Characterization of the aminocarboxycyclopropane-forming enzyme CmaC.
The biosynthesis of the coronamic acid fragment of the pseudomonal phytotoxin coronatine involves construction of the cyclopropane ring from a gamma-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 gamma-Cl group by the alpha carbon. CmaC can be isolated as a Zn2+ protein with about 10-fold higher activity over the apo form. CmaC will not cyclize free gamma-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 gamma-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 gamma-Cl-aminoacyl-S-CmaD, whereas the product ACC-S-CmaD lacked the deuterium, consistent with a competition for a gamma-Cl-aminobutyryl alpha-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 gamma,gamma-dichloroaminobutyryl to the Cl-ACC product but did not cyclize delta- or epsilon-chloroaminoacyl-S-CmaD substrates.
Kelly
Wendy L
WL
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Boyne
Michael T
MT
Yeh
Ellen
E
Vosburg
David A
DA
Galonić
Danica P
DP
Kelleher
Neil L
NL
Walsh
Christopher T
CT
D000596:Amino Acids / Q000096:biosynthesis* / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D044224:Pseudomonas syringae / Q000201:enzymology; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; C043950:1-amino-2-ethylcyclopropane-1-carboxylic acid; D013869:Thiolester Hydrolases