme to enable substrate access to the active
site. Molecules in C are rotated  90° toward the viewer
along a horizontal axis in the plane of drawing when compared
with A and B. The F helix is not seen in this orientation.
Closed conformation is related within r.m.s. deviations of 0.58
Å for C  atoms to catalytically
relevant YC-17- and narbomycin-bound forms. The heme co-factor
is shown in red.
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Figure 6.
FIGURE 6. Functional activity of PikC mutants. High
pressure liquid chromatography analyses of PikC-catalyzed
reactions using YC-17 (A series) and narbomycin (B series) as
substrate are shown. A1/B1, negative control in the absence of
PikC. A2/B2, PikC wild type (PikC-wt). Mutants are used as
indicated in the figure. Compound identities are as follows: 1,
YC-17; 2, neomethymycin; 3, methymycin; 4, narbomycin; 5,
pikromycin. Conversion of narbomycin at low efficiency is
probably due to use of exogenous redox partners (e.g. spinach
ferredoxin reductase) in P450 reconstitution assays.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
26289-26297)
copyright 2006.
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