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PDBsum entry 1bwk
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Oxidoreductase
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PDB id
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1bwk
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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On the active site of old yellow enzyme. Role of histidine 191 and asparagine 194.
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Authors
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B.J.Brown,
Z.Deng,
P.A.Karplus,
V.Massey.
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Ref.
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J Biol Chem, 1998,
273,
32753-32762.
[DOI no: ]
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PubMed id
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Abstract
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Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500-800
nm) charge-transfer complexes. The enzyme is reduced by NADPH, and oxygen,
quinones, and alpha,beta-unsaturated aldehydes and ketones can act as electron
acceptors to complete catalytic turnover. Solution of the crystal structure of
OYE1 from brewer's bottom yeast (Fox, K. M., and Karplus, P. A. (1994) Structure
2, 1089-1105) made it possible to identify histidine 191 and asparagine 194 as
amino acid residues that hydrogen-bond with the phenolic ligands, stabilizing
the anionic form involved in charge-transfer interaction with the FMN prosthetic
group. His-191 and Asn-194 are also predicted to interact with the nicotinamide
ring of NADPH in the active site. Mutations of His-191 to Asn, Asn-194 to His,
and a double mutation, H191N/N194H, were made of OYE1. It was not possible to
isolate the N191H mutant enzyme, but the other two mutant forms had the expected
effect on phenolic ligand binding, i.e. decreased binding affinity and decreased
charge-transfer absorbance. Reduction of the H191N mutant enzyme by NADPH was
similar to that of OYE1, but the reduction rate constant for NADH was greatly
decreased. The double mutant enzyme had an increased rate constant for reduction
by NADPH, but the reduction rate constant with NADH was lower by a factor of 15.
The reactivity of OYE1 and the mutant enzymes with oxygen was similar, but the
reactivity of 2-cyclohexenone was greatly decreased by the mutations. The
crystal structures of the two mutant forms showed only minor changes from that
of the wild type enzyme.
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Figure 5.
Fig. 5. Oxidation of OYE1 by 2-cyclohexenone. OYE1 was
reduced by an NADPH-generating system consisting of
glucose-6-phosphate (0.625 mM), glucose-6-phosphate
dehydrogenase (5 µl), and NADP (0.625 µM). Reduction
of the enzyme was followed spectrally and was complete at 84
min. The reduced enzyme was mixed in the stopped flow apparatus
with the concentrations of cyclohexenone shown, and the reaction
traces at 460 nm were used to calculate the values of k[obs]
shown.
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Figure 6.
Fig. 6. Active site of OYE1. Overlay of four structures
around the active sites: PHB bound wild type OYE1 as the
thickest lines (PDB entry code 1OYB), empty wild type structure
(PDB entry code 1OYA) as medium thickness lines, H191N as dashed
lines, and H191N/N194H as thin lines.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(1998,
273,
32753-32762)
copyright 1998.
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