Purification of and kinetic studies on a cloned protoporphyrinogen oxidase from the aerobic bacterium Bacillus subtilis

The previously cloned and expressed protoporphyrinogen oxidase from Bacillus subtilis has been purified to homogeneity by Ni2+ affinity chromatography using a His6 tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a pI of 7.5, a pH optimum (protoporphyrinogen)...

Full description

Saved in:

Bibliographic Details
Published in:	Archives of biochemistry and biophysics Vol. 358; no. 2; pp. 251 - 256
Main Authors:	Corrigall, A V, Siziba, K B, Maneli, M H, Shephard, E G, Ziman, M, Dailey, T A, Dailey, H A, Kirsch, R E, Meissner, P N
Format:	Journal Article
Language:	English
Published:	United States 15-10-1998
Subjects:	Aerobiosis Bacillus subtilis - enzymology Cloning, Molecular Enzyme Inhibitors - pharmacology Isoelectric Focusing Kinetics Oxidoreductases - antagonists & inhibitors Oxidoreductases - genetics Oxidoreductases - isolation & purification Oxidoreductases Acting on CH-CH Group Donors Protoporphyrinogen Oxidase
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	The previously cloned and expressed protoporphyrinogen oxidase from Bacillus subtilis has been purified to homogeneity by Ni2+ affinity chromatography using a His6 tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a pI of 7.5, a pH optimum (protoporphyrinogen) of 8.7, and a noncovalently bound flavine adenine dinucleotide cofactor. The Michaelis constants (Km) for protoporphyrinogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5.29, and 4.92 microM, respectively. Polyclonal antibody to B. subtilis protoporphyrinogen oxidase demonstrated weak cross-reactivity with both human and Myxococcus xanthus protoporphyrinogen oxidase. B. subtilis protoporphyrinogen oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 microM and is weakly inhibited by methylacifluorfen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0003-9861
DOI:	10.1006/abbi.1998.0834