The nonribosomal peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of yersiniabactin, an iron-chelating virulence factor of Yersinia pestis.
Pathogenic Yersinia species have been shown to synthesize a siderophore molecule, yersiniabactin, as a virulence factor during iron starvation. Here we provide the first biochemical evidence for the role of the Yersinia pestis high molecular weight protein 2 (HMWP2), a nonribosomal peptide synthetase homologue, and YbtE in the initiation of yersiniabactin biosynthesis. YbtE catalyzes the adenylation of salicylate and the transfer of this activated salicyl group to the N-terminal aryl carrier protein domain (ArCP; residues 1-100) of HMWP2. A fragment of HMWP2, residues 1-1491, can adenylate cysteine and with the resulting cysteinyl-AMP autoaminoacylate the peptidyl carrier protein domain (PCP1; residues 1383-1491) either in cis or in trans. Catalytic release of hydroxyphenylthiazoline carboxylic acid (HPT-COOH) and/or N-(hydroxyphenylthiazolinylcarbonyl)cysteine (HPT-cys) is observed upon incubation of YbtE, HMWP2 1-1491, L-cysteine, salicylate, and ATP. These products presumably arise from nucleophilic attack by water or cysteine of a stoichiometric hydroxyphenylthiazolinylcarbonyl-S-PCP1-HMWP2 intermediate. Detection of the heterocyclization capacity of HMWP2 1-1491 implies salicyl-transferring and thiazoline-forming activity for the HMWP2 condensation domain (residues 101-544) and is the first demonstration of such heterocyclization ability in a nonribosomal peptide synthetase enzyme.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
A M
AM
Gehring
I
I
Mori
R D
RD
Perry
C T
CT
Walsh
D000249:Adenosine Monophosphate / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D033862:Iron-Binding Proteins; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D033902:Periplasmic Binding Proteins; D010636:Phenols; D017434:Protein Structure, Tertiary; D017262:Siderophores / Q000378:metabolism*; D013844:Thiazoles / Q000378:metabolism*; D014774:Virulence; D015010:Yersinia pestis / Q000201:enzymology* / Q000472:pathogenicity*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D010446:Peptide Fragments; D033902:Periplasmic Binding Proteins; D010636:Phenols; D017262:Siderophores; D013844:Thiazoles; C104398:yersiniabactin; D000249:Adenosine Monophosphate; D007501:Iron; D010453:Peptide Synthases; D003545:Cysteine