Protein assembly line components in prodigiosin biosynthesis: characterization of PigA,G,H,I,J.
The red streptomycete metabolite prodigiosin has a unique tripyrrolic structure with two of the three pyrrolyl moieties in tandem. Five enzymes, PigA,G,H,I, and J, are involved in dipyrrole (rings A and B) formation. We have heterologously expressed and purified from Escherichia coli these five enzymes. At first, pyrrole ring A is formed on the peptidyl carrier protein PigG by one of two possible ways: (i) by action of the adenylation domain PigI that transforms l-proline into l-prolyl-AMP and by the flavoprotein dehydrogenase PigA responsible for the four-electron oxidation reaction; (ii) by loading with the pyrrolyl-2-carboxyl-(S)-pantetheinyl moiety from synthetic pyrrolyl-CoA using the phosphopantetheinyl transferase Sfp. Subsequently, pyrrole ring B is constructed by PigH after the transfer of ring A to the ketosynthase of PigJ. PigH consists of three domains: two acyl carrier proteins (ACPs) and a seryltransferase (SerT). Using HPLC and nanospray-Fourier Transform Mass Spectrometry (nFTMS), we established that all three domains of PigH undergo post-translational modifications and gained insight into the machinery involved in 2,2-dipyrrole biosynthesis.
Garneau-Tsodikova
Sylvie
S
Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Dorrestein
Pieter C
PC
Kelleher
Neil L
NL
Walsh
Christopher T
CT
D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D013058:Mass Spectrometry; D010666:Phenylpropionates; D011353:Prodigiosin / Q000096:biosynthesis*; D011392:Proline / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012705:Serratia / Q000201:enzymology* / Q000378:metabolism
D002352:Carrier Proteins; D010666:Phenylpropionates; D011758:Pyrroles; D011994:Recombinant Proteins; C001418:4-hydroxyphenyllactic acid; D011392:Proline; D011353:Prodigiosin