Tunable, post-translational hydroxylation of collagen Domains in Escherichia coli.
ACS Chem Biol
; 6(4): 320-4, 2011 Apr 15.
Article
em En
| MEDLINE
| ID: mdl-21210682
ABSTRACT
Prolyl 4-hydroxylases are ascorbate-dependent oxygenases that play key roles in a variety of eukaryotic biological processes including oxygen sensing, siRNA regulation, and collagen folding. They perform their functions by catalyzing the post-translational hydroxylation of specific proline residues on target proteins to form (2S,4R)-4-hydroxyproline. Thus far, the study of these post-translational modifications has been limited by the lack of a prokaryotic recombinant expression system for producing hydroxylated proteins. By introducing a biosynthetic shunt to produce ascorbate-like molecules in Eschericia coli cells that heterologously express human prolyl 4-hydroxylase (P4H), we have created a strain of E. coli that produces collagenous proteins with high levels of (2S,4R)-4-hydroxyproline. Using this new system, we have observed hydroxylation patterns indicative of a processive catalytic mode for P4H that is active even in the absence of ascorbate. Our results provide insights into P4H enzymology and create a foundation for better understanding how post-translational hydroxylation affects proteins.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Ácido Ascórbico
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Prolina
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Engenharia Genética
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Colágeno
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Pró-Colágeno-Prolina Dioxigenase
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Escherichia coli
Limite:
Humans
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article