Elucidating the substrate specificity and condensation domain activity of FkbP, the FK520 pipecolate-incorporating enzyme

Gregory J. Gatto, Shaun M. McLoughlin, Neil L. Kelleher, Christopher T. Walsh*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

Abstract

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-5-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.

Original languageEnglish (US)
Pages (from-to)5993-6002
Number of pages10
JournalBiochemistry
Volume44
Issue number16
DOIs
StatePublished - Apr 26 2005

ASJC Scopus subject areas

  • Biochemistry

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