Direction of Chain Growth and Substrate Preferences of Shape

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The direction of chain growth and substrate preferences of SEDS-family peptidoglycan glycosyltransferases Michael A. Welsh, Kaitlin Schaefer, Atsushi Taguchi, Daniel Kahne, and Suzanne Walker J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06358 • Publication Date (Web): 06 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019

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Journal of the American Chemical Society

The direction of chain growth and substrate preferences of SEDSfamily peptidoglycan glycosyltransferases Michael A. Welsh,1,‡,† Kaitlin Schaefer,1,2‡ Atsushi Taguchi,1 Daniel Kahne,2 and Suzanne Walker*1 1Department 2Department

of Microbiology, Harvard Medical School, Boston, Massachusetts 02115, United States of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States

Supporting Information Placeholder ABSTRACT: The bacterial cell wall is composed of pep-

tidoglycan, and its biosynthesis is an established target for antibiotics. Peptidoglycan is assembled from a glycopeptide precursor, Lipid II, that is polymerized by peptidoglycan glycosyltransferases into glycan strands that are subsequently crosslinked to form the mature cell wall. For decades bacteria were thought to contain only one family of enzymes that polymerize Lipid II, but recently, the ubiquitous SEDS-family proteins RodA and FtsW were shown to be peptidoglycan polymerases. Because RodA and FtsW are essential in nearly all bacteria, these enzymes are promising targets for new antibiotics. However, almost nothing is known about the mechanisms of these polymerases. Here, we report that SEDS proteins synthesize peptidoglycan by adding new Lipid II monomers to the reducing end of the growing glycan chain. Using substrates that can only react at the reducing end, we also show that the glycosyl donor and acceptor in the polymerization reaction have distinct lipid requirements. These findings provide the first fundamental insights into the mechanism of SEDS-family polymerases and lay the groundwork for future biochemical and structural studies.

The bacterial cell wall is composed of peptidoglycan, which consists of polymeric glycan strands that are crosslinked by peptide bridges (Figure 1a).1, 2 The structural integrity of the cell wall is essential for bacterial viability, and drugs that inhibit peptidoglycan biosynthesis have been among the most successful antibiotics.3 Bacterial resistance to these compounds necessitates a better understanding of peptidoglycan synthesis so that new antibiotics can be designed. Peptidoglycan glycosyltransferases (PGTs) catalyze polymerization of the lipid-linked cell wall precursor Lipid II, 1, into glycan strands (Figure 1a,b).4 The peptidoglycan glycosyltransferase domains of class A penicillin-binding proteins (aPBPs) and the closely related monofunctional glycosyltransferases are well-

Figure 1: The SEDS proteins FtsW and RodA are peptidoglycan polymerases. a) Schematic of peptidoglycan structure and synthesis by SEDS-bPBP complexes. FtsW and RodA synthesize nascent glycan strands that are crosslinked into the existing peptidoglycan by cognate bPBPs. b) Structures of the Lipid II and peptidoglycan oligomer substrates used in this study.

characterized.5-14 These PGTs were long thought to be the only enzymes responsible for peptidoglycan polymerization, but the observation that some bacteria can make peptidoglycan when their aPBPs are deleted suggested that there must be another family of enzymes that polymerize Lipid II.15-17 Recently, the Shape, Elongation, Division, and Sporulation (SEDS)-family

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Figure 2: SEDS proteins add Lipid II monomers to the reducing end of peptidoglycan. a) Schematic of the PAGEautoradiography assays used to discriminate reducing end and nonreducing end addition. b) PAGE analysis of the products of S. aureus FtsW-PBP1 reactions with [14C]-Gal-capped (Gal*) donor substrates. See Methods for reaction conditions. PBP1S314A is an inactive point mutant. Gels are representative of at least three independent experiments.

