C-methyltransferase and cyclization domain activity at the intraprotein PK/NRP switch point of yersiniabactin synthetase.

Some of the most therapeutically interesting natural products are mixed polyketide/nonribosomal peptide molecules, including bleomycin and epothilones (anticancer agents) and FK506 and rapamycin (immunosuppressive drugs).1-3 These enzymatically assembled molecules are encoded by multimodular protein arrays that, through their chain-ordering, determine the structure of the natural product elongated and released. Among the fundamental questions in such hybrid nonribosomal peptide synthetase (NRPS)/ polyketide synthase (PKS) and PKS/NRPS assembly lines are how elongating chains are switched from amide synthetase chemistry (by condensation domains, NRPS logic) to Claisen condensation chemistry (by ketosynthase domains, PKS logic) and back as the chains move through switch-point module interfaces. This study describes the first in vitro assay of one such switch point that also involves a concomitant C-methylation of the growing acyl chain. The bacteria that are the causative agents of the plague, Yersinia pestis, elaborate an iron-chelating virulence factor, yersiniabactin, when in iron-deficient microenvironments. Yersiniabactin (Ybt, 1)4 (Figure 1) is a nonribosomal peptide in which a salicyl N-cap has been added to three cysteines that have been cyclized to thiazolines, then C-methylated and reduced to adjust the oxidation level. Between the second and third five-membered rings is a four-carbon fragment derived from two C-methylations and one malonyl unit.5-7 Thus, Ybt is presumably derived from an NRP/ PK/NRP hybrid. This prediction has been validated by the sequencing of genes for two high-molecular weight proteins, HMWP1 and HMWP2, from a Y. pestis high-pathogenicity island.8 The HMWP2/HMWP1 subunits form a 15-domain assembly line, with two NRPS modules in the 230 kDa HMWP29 and then a PKS module and an NRPS module in the 350 kDa HMWP1 subunit. The first NRPS/PKS module interface is at the junction of HMWP2/ HMWP1 where the elongating chain moves across the interprotein boundary. The second switch point, now a PKS/NRPS intraprotein switch, is where the chain passes from the five-domain PKS module of HMWP1 to the four-domain NRPS module. The 3-(hydrophenylthiazolinyl-thiazolidinyl)-3-hydroxy-2,2-dimethyl acyl chain 2 tethered in thioester linkage on the acyl carrier protein (ACP) domain of the PKS module is the proposed donor, and the cysteinyl-S-peptidyl carrier protein (PCP3) domain in the downstream NRPS module is the proposed acceptor at the PKS/ NRPS switch point. This should yield the desmethyl Ybt chain that subsequently undergoes C-methylation by the methyl transferase domain (MT2) and hydrolytic release by the thioesterase (TE) to yield Ybt. To evaluate the catalytic capacity of the HMWP1 NRPS fourdomain module to effect the PK-to-NRP chain-switching step, we have purified the 144 kDa fragment, residues 1896-3163, after expression in Escherichia coli, primed it with phosphopantetheine on the apo PCP3 domain, using Sfp,10 a member of the phosphopantetheine transferase (PPTase) family, and CoASH as substrate, to create the holo form competent to be cysteinylated on the pantetheine terminal SH by the adenylation domain of partner subunit HMWP2 (fragment 1-1382), using ATP and [35S]cysteine.11 Since the complex natural acyl donor, an acyl-S-ACP domain embedded within the large PKS module 2, was not available, we have prepared the simple 2,2-dimethyl-3-hydroxypropionyl-S-pantetheinyl thioester (DHPP) 3 as a soluble surrogate substrate (Figure 2), which simplifies both the acyl chain donor and the pantetheinyl-ACP scaffold.12 When 3 was incubated with [35S]-Cys-S-[1896-3163] fragment of HMWP1, the released products could be analyzed by radioactive HPLC analysis.13 Two new peaks were detected dependent on 3 and cysteinylation of ‡ These authors contributed equally to this work. (1) Du, L.; Sanchez, C.; Shen, B. Metab. Eng. 2001, 3, 78-95. (2) Du, L.; Shen, B. Curr. Opin. Drug DiscoVery DeV. 2001, 4, 215-228. (3) Keating, T. A.; Walsh, C. T. Curr. Opin. Biochemistry 1999, 3, 598606. (4) Ino, A.; Murabayashi, A. Tetrahedron 2001, 57, 1897-1902. (5) Gehring, A. M.; DeMoll, E.; Fetherston, J. D.; Mori, I.; Mayhew, G. F.; Blattner, F. R.; Walsh, C. T.; Perry, R. D. Chem. Biol. 1998, 5, 573-586. (6) Gehring, A. M.; Mori, I.; Perry, R. D.; Walsh, C. T. Biochemistry 1998, 37, 11637-11650. (7) Perry, R. D.; Balbo, P. B.; Jones, H. A.; Fetherston, J. D.; DeMoll, E. Microbiology 1999, 145, 1181-1190. (8) Pelludat, C.; Rakin, A.; Jacobl, C. A.; Schubert, S.; Heesemann, J. J. Bacteriol. 1998, 180, 538-546. (9) Keating, T. A.; Miller, D. A.; Walsh, C. T. Biochemistry 2000, 39, 4729-4739. (10) Lambalot, R. H.; Gehring, A. M.; Flugel, R. S.; Zuber, P.; LaCelle, M.; Marahiel, M. A.; Reid, R.; Khosla, C.; Walsh, C. T. Chem. Biol. 1996, 3, 923-936. (11) Suo, Z.; Walsh, C. T.; Miller, D. A. Chem. Biol. 1999, 38, 1402314035. Figure 1. Structure of Ybt and HMWP1 acyl intermediates [drawn covalently tethered to the appropriate carrier protein domains through a 4′-phosphopantetheine (wavy line)]. KS ) ketosynthase; AT ) acyltransferase; MT ) methyltransferase; KR ) ketoreductase; ACP ) acyl carrier protein; Cy ) cyclization; PCP ) peptidyl carrier protein; TE ) thioesterase.