Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion.
J Biol Chem
; 290(25): 15730-15745, 2015 Jun 19.
Article
en En
| MEDLINE
| ID: mdl-25931126
ABSTRACT
To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Sulfatasas
/
Proteínas Bacterianas
/
Coenzimas
/
Cobre
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Streptomyces coelicolor
Límite:
Humans
Idioma:
En
Revista:
J Biol Chem
Año:
2015
Tipo del documento:
Article