PDBsum entry 1bwk

Oxidoreductase

PDB id: 1bwk

Contents

Protein chain

399 a.a. *

Ligands

FMN

Waters ×127

* Residue conservation analysis

PDB id:

1bwk

Name:

Oxidoreductase

Title:

Old yellow enzyme (oye1) mutant h191n

Structure:

Protein (NADPH dehydrogenase 1). Chain: a. Synonym: old yellow enzyme 1, oye1. Engineered: yes. Mutation: yes

Source:

Candida albicans. Organism_taxid: 5476. Gene: oye1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.30Å R-factor: 0.180 R-free: 0.254

Authors:

B.J.Brown,Z.Deng,P.A.Karplus,V.Massey

Key ref:

B.J.Brown et al. (1998). On the active site of Old Yellow Enzyme. Role of histidine 191 and asparagine 194. J Biol Chem, 273, 32753-32762. PubMed id: 9830019 DOI: 10.1074/jbc.273.49.32753

Date:

24-Sep-98 Release date: 30-Sep-98

Protein chain

Q02899  (OYE1_SACPS) -  NADPH dehydrogenase 1 from Saccharomyces pastorianus

Seq: 400 a.a.

Struc: 399 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.6.99.1 - Nadph dehydrogenase.
• Reaction: A + NADPH + H⁺ = AH2 + NADP⁺
• Cofactor: FMN or FAD

FMN (Bound ligand (Het Group name = FMN) corresponds exactly) or FAD

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.273.49.32753

J Biol Chem 273:32753-32762 (1998)

PubMed id: 9830019

On the active site of Old Yellow Enzyme. Role of histidine 191 and asparagine 194.

B.J.Brown, Z.Deng, P.A.Karplus, V.Massey.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (1998, 273, 32753-32762) copyright 1998.

Figures were selected by an automated process.