Thioesterase portability and peptidyl carrier protein swapping in yersiniabactin synthetase from Yersinia pestis.
Multimodular enzymes, including polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and mixed PKS/NRPS systems, contain functional domains with similar functions. Domain swapping and module fusion are potential powerful strategies for creating hybrid enzymes to synthesize modified natural products. To explore these strategies, yersiniabactin (Ybt) synthetase containing two subunits, HMWP2 [two NRPS modules (N-terminus-ArCP-Cy1-A-PCP1 and Cy2-PCP2-C-terminus)] and HMWP1 [one PKS (N-terminus-KS-AT-MT1-KR-ACP) one NRPS module (Cy3-MT2-PCP3-TE-C-terminus)], was used as a model system to study peptidyl carrier protein (PCP) domain swapping, thioesterase (TE) portability, and module-module fusion. The PCP1 domain of the N-terminal NRPS module of HMWP2 was swapped with either PCP2 or PCP3. The fusion proteins were 3-8-fold less active than the wild-type protein. The swapping of PCP2 of HMWP2 abolished the heterocyclization activity of the Cy2 domain while retaining its condensation function. When the two PCPs of HMWP2 were swapped by PCP3TE, it created two active fusion proteins: one or two NRPS modules fused to the TE domain. The internal TE domain of the two fusion proteins catalyzed the hydrolysis of enzyme-bound intermediates HPT-S-PCP3 to form HPT-COOH and HPTT-S-PCP3 to form HPTT-COOH. The TE activity was eliminated by the S2980A point mutation at its active site. Therefore, the three PCPs of the Ybt synthetase were swappable, and its lone TE domain was portable. The reasons for the observed low activities of the fusion proteins and lessons for protein engineering in generating novel modular enzymes were discussed.
Suo
Zucai
Z
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA. suo.3@osu.edu
D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D020035:Holoenzymes / Q000378:metabolism; D033862:Iron-Binding Proteins; D008969:Molecular Sequence Data; D010170:Palmitoyl-CoA Hydrolase / Q000737:chemistry* / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D033902:Periplasmic Binding Proteins; D010636:Phenols / Q000737:chemistry / Q000378:metabolism; D048630:Polyketide Synthases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism; D015010:Yersinia pestis / Q000201:enzymology*
D000213:Acyl Carrier Protein; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D020035:Holoenzymes; D033862:Iron-Binding Proteins; D033902:Periplasmic Binding Proteins; D010636:Phenols; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; C104398:yersiniabactin; D048630:Polyketide Synthases; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D010170:Palmitoyl-CoA Hydrolase; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase