The dimerization of Pseudomonas putida cytochrome P450cam: practical consequences and engineering of a monomeric enzyme.
Protein Eng
; 10(12): 1357-61, 1997 Dec.
Article
em En
| MEDLINE
| ID: mdl-9542996
ABSTRACT
Cytochrome P450cam dimerizes via the formation of an intermolecular disulfide bond, complicating the storage and handling of the enzyme, particularly at higher concentrations. The dimeric enzyme is 14% less active than the monomer and forms at a slow but significant rate even at 4 degrees C [k = 1.09 x 10(-3) mM(-1) h(-1)]. To eliminate any ambiguity introduced by dimer formation and to simplify handling and storage of the enzyme, site-directed mutagenesis was used to identify C334 as the single cysteine residue responsible for the formation of the disulfide linkage and to engineer a monomeric enzyme by substituting an alanine in its place. The C334A mutant is identical with the wild-type P450cam monomer in terms of optical spectra, camphor binding and turnover activity, but shows no evidence of dimerization and aggregation even at millimolar concentrations. Preliminary 1H NMR investigations also indicate a significant improvement in the quality of spectra obtained with this mutant. (C334A)P450cam is therefore proposed as an alternative to the wild-type enzyme-a base mutant otherwise identical with the wild-type but with improved handling characteristics.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Engenharia de Proteínas
/
Pseudomonas putida
/
Cânfora 5-Mono-Oxigenase
/
Dimerização
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Protein Eng
Assunto da revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Reino Unido