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. 3 H]allo-isoleucine, followed by sodium dodecyl sulfatepolyacrylamide 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.

Nonribosomal peptide synthesis in Aspergillus fumigatus and other fungi

“…Nonetheless, as a general rule, all bioinformatic predictions should be confirmed by in vitro studies . Thus, the unambiguous assessment of fungal adenylation domain specificity requires direct biochemical analysis whereby the substrate amino acid binding specificity of individual recombinant adenylation domains is assessed by amino-acid-dependent ATP/PP i exchange reaction (Couch et al, 2004;May et al, 2005). In addition, expression of functional apo-adenylation-thiolation domains, in vitro 49-phosphopantetheinylation to the holo-form followed by incubation with relevant amino acid substrates should facilitate the identification of the bound amino acid -by either whole protein or peptide mass spectrometry .…”

Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 Family Member, Is Needed To Produce l - allo -Isoleucine, a Precursor for the Phytotoxin Coronatine

“…These include the formation of COR by the ligation of two independently produced moieties (CFA and CMA), the synthesis of CMA by cryptic halogenation using a new type of nonheme-Fe 2ϩ -ketoglutarate-dependent halogenase, and the incorporation of an uncommon amino acid in CMA biosynthesis-Lallo-isoleucine, a stereoisomer of L-isoleucine at the beta carbon (16,18,34,54). We have found evidence that CmaL, a DUF1330 protein encoded within a cluster of type III effector genes in P. syringae pv.…”

“…The biosynthesis of CMA begins with the activation of L-allo-isoleucine by the nonribosomal peptide synthetase adenylation domain of CmaA (34). The complete CmaA/CmaB/CmaC/CmaD/ CmaE/CmaT biosynthetic pathway utilizes cryptic chlorination of thioester-tethered L-allo-isoleucine to produce the cyclopropyl ring in CMA.…”

“…The complete CmaA/CmaB/CmaC/CmaD/ CmaE/CmaT biosynthetic pathway utilizes cryptic chlorination of thioester-tethered L-allo-isoleucine to produce the cyclopropyl ring in CMA. While L-allo-isoleucine is strongly preferred over L-isoleucine as a substrate for the CMA pathway (34,35), the source of L-allo-isoleucine is unknown (16).…”

Draft Genome Sequence of Pseudomonas syringae Pathovar Syringae Strain FF5, Causal Agent of Stem Tip Dieback Disease on Ornamental Pear

“…syringae strain FF5 was originally isolated (15) in Oklahoma, where it was causing stem tip dieback disease on ornamental pear (Pyrus calleryana). It is a common laboratory strain used in molecular studies (1,4,7,(10)(11)(12)(14)(15)(16)(17)(18)(19).…”