proteins RodA and FtsW were shown to be peptidoglycan polymerases (Figure 1a).18-21 These integral membrane proteins function in complex with a cognate class B penicillin-binding protein (bPBP) transpeptidase, which serves both to stimulate polymerase activity and crosslink the resulting glycan strands.21, 22 The RodAbPBP complex makes peptidoglycan during cell elongation while the FtsW-bPBP complex makes peptidoglycan at the septum during cell division. RodA and FtsW share highly conserved folds and putative catalytic residues, and one or both is essential for cell growth and viability in all bacteria.18, 21 Thus, SEDS proteins are attractive antibiotic targets, but there is very little information available regarding their mechanism, structure, and substrate binding modes. Such knowledge is required before inhibitors can be developed. A fundamental property of the mechanism of all glycan polymerases is the direction of substrate polymerization.23 Glycan polymerases may add new monomer units to either the nonreducing end or the reducing end of the growing sugar polymer (Figure 2a). Establishing the direction of chain elongation for a PGT requires having substrates that can react at only one end. Lipid II and short oligomeric peptidoglycan substrates that are blocked at the nonreducing end can be obtained using bovine galactosyltransferase (GalT), which transfers galactose to the 4’-OH of GlcNAc (Figure 1b).12, 24 These “donor only” Gal-Lipid II substrates can only elongate through reducing end addition of a glycosyl acceptor substrate. We reasoned that we could distinguish a reducing end addition mechanism from a nonreducing end addition mechanism for SEDS proteins using [14C]Gal-capped (Gal*) donor substrates and a PAGE autoradiography assay (Figure 2a).9, 12 Reducing end addition is evidenced by elongation of Gal*-Lipid II or Gal*-capped peptidoglycan oligomers when non-radiolabeled (“cold”) Lipid II is provided. In contrast, the transfer of Gal*-Lipid II to cold peptidoglycan oligomers is indicative of nonreducing end addition.

We first incubated the purified Staphylococcus aureus FtsW-PBP1 complex with Gal*-Lipid II (Gal*-2, Figure 1b) and added cold Lipid II (2) as an acceptor. We expected that if Gal*-2 elongated from the reducing end, a ladder of radioactive bands would appear higher in the gel. In accordance with our expectation, Gal*-2 alone did not react, but when 2 was provided, we observed a ladder of radioactive products (Figure S1). This result indicates that FtsW can use Gal*-2 as a glycosyl donor, but it does not establish the direction of polymerization because it is possible that Gal*-2 is transferred to the nonreducing end of polymers generated first by coupling cold 2. To exclude this possibility, we incubated S. aureus FtsW-PBP1 with short peptidoglycan oligomers capped at the nonreducing end with Gal* (Figure 2a). When cold Lipid II was added to the reaction, we observed an upward shift in the distribution of polymer bands (Figure 2b, S2). In contrast, when we incubated S. aureus FtsW-PBP1 with Gal*-2 and cold peptidoglycan oligomers generated from 2 as the acceptor substrate (Figure S3), we observed no radioactive polymer bands after product separation by SDS-PAGE (Figure 2b, S2). From these results, we conclude that FtsW adds new Lipid II monomers to the reducing end of the growing peptidoglycan polymer. We conducted analogous experiments with a purified RodA-bPBP complex and found that RodA also uses reducing end addition for chain elongation (Figure S4). Thus, this mechanism is general for SEDS proteins. Our finding that SEDS proteins elongate peptidoglycan by reducing end addition allows us to design experiments to probe other aspects of the polymerization mechanism. In principle, glycosyltransferases that use lipid-linked substrates may have two hydrophobic binding sites where the donor and acceptor substrate lipids reside during coupling.24 Two lipids were found to be bound to RodA in a recently published crystal structure (Figure S5),25 and it is tempting to speculate that these lipids reveal where the hydrophobic chains of the


