# Glycosynthase-Based Synthesis of Peptidoglycan Oligosaccharides to Address Bacterial Cell-Wall Elongation Processes

Antoine Rousseau, Dindet Steve-Evanes Koffi Teki, Célia Boyat, Emeline Richard, Stéphanie Pradeau, Louis Brigandat, Isabel Ayala, Jean-Pierre Simorre, Sylvie Armand, Sylvain Cottaz, et al.

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## ► To cite this version:

Antoine Rousseau, Dindet Steve-Evanes Koffi Teki, Célia Boyat, Emeline Richard, Stéphanie Pradeau, et al.. Glycosynthase-Based Synthesis of Peptidoglycan Oligosaccharides to Address Bacterial Cell-Wall Elongation Processes. ACS Chemical Biology, 2025, 20 (12), pp.2999-3008. 10.1021/acschembio.5c00792. hal-05527575

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Submitted on 25 Feb 2026

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Glycosynthase-based synthesis of peptidoglycan oligosaccharides to address
bacterial cell-wall elongation processes

Antoine Rousseau,<fnref n="1" /> Dindet Steve-Evanes Koffi Teki,<fnref n="1" /> Célia Boyat,<fnref n="2" /> Emeline Richard,<fnref n="1" />

Stéphanie Pradeau,<fnref n="1" /> Louis Brigandat,<fnref n="2" /> Isabel Ayala,<fnref n="2" /> Jean-Pierre Simorre,<fnref n="2" /> Sylvie Armand,<fnref n="1" />

Sylvain Cottaz,<fnref n="1" /> Catherine Bougault<fnref n="2" />* and Sébastien Fort<fnref n="1" />*

ABSTRACT

Most antibiotics used in human medicine inhibit the biosynthesis of peptidoglycan (PG), a
vital component of the bacterial cell wall. However, the rapid rise in antimicrobial resistance
(AMR) represents a major global health threat. Gaining deeper insight into PG metabolism is
a crucial step in combating AMR, an objective achievable only through the use of well-
defined molecular probes. In this study, we developed a chemo-enzymatic method to
synthesize PG oligosaccharides using a lysozyme-derived glycosynthase. To prevent
unwanted polymerization and enable precise control over product size, a glycosyl fluoride
donor bearing a terminal galactosyl unit was designed. The trisaccharide Gal-β-(1→4)-
GlcNAc-β-(1→4)-1,6-anhydro-MurNAc was biosynthesized in metabolically engineered
Escherichia coli cells and subsequently fluorinated at the reducing end via chemical
modification. Successive glycosylation and degalactosylation steps using this donor, starting
from two disaccharide acceptors, led to the synthesis of PG tetra-, hexa-, and octasaccharides,
with 60–70% yield at each cycle. These compounds were used to probe the specificity of E.
coli DedD, a SPOR-domain-containing protein involved in bacterial cell division. NMR

<footnote n="1">¹Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France</footnote>

<footnote n="2">²Univ. Grenoble Alpes, CNRS, CEA, IBS, 38044 Grenoble Cedex 9, France</footnote>
spectroscopy and NMR-restraint-driven molecular dynamics provide new insights into the
role of this protein relative to other *E. coli* SPOR domain-containing proteins.

INTRODUCTION

Peptidoglycan (PG) is an essential and unique component of the bacterial cell wall, with no counterpart in humans. For this reason, it has long been an attractive and successful target for antibacterial drug development.¹ However, antimicrobial resistance (AMR) is rising to dangerously high levels and now represents one of the greatest threats to global health. Bacterial infections are becoming more severe and, in some cases, untreatable due to the declining effectiveness of antibiotics. In 2016, the O’Neill Review on AMR² estimated that annual deaths caused by AMR may rise from approximately 700,000 to nearly 10 million by 2050, surpassing cancer as a leading cause of mortality. Developing new, effective antibacterial treatments therefore requires a deeper understanding of PG metabolism and maturation, as well as of antibiotic resistance mechanisms.³

PG is a complex macromolecule that forms a three-dimensional, net-like structure, providing structural strength to the cell, protecting the