Purification and analysis of streptococcal NADH peroxidase expressed in Escherichia coli.
Using the T7 RNA polymerase expression system, a modified plasmid vector has been developed which gives reliable, high level expression in Escherichia coli of the gene encoding streptococcal NADH peroxidase. The recombinant enzyme has been purified to homogeneity using a revised protocol which yields over 35 mg of pure protein per liter of culture. Recombinant NADH peroxidase is fully active and exhibits spectroscopic and redox properties identical to those for the enzyme purified from Streptococcus faecalis 10C1. Reductive titrations and thiol analyses confirm the presence of the unusual cysteine-sulfenic acid (Cys-SOH) redox center identified previously. N-terminal sequence analysis, analytical gel filtration, and preliminary x-ray diffraction data all confirm the structural identity of the recombinant and S. faecalis enzymes. Steady-state kinetic analysis of the peroxidase, coupled with results from static titration experiments is consistent with a limiting type of ternary complex mechanism and allows the determination of many of the corresponding kinetic constants. In addition, preliminary 1H NMR spectra of the enzyme at millimolar concentrations show good dispersion in the amide region and indicate that the recombinant peroxidase is suitable for one-dimensional NMR work with labeled amino acids.
Parsonage
D
D
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-1016.
Miller
H
H
Ross
R P
RP
Claiborne
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010544:Peroxidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010544:Peroxidases; C021845:NAD+ peroxidase