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Figures 3: S. aureus FtsW has distinct lipid substrate requirements at the glycosyl donor and acceptor sites. FtsWPBP1S314A complexes were incubated with Gal-2 (a,b) or Gal3 (c,d) and the indicated Lipid II acceptor. The reaction products were analyzed by LC-MS after enzymatic cleavage of the lipids (see Methods, Figure S8). The resulting mass spectra were summed over a 30 s window containing the co-eluting Gal-capped products. Roman numerals indicate the number of sugars in the polymer product. Spectra are representative of at least two independent experiments.

substrates bind. Knowing the lipid substrate requirements for binding at these sites is a prerequisite for designing future biochemical and kinetic experiments. To determine the minimum lipid length required for polymerization, we prepared radiolabeled Lipid II bearing lipids between 20 and 55 carbons in length (Figure S6) and incubated these substrates with S. aureus FtsWPBP1. We found that S. aureus FtsW polymerized substrates having at least 30 carbons in their lipid chains (Figure S6). Lipid II with a 20-carbon lipid (3, Figure 1b) did not react under any condition we tested (Figure S7). The competent C30 substrate we tested has the same double bond configuration over its first four isoprene units as the unreactive C20 variant 3 (Figure S6), so we conclude that the reactivity differences are due to lipid length. We were interested to know if this strict length

requirement was due to lipid substrate preferences at the FtsW glycosyl donor or acceptor binding sites. We reasoned that the Gal-capped, “donor only” substrates we identified would allow us to tease out these requirements. We first incubated the S. aureus FtsW-PBP1 complex with Gal-2, which can only act as a glycosyl donor, and 2 then analyzed the reaction products by LC-MS after enzymatic removal of the lipid chains. We observed two distinct populations of peptidoglycan products, each up to 10 sugars long (Figure 3a, S8). One set of polymers was capped with Gal from reducing end elongation of Gal-2. The other polymers did not contain Gal and resulted from homo-coupling of 2. This data is consistent with the C35 lipid being both a suitable glycosyl donor and acceptor substrate for FtsW. When we conducted the same experiment using 3 as the acceptor, we observed only one product, Gal-IV, resulting from exactly one coupling of Gal-2 with 3 (Figure 3b). This result shows that FtsW can use 3 as a glycosyl acceptor, but it also suggests that the C20 lipid cannot serve as a donor substrate as no further turnover products were observed. To test if the C20 lipid could serve as a donor, we prepared Gal-3 and subjected it to the same set of experiments. We observed no peptidoglycan products containing Gal when either 2 or 3 was provided as the acceptor (Figure 3c,d). In total, these results show that S. aureus FtsW has distinct lipid substrate binding requirements at the donor and acceptor sites. A longer lipid (at least 30 carbons) is necessary to anchor the growing peptidoglycan chain in the donor site. In contrast, the acceptor site can tolerate shorter lipids. In summary, we have elucidated the direction of peptidoglycan chain elongation by SEDS proteins and determined the substrate-binding preferences at the glycosyl donor and acceptor sites. Our finding that Gal-2 binds at the donor site of FtsW, but does not react with itself, should prove broadly useful in other biochemical contexts. The published structures of SEDS proteins exist only in the apo form.25 The substrates reported here should enable future crystallography work, where having defined substrates with the minimum length lipid is especially beneficial. In addition, blocking Lipid II with GalNAz, an azido sugar that can also be introduced with GalT,26 would generate a donor only substrate that could be derivatized, enabling fluorescence experiments or immobilization and pulldown of SEDS-bPBP complexes. The reducing end addition mechanism for SEDS proteins is shared with aPBPs,12 the other principle peptidoglycan synthases, but there are also examples of bacterial cell envelope polymers that grow from the nonreducing end.27-29 Why might PGTs use reducing end addition? Nascent glycan strands synthesized by PGTs are crosslinked into the existing cell wall by transpeptidases (aPBPs and bPBPs). Allowing the polymer to grow at the same end as the lipid anchor while continuously feeding the free, nonreducing end up to a waiting enzyme



may allow the most efficient coupling of polymer synthesis with crosslinking or other cell wall modifications. ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Full Methods, bacterial strains, plasmids, and supporting figures and tables.

