Elucidating the substrate specificity and condensation domain activity of FkbP, the FK520 pipecolate-incorporating enzyme.
Rapamycin, FK506, and FK520 are potent immunosuppressant natural product macrocycles generated by hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) systems in streptomycetes. An important functional element within these molecules is an l-pipecolate moiety that is incorporated into the completed polyketide chain by the action of RapP/FkbP, a four-domain NRPS that also putatively serves to cyclize the chain after amino acid insertion. Here we report the expression and purification of recombinant FkbP from the FK520 biosynthetic pathway. Using a combination of radioassays and Fourier transform mass spectrometry, we demonstrate that once FkbP has been phosphopantetheinylated in vitro, its peptidyl carrier protein domain can be successfully loaded with l-pipecolic acid and, to a lesser extent, l-proline. The first condensation domain of FkbP is shown to be active through the successful acetylation of aminoacyl-S-FkbP using the appropriately loaded terminal acyl carrier protein from the PKS array, FkbA, as the chain donor. Site-directed mutagenesis confirmed that the N-terminal condensation domain catalyzes the transfer reaction. Acetylation of prolyl-S-FkbP was more rapid and occurred to a greater extent than that of pipecolyl-S-FkbP, a trend which was also observed with alternative acyl chain donors. These observations suggest that the adenylation domain of FkbP serves as the primary selectivity filter for pipecolate incorporation.
Gatto
Gregory J
GJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
McLoughlin
Shaun M
SM
Kelleher
Neil L
NL
Walsh
Christopher T
CT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005583:Fourier Analysis; D005798:Genes, Bacterial; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D015394:Molecular Structure; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010875:Pipecolic Acids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007166:Immunosuppressive Agents; D010875:Pipecolic Acids; D011994:Recombinant Proteins; C058028:immunomycin; D010453:Peptide Synthases; C107614:pipecolate-incorporating enzyme, Streptomyces; C031345:pipecolic acid; D020123:Sirolimus; D016559:Tacrolimus