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

• Published in: Journal of the American Chemical Society 2006-03, Vol.128 (11), p.3838-3847
• Main Authors: Gatto, Gregory J, Boyne, Michael T, Kelleher, Neil L, Walsh, Christopher T
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Ammonia-Lyases - antagonists & inhibitors; Ammonia-Lyases - genetics; Ammonia-Lyases - isolation & purification; Ammonia-Lyases - metabolism; Enzyme Inhibitors - pharmacology; Escherichia coli - genetics; Escherichia coli - metabolism; Kinetics; Molecular Sequence Data; Nipecotic Acids - pharmacology; Pipecolic Acids - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sirolimus - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0587603

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 α-amine, internal cyclization, and subsequent re-reduction of the cyclic Δ1-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 K m of 2.3 μM 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 α-amine and retention of the hydrogen atom at the α-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.