title: Characterization of CmaA, an adenylation-thiolation didomain enzyme involved in the biosynthesis of coronatine.
abstract: Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-(32)PP(i) exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and L-[G-(3)H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.
authors.affiliation: Department of Chemistry, Rice University, Houston, Texas 77005, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Couch
authors.forename: Sarah E
authors.initials: SE
authors.lastname: O'Connor
authors.forename: Heather
authors.initials: H
authors.lastname: Seidle
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
authors.forename: Ronald
authors.initials: R
authors.lastname: Parry
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000096:biosynthesis* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007192:Indenes; D007532:Isoleucine / Q000378:metabolism; D008969:Molecular Sequence Data; D044224:Pseudomonas syringae / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; C091077:CmaA protein, Pseudomonas syringae; D007192:Indenes; D013438:Sulfhydryl Compounds; D007532:Isoleucine; D000249:Adenosine Monophosphate; C058487:coronatine; C043950:1-amino-2-ethylcyclopropane-1-carboxylic acid