Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235 .fwdarw. Asn-235 mutant

Biochemistry ◽  
1990 ◽  
Vol 29 (37) ◽  
pp. 8797-8804 ◽  
Author(s):  
James D. Satterlee ◽  
James E. Erman ◽  
J. Matthew Mauro ◽  
Joseph Kraut
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Proton Nmr ◽  
Cytochrome C Peroxidase ◽  
Recombinant Enzyme ◽  
Nmr Analysis ◽  
Wild Type

Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast ferricytochromes c

Biochemistry ◽  
1992 ◽  
Vol 31 (14) ◽  
pp. 3661-3670 ◽  
Author(s):  
Susan J. Moench ◽  
Stamatia Chroni ◽  
Bih Show Lou ◽  
James E. Erman ◽  
James D. Satterlee
Keyword(s):  
Cytochrome C ◽  
Proton Nmr ◽  
Cytochrome C Peroxidase

scholarly journals The Effect of the Asn82Asp Mutation in Yeast Cytochrome c Peroxidase Studied by Proton NMR Spectroscopy

1994 ◽  
Vol 224 (1) ◽  
pp. 81-87 ◽  
Author(s):  
James D. Satterlee ◽  
Steve L. Alam ◽  
J. Matthew Mauro ◽  
James E. Erman ◽  
Thomas L. Poulos
Keyword(s):  
Nmr Spectroscopy ◽  
Cytochrome C ◽  
Proton Nmr ◽  
Cytochrome C Peroxidase ◽  
Proton Nmr Spectroscopy

scholarly journals Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

2000 ◽  
Vol 352 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Claire LEADBEATER ◽  
Lisa MCIVER ◽  
Dominic J. CAMPOPIANO ◽  
Scott P. WEBSTER ◽  
Robert L. BAXTER ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Binding Site ◽  
Tertiary Structure ◽  
Reductase Activity ◽  
Close Approach ◽  
Pyridine Nucleotide ◽  
Phosphate Group ◽  
Site Directed Mutagenesis ◽  
Wild Type

The structure of the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6min-1; R174A, 132.1min-1; R184A, 305.5min-1 versus wild type, 338.9min-1) and increased Km for NADPH (R144A, 5.3µM; R174A, 20.2µM; R184A, 54.4µM versus wild type, 3.9µM). The kcat value for NADH-dependent cytochrome c reduction was increased for R174A (42.3min-1) and R184A (50.4min-1) compared with the wild type (33.0min-1), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2′-phosphate. Stopped-flow studies indicated that affinity (Kd) for NADPH was markedly reduced in mutants R144A (635µM) and R184A (2.3mM) compared with the wild type (< 5µM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH Kd ratio > 175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (kred = 8.82s-1 versus 22.63s-1 for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2′-phosphate group, and R144 likely to interact with the nicotinamide ribose 5′-phosphate group.

Yeast Cytochrome c Peroxidase Expression in Escherichia coli and Rapid Isolation of Various Highly Pure Holoenzymes

2000 ◽  
Vol 19 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Jennifer G. Teske ◽  
Marina I. Savenkova ◽  
J.Matthew Mauro ◽  
James E. Erman ◽  
James D. Satterlee
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Cytochrome C Peroxidase ◽  
Rapid Isolation ◽  
Expression In Escherichia Coli

Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I

Biochemistry ◽  
1987 ◽  
Vol 26 (2) ◽  
pp. 351-360 ◽  
Author(s):  
Laurence A. Fishel ◽  
Jesus E. Villafranca ◽  
J. Matthew Mauro ◽  
Joseph Kraut
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Cytochrome C Peroxidase ◽  
Compound I ◽  
Radical Site ◽  
Expression In Escherichia Coli
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