Dimeric structure of the six-domain VibF subunit of vibriobactin synthetase: mutant domain activity regain and ultracentrifugation studies.
Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide sythases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis. Previous data on FAS and PKS subunits have indicated that they are homodimers and that some of their catalytic functions can work in trans. When NRPS assembly lines have been probed for comparable formation of stable oligomers, no evidence had been forthcoming that species other than monomer forms were active. In this work we focus on the six-domain (Cy1-Cy2-A-C1-PCP-C2) enzyme VibF from the vibriobactin synthetase assembly line, which contains three other proteins, VibB, VibE, and VibH, that--when purified and mixed with VibF and the substrates ATP, threonine, 2,3-dihydroxybenzoate (DHB), and norspermidine--produce the iron chelator vibriobactin. Using a deletion of the Cy1 domain and separate inactivating mutations in the Cy2, A, PCP, and C2 domains of VibF, we report regain of catalytic activity upon mutant protein mixing that argues for heterodimer formation, stable for hundreds to thousands of catalytic cycles, with acyl chain processing and transfer around blocked domains. Ultracentrifugation data likewise confirm a dimeric structure for VibF and establish that domains within NRPS dimeric modules can act on acyl chains in trans. The results described here are the first indication for an NRPS subunit that homodimerization can occur and that there is a continuum of functional oligomerization states between monomers and dimers in nonribosomal peptide synthetases.
Hillson
Nathan J
NJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walsh
Christopher T
CT
D000215:Acylation; D000255:Adenosine Triphosphate / Q000032:analysis; D001426:Bacterial Proteins; D002384:Catalysis; D002396:Catechols / Q000378:metabolism*; D002850:Chromatography, Gel; D019281:Dimerization; D011756:Diphosphates / Q000032:analysis; D004789:Enzyme Activation / Q000235:genetics; D005816:Genetic Complementation Test; D010080:Oxazoles; D010453:Peptide Synthases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D017384:Sequence Deletion; D013997:Time Factors; D017186:Titrimetry; D014461:Ultracentrifugation / Q000379:methods
D001426:Bacterial Proteins; D002396:Catechols; D011756:Diphosphates; D010080:Oxazoles; D021122:Protein Subunits; C040227:vibriobactin; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C405545:VibF protein, bacteria