| Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster. | |
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MedLine Citation:
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PMID: 16536560 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Gregory J Gatto; Michael T Boyne; Neil L Kelleher; Christopher T Walsh |
Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural |
Journal Detail:
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Title: Journal of the American Chemical Society Volume: 128 ISSN: 0002-7863 ISO Abbreviation: J. Am. Chem. Soc. Publication Date: 2006 Mar |
Date Detail:
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Created Date: 2006-03-15 Completed Date: 2006-05-25 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 7503056 Medline TA: J Am Chem Soc Country: United States |
Other Details:
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Languages: eng Pagination: 3838-47 Citation Subset: IM |
Affiliation:
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Ammonia-Lyases / antagonists & inhibitors, genetics, isolation & purification, metabolism* Enzyme Inhibitors / pharmacology Escherichia coli / genetics, metabolism Kinetics Molecular Sequence Data Nipecotic Acids / pharmacology Pipecolic Acids / metabolism* Recombinant Proteins / genetics, metabolism Sirolimus / metabolism |
| Grant Support | |
ID/Acronym/Agency:
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F32GM069169/GM/NIGMS NIH HHS; GM020011/GM/NIGMS NIH HHS; GM067725/GM/NIGMS NIH HHS; T32GM07283/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Enzyme Inhibitors; 0/Nipecotic Acids; 0/Pipecolic Acids; 0/Recombinant Proteins; 53123-88-9/Sirolimus; 535-75-1/pipecolic acid; EC 4.3.1.-/Ammonia-Lyases; EC 4.3.1.-/lysine cyclodeaminase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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