Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster

Journal of the American Chemical Society
Gregory J GattoChristopher T Walsh

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 o...Continue Reading

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Citations

Jun 20, 2013·Journal of Industrial Microbiology & Biotechnology·Di HuangXiaoqiang Jia
Jun 21, 2011·Chemical Reviews·Nicholas J Turner
Apr 3, 2012·Journal of the American Chemical Society·Tejas P Pathak, Scott J Miller
Feb 4, 2010·Journal of the American Chemical Society·Christopher T Walsh, Michael A Fischbach
Sep 11, 2013·Journal of Natural Products·Christopher C ThornburgKerry L McPhail
Jul 1, 2010·The Journal of Antibiotics·Sung Ryeol ParkYeo Joon Yoon
Nov 14, 2013·Journal of Amino Acids·Amina Sadiq, Norbert Sewald
Jan 15, 2013·Neurochemical Research·André HallenArthur J L Cooper
May 14, 2014·Marine Drugs·Javier FernándezFelipe Lombó
Oct 12, 2013·Chirality·Valerie VranovaPavel Formanek
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Mar 31, 2012·Experimental Parasitology·S UmairH V Simpson
Jun 28, 2016·Applied Microbiology and Biotechnology·Fernando Pérez-GarcíaVolker F Wendisch
Aug 16, 2016·Neurochemical Research·André Hallen, Arthur J L Cooper
Sep 11, 2016·Journal of Industrial Microbiology & Biotechnology·Young Ji YooYeo Joon Yoon
Oct 21, 2016·Journal of Industrial Microbiology & Biotechnology·Xiao-Sheng ZhangYong-Quan Li
Oct 25, 2016·Biotechnology Letters·Li-Feng FuHui Jiang
Dec 13, 2016·Chemical Reviews·Man-Cheng TangYi Tang
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Jun 26, 2020·Natural Product Reports·Silja Mordhorst, Jennifer N Andexer
Sep 25, 2019·Nature Chemical Biology·Monica E NeugebauerMichelle C Y Chang

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