Biosynthesis of Pipecolic Acid by RapL, a Lysine Cyclodeaminase Encoded in the Rapamycin Gene Cluster

Abstract

Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner.

Improvement of FK506 Production in Streptomyces tsukubaensis by Genetic Enhancement of the Supply of Unusual Polyketide Extender Units via Utilization of Two Distinct Site-Specific Recombination Systems

“…In regard to FK506 production, the fermentation culture of S. tsukubaensis in this study contained soybean oil and L-lysine, which provide the precursors for FK506 biosynthesis through cellular metabolism, including M-CoA, MM-CoA, and L-pipecolic acid (7,16,32,36). Glucose, one of the most commonly used carbon sources for fermentative production, also serves as a direct source to biosynthesize the precursors of various secondary metabolites.…”

Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis

“…On this basis we designed a set of probes using degenerate primers based on A-domains of streptomycete origin using the CODEHOP software (20); priming was targeted against the core A3 and A6 motifs. Furthermore, from the structure of 1 we predicted that the starter unit likely involves pipecolic acid, which is known to be formed from lysine via the action of a lysine cyclodeaminase (21). As cyclodeaminase genes are relatively rare in bacterial genomes, this gene was used to design a second probe to identify the get cluster (22).…”

Biosynthesis of rapamycin and its regulation: past achievements and recent progress

“…20 In addition, heterologous expression and purification of recombinant RapL from Escherichia coli also characterized the in vitro enzymatic activity of RapL. 51 Modifications of the 31-memebered lactone ring by oxidation and O-methylation are thought to be the final steps in the biosynthesis of rapamycin. Recently, the Sheridan group identified the functions of several gene products involved in post-PKS tailoring by biotransformation and gene complementation.…”