Tandem heterocyclization activity of the multidomain 230 kDa HMWP2 subunit of Yersinia pestis yersiniabactin synthetase: interaction of the 1-1382 and 1383-2035 fragments.
The six-domain, 2035-amino acid subunit high-molecular weight protein 2 (HMWP2) activates salicylate and two cysteines and loads them covalently on its three carrier protein domains during assembly of the iron-chelating virulence factor, yersiniabactin of the plague bacterium Yersinia pestis. The 1-1382 fragment of HMWP2 (ArCP-Cy1-A), overproduced in Escherichia coli, contains the first three domains: the aryl carrier protein (ArCP) domain, the cysteine specific adenylation domain (A), and the first condensation/cyclization domain (Cy1). The ArCP can be posttranslationally phosphopantetheinylated on Ser52 and then loaded with a salicyl group on the phosphopantetheine (Ppant) thiol by action of the YbtE, a salicyl-AMP ligase. The HMWP2 1-1382 fragment can activate L-cysteine as Cys-AMP. The HMWP2 1383-2035 fragment contains the remaining three domains: two peptidyl carrier proteins (PCP1 and PCP2) separated by a second condensation/cyclization domain (Cy2). Phosphopantetheinylation of the HMWP2 1383-2035 fragment at Ser1439 (PCP1) and Ser1977 (PCP2) facilitates cysteinylation of both thiols by HMWP2 1-1382. When the holo 1-1382 and bis-holo 1383-2035 protein fragments are mixed with ATP, salicylate, and cysteine, four products are slowly released [salicylcysteine (Sal-Cys), (hydroxyphenylthiazolinyl)cysteine (HPT-Cys), HPT-Cys-Cys, and the bisheterocyclic HPTT-Cys], reflecting thiolytic rerouting by cysteine in solution of elongating acyl-S-enzyme intermediates tethered at ArCP, PCP1, and PCP2 carrier protein domains, respectively. Conducting the in trans reconstitution with the S1439A mutant of HMWP2 1383-2035 releases only Sal-Cys, while the S1977A mutant leads to HPT-Cys formation but not HPT-Cys-Cys or HPTT-Cys. These results suggest localization of particular acyl-S-enzyme intermediates to each of the three carrier protein regions and also establish the sequential action of Cy1 and Cy2, with the latter producing the tandem 4,2-bisheterocyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) moiety characteristic of this class of siderophores.
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walsh
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Miller
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D000213:Acyl Carrier Protein / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D033862:Iron-Binding Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D033902:Periplasmic Binding Proteins; D010636:Phenols; D017434:Protein Structure, Tertiary / Q000235:genetics; D017262:Siderophores / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013844:Thiazoles; D015010:Yersinia pestis / Q000201:enzymology*
D000213:Acyl Carrier Protein; 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; D013438:Sulfhydryl Compounds; D013844:Thiazoles; C104398:yersiniabactin; D003545:Cysteine; D004229:Dithiothreitol