Portability of oxidase domains in nonribosomal peptide synthetase modules.
Oxazole and thiazole rings are present in numerous nonribosomal peptide natural products. Oxidase domains are responsible for catalyzing the oxidation of thiazolines and oxazolines to yield fully aromatic heterocycles. Unlike most domains, the placement of oxidase domains within assembly line modules varies. Noting this tolerance, we investigated the portability of an oxidase domain to a heterologous assembly line. The epimerase domain of PchE, involved in pyochelin biosynthesis, was replaced with the oxidase domain from MtaD, involved in myxothiazol biosynthesis. The chimeric module was expressed in soluble form as a flavin mononucleotide-containing flavoprotein. The functionality of the inserted oxidase domain was assayed within PchE and in transfer of the growing siderophore acyl chain from PchE to the next downstream module. While pyochelin-like product release was not observed downstream, the robust activity of the transplanted oxidase domain and the ability of the chimeric module to produce an advanced intermediate bound to the synthetase underscore the possibility of future engineering within nonribosomal peptide synthetase pathways using oxidase domains.
Schneider
Tanya L
TL
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walsh
Christopher T
CT
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005486:Flavin Mononucleotide / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; D010453:Peptide Synthases; C401840:aryl-4,2-bisthiazoline synthetase, Pseudomonas aeruginosa