Acyl-CoA subunit selectivity in the pikromycin polyketide synthase PikAIV: steady-state kinetics and active-site occupancy analysis by FTICR-MS.
Chem Biol
; 18(9): 1075-81, 2011 Sep 23.
Article
em En
| MEDLINE
| ID: mdl-21944746
ABSTRACT
Polyketide natural products generated by type I modular polyketide synthases (PKSs) are vital components in our drug repertoire. To reprogram these biosynthetic assembly lines, we must first understand the steps that occur within the modular "black boxes." Herein, key steps of acyl-CoA extender unit selection are explored by in vitro biochemical analysis of the PikAIV PKS model system. Two complementary approaches are employed a fluorescent-probe assay for steady-state kinetic analysis, and Fourier Transform Ion Cyclotron Resonance-mass spectrometry (FTICR-MS) to monitor active-site occupancy. Findings from five enzyme variants and four model substrates have enabled a model to be proposed involving catalysis based upon acyl-CoA substrate loading followed by differential rates of hydrolysis. These efforts suggest a strategy for future pathway engineering efforts using unnatural extender units with slow rates of hydrolytic off-loading from the acyltransferase domain.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
/
Acil Coenzima A
/
Macrolídeos
/
Policetídeo Sintases
/
Análise de Fourier
/
Antibacterianos
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Chem Biol
Assunto da revista:
BIOLOGIA
/
BIOQUIMICA
/
QUIMICA
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Estados Unidos