Posttranslational modification of myxobacterial carrier protein domains in Pseudomonas sp. by an intrinsic phosphopantetheinyl transferase.
We demonstrate the ability of Pseudomonas putida KT2440, Pseudomonas syringae pv. tomato DC3000 and Pseudomonas stutzeri DSM10701 to posttranslationally activate carrier protein (CP) domains of various polyketide synthases, nonribosomal peptide synthetases, and fatty acid synthase by their intrinsic phosphopantetheinyl transferase. The apo-form is modified to the holo-form of the CP by attaching a phosphopantetheine moiety from coenzymeA to a conserved serine residue. The coding regions of the respective domains were cloned in order to generate C-terminal fusions with intein-chitin. The constructs were subcloned into a broad host range vector and transferred into the three pseudomonad hosts. The resulting recombinant pseudomonad strains were cultivated and each fusion protein was purified by affinity chromatography. Each purified CP was analysed using MALDI/TOF for the expected mass increase. Of the seven CPs tested, six could be purified from P. putida, which was chosen as the general host strain. Out of the six domains, five were completely activated, whereas only 5% of the protein of the sixth domain was in holo-form. Four domains were also expressed in the other hosts.
Gross
Frank
F
Institut für Pharmazeutische Biologie, Technische Universität Carolo-Wilhelmina, Mendelssohnstrasse 1, 38106 Braunschweig, Germany.
Gottschalk
Daniela
D
Müller
Rolf
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D064429:Fatty Acid Synthases / Q000378:metabolism; D008969:Molecular Sequence Data; D009231:Myxococcales / Q000378:metabolism*; D010453:Peptide Synthases / Q000378:metabolism; D048630:Polyketide Synthases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; D048630:Polyketide Synthases; D064429:Fatty Acid Synthases; D017855:Transferases (Other Substituted Phosphate Groups); D010453:Peptide Synthases