AUTHOR INFORMATION Corresponding Author

* [email protected] Present Addresses

† Chemistry Department, Hamilton College, Clinton, NY, 13323 Author Contributions

‡ M.A.W. and K.S. contributed equally. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by NIH grants R01GM76710, P01AI083214, and F32GM123579 (to M.A.W.). We thank S. Trauger and the staff of the Harvard Small Molecule Mass Spectrometry Facility for assistance with MS. We thank A. Mollo for providing ColM. We thank Z. Levine and C. Joiner for critical reading of the manuscript.

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10. Lovering, A. L.; de Castro, L. H.; Lim, D.; Strynadka, N. C. J. Structural insight into the transglycosylation step of bacterial cellwall biosynthesis. Science 2007, 315, 1402-1405. 11. Yuan, Y.; Barrett, D.; Zhang, Y.; Kahne, D.; Sliz, P.; Walker, S. Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 5348-5353. 12. Perlstein, D. L.; Zhang, Y.; Wang, T.-S.; Kahne, D.; Walker, S. The direction of glycan chain elongation by peptidoglycan glycosyltransferases. J. Am. Chem. Soc. 2007, 129, 12674-12675. 13. Sung, M.-T.; Lai, Y.-T.; Huang, C.-Y.; Chou, L.-Y.; Shih, H.-W.; Cheng, W.-C.; Wong, C.-H.; Ma, C. Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 8824-8829. 14. Wang, T. S.; Lupoli, T. J.; Sumida, Y.; Tsukamoto, H.; Wu, Y.; Rebets, Y.; Kahne, D. E.; Walker, S. Primer preactivation of peptidoglycan polymerases. J. Am. Chem. Soc. 2011, 133, 8528-8530. 15. McPherson, D. C.; Popham, D. L. Peptidoglycan synthesis in the absence of class A penicillin-binding proteins in Bacillus subtilis. J. Bacteriol. 2003, 185, 1423-1431. 16. Arbeloa, A.; Segal, H.; Hugonnet, J. E.; Josseaume, N.; Dubost, L.; Brouard, J. P.; Gutmann, L.; Mengin-Lecreulx, D.; Arthur, M. Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis. J. Bacteriol. 2004, 186, 1221-1228. 17. Rice, L. B.; Carias, L. L.; Rudin, S.; Hutton, R.; Marshall, S.; Hassan, M.; Josseaume, N.; Dubost, L.; Marie, A.; Arthur, M. Role of class A penicillin-binding proteins in the expression of beta-lactam resistance in Enterococcus faecium. J. Bacteriol. 2009, 191, 3649-3656. 18. Meeske, A. J.; Riley, E. P.; Robins, W. P.; Uehara, T.; Mekelanos, J. J.; Kahne, D.; Walker, S.; Kruse, A. C.; Bernhardt, T. G.; Rudner, D. Z. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature 2016, 537, 634-638. 19. Cho, H.; Wivagg, C. N.; Kapoor, M.; Barry, Z.; Rohs, P. D. A.; Suh, H.; Marto, J. A.; Garner, E. C.; Bernhardt, T. G. Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously. Nat. Microbiol. 2016, 1, 16172. 20. Emami, K.; Guyet, A.; Kawai, Y.; Devi, J.; Wu, L. J.; Allenby, N.; Daniel, R. A.; Errington, J. RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway. Nat. Microbiol. 2017, 2, 16253. 21. Taguchi, A.; Welsh, M. A.; Marmont, L. S.; Lee, W.; Sjodt, M.; Kruse, A. C.; Kahne, D.; Bernhardt, T. G.; Walker, S. FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein. Nat. Microbiol. 2019, 4, 587-594. 22. Reichmann, N. T.; Tavares, A. C.; Saraiva, B. M.; Jousselin, A.; Reed, P.; Pereira, A. R.; Monteiro, J. M.; Sobral, R. G.; VanNieuwenhze, M. S.; Fernandes, F.; Pinho, M. G. SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in Staphylococcus aureus. Nat. Microbiol. 2019, 284, 851. 23. Lairson, L. L.; Henrissat, B.; Davies, G. J.; Withers, S. G. Glycosyltransferases: Structures, Functions, and Mechanisms. Annu. Rev. Biochem. 2008, 77, 521-555. 24. Perlstein, D. L.; Wang, T.-S. A.; Doud, E. H.; Kahne, D.; Walker, S. The role of the substrate lipid in processive glycan polymerization by the peptidoglycan glycosyltransferases. J. Am. Chem. Soc. 2010, 132, 48-49. 25. Sjodt, M.; Brock, K.; Dobihal, G.; Rohs, P. D. A.; Green, A. G.; Hopf, T. A.; Meeske, A. J.; Srisuknimit, V.; Kahne, D.; Walker, S.; Marks, D. S.; Bernhardt, T. G.; Rudner, D. Z.; Kruse, A. C. Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis. Nature 2018, 556, 118-121. 26. Ramakrishnan, B.; Qasba, P. K. Structure-based design of beta 1,4-galactosyltransferase I (beta 4Gal-T1) with equally efficient Nacetylgalactosaminyltransferase activity: point mutation broadens beta 4Gal-T1 donor specificity. J. Biol. Chem. 2002, 277, 20833-20839. 27. Brown, S.; Zhang, Y. H.; Walker, S. A revised pathway proposed for Staphylococcus aureus wall teichoic acid biosynthesis based on in vitro reconstitution of the intracellular steps. Chem. Biol. 2008, 15, 1221. 28. May, J. F.; Splain, R. A.; Brotschi, C.; Kiessling, L. L. A tethering mechanism for length control in a processive carbohydrate polymerization. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 11851-11856.


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29. Troutman, J. M.; Imperiali, B. Campylobacter jejuni PglH is a single active site processive polymerase that utilizes product

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Figure 1: The SEDS proteins FtsW and RodA are peptidoglycan polymerases. a) Schematic of peptidoglycan structure and synthesis by SEDS-bPBP complexes. FtsW and RodA synthesize nascent glycan strands that are crosslinked into the existing peptidoglycan by cognate bPBPs. b) Structures of the Lipid II and peptidoglycan oligomer substrates used in this study. 83x108mm (300 x 300 DPI)



Figure 2: SEDS proteins add Lipid II monomers to the reducing end of peptidoglycan. a) Schematic of the PAGE-autoradiography assays used to discriminate reducing end and nonreducing end addition. b) PAGE analysis of the products of S. aureus FtsW-PBP1 reactions with [14C]-Gal-capped (Gal*) donor substrates. See Methods for reaction conditions. PBP1S314A is an inactive point mutant. Gels are representative of at least three independent experiments. 174x60mm (300 x 300 DPI)


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Figures 3: S. aureus FtsW has distinct lipid substrate requirements at the glycosyl donor and acceptor sites. FtsW-PBP1S314A complexes were incubated with Gal-2 (a,b) or Gal-3 (c,d) and the indicated Lipid II acceptor. The reaction products were analyzed by LC-MS after enzymatic cleavage of the lipids (see Methods, Figure S7). To generate the mass spectra shown, the resulting total ion chromatograms were summed over a 30 s window containing the co-eluting Gal-capped products. Roman numerals indicate the number of sugars in the polymer product. Spectra are representative of at least two independent experiments. 83x128mm (300 x 300 DPI)



Table of Contents Graphic 40x28mm (300 x 300 DPI)


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Direction of Chain Growth and Substrate Preferences of Shape

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