Catalytically inactive condensation domain C1 is responsible for the dimerization of the VibF subunit of vibriobactin synthetase.
Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide synthases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis. The oligomeric structure of these assembly line enzymes has been a topic of interest because higher order oligomeric quaternary structural arrangements allow for alternate paths of acyl intermediate elongation and present unique challenges for the chimeric engineering of hybrid assembly lines. Unlike other NRPS systems that in general appear to be monomeric, the six domain (Cy1-Cy2-A-C1-PCP-C2) VibF subunit of vibriobactin synthetase has previously been shown to be dimeric, the same oligomeric state as that observed for FAS and PKS assembly lines. It has been demonstrated that the C1 domain within VibF is catalytically inactive and is not required for vibriobactin production. Utilizing sedimentation equilibrium analytical ultracentrifugation experiments to determine the oligomeric states of several VibF subfragments, we report that the C1 domain is largely responsible for VibF dimerization. Comparative rates of vibriobactin production, coupled with dissociation constants for VibF subfragment pair heterocomplexes, suggest that the mere presence of C1 does not detectably enhance the catalytic rates of neighboring domains, but it may properly orient Cy1-Cy2-A relative to PCP-C2.
Hillson
Nathan J
NJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Balibar
Carl J
CJ
Walsh
Christopher T
CT
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D020134:Catalytic Domain; D002396:Catechols / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D010080:Oxazoles; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000302:isolation & purification; D010453:Peptide Synthases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017262:Siderophores / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation; D014734:Vibrio cholerae / Q000201:enzymology*
D001426:Bacterial Proteins; D002396:Catechols; D046911:Macromolecular Substances; D010080:Oxazoles; D010446:Peptide Fragments; D021122:Protein Subunits; D017262:Siderophores; C040227:vibriobactin; D010453:Peptide Synthases; C405545:VibF protein, bacteria