Expression, purification and characterization of the acyl carrier protein phosphodiesterase from Pseudomonas Aeruginosa.
Acyl carrier protein phosphodiesterases (AcpH) are the only enzymes known to remove the 4'-phosphopantetheinyl moiety from holo acyl carrier proteins (ACP), which are a large family of proteins essential for the biosynthesis of lipid and other cellular metabolites. Here we report that the AcpH (paAcpH) from Pseudomonas aeruginosa can be overexpressed in Escherichia coli as a soluble and stable protein after optimization of the expression and purification conditions. This marks an improvement from the aggregation-prone E. coli AcpH that could only be obtained by refolding the polypeptide obtained from the inclusion body. With the soluble recombinant protein, we found that PaAcpH exhibits preferred substrate specificity towards the ACPs from the fatty acid synthesis pathway among eight carrier proteins. We further showed that PaAcpH hydrolyzes and releases the 4'-phosphopantetheinyl group-linked products from a multidomain polyketide synthase, demonstrating that the enzyme is fully capable of hydrolyzing acylated ACP substrates.
Murugan
Elavazhagan
E
Division of Chemical Biology & Biotechnology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, Singapore.
Kong
Rong
R
Sun
Huihua
H
Rao
Feng
F
Liang
Zhao-Xun
ZX
D000213:Acyl Carrier Protein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D000213:Acyl Carrier Protein; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; C018754:(acyl-carrier-protein) phosphodiesterase