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Characterization of the Aminocarboxycyclopropane-Forming Enzyme CmaC

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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and Department of Chemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801
Cite this: Biochemistry 2007, 46, 2, 359–368
Publication Date (Web):December 16, 2006
Copyright © 2007 American Chemical Society

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    The biosynthesis of the coronamic acid fragment of the pseudomonal phytotoxin coronatine involves construction of the cyclopropane ring from a γ-chloro-l-allo-Ile intermediate while covalently tethered as a phosphopantetheinyl thioester to the carrier protein CmaD. The cyclopropane-forming catalyst is CmaC, catalyzing an intramolecular displacement of the γ-Cl group by the α carbon. CmaC can be isolated as a Zn2+ protein with about 10-fold higher activity over the apo form. CmaC will not cyclize free γ-chloro amino acids or their S-N-acetylcysteamine (NAC) thioester derivatives but will recognize some other carrier protein scaffolds. Turnover numbers of 5 min-1 are observed for Zn−CmaC, acting on γ-chloro-l-aminobutyryl-S-CmaD, generating 1-aminocyclopropane-1-carbonyl (ACC)-S-CmaD. Products were detected either while still tethered to the phosphopantetheinyl prosthetic arm by mass spectrometry or after thioesterase-mediated release and derivatization of the free amino acid. In D2O, CmaC catalyzed exchange of one deuterium into the aminobutyryl moiety of the γ-Cl-aminoacyl-S-CmaD, whereas the product ACC-S-CmaD lacked the deuterium, consistent with a competition for a γ-Cl-aminobutyryl α-carbanion between reprotonation and cyclization. CmaC-mediated cyclization yielded solely ACC, resulting from C−C bond formation and no azetidine carboxylate from an alternate N−C cyclization. CmaC could cyclize γ,γ-dichloroaminobutyryl to the Cl-ACC product but did not cyclize δ- or ε-chloroaminoacyl-S-CmaD substrates.

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     This work was supported in part by the National Institutes of Health GM 20011 (to C.T.W.), F32GM7215 (to W.L.K.), and GM 067725 (to N.L.K.), the Jane Coffin Childs Memorial Fund (to D.A.V.), and the Damon Runyon Cancer Research Foundation Postdoctoral Fellowship (DRG-1893-05 to D.G.).

     These authors contributed equally to this work.


     Harvard Medical School.

     Present address:  School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332.

     University of Illinois at Urbana−Champaign.


     Present address:  Department of Chemistry, Harvey Mudd College, Claremont, California 91711.


     To whom correspondence should be addressed. E-mail: [email protected]. Telephone:  (617) 432-1715. Fax:  (617) 432-0438.

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