Salicylate biosynthesis: overexpression, purification, and characterization of Irp9, a bifunctional salicylate synthase from Yersinia enterocolitica.
In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K(m) for chorismate of 4.2 microM and a k(cat) of 8 min(-1). The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1H NMR spectroscopy.
Kerbarh
Olivier
O
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Ciulli
Alessio
A
Howard
Nigel I
NI
Abell
Chris
C
D002827:Chorismic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008190:Lyases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000378:metabolism; D012459:Salicylates / Q000378:metabolism*; D017262:Siderophores / Q000378:metabolism; D015008:Yersinia enterocolitica / Q000201:enzymology*
D011994:Recombinant Proteins; D012459:Salicylates; D017262:Siderophores; D008190:Lyases; C022620:salicylate synthetase; D002827:Chorismic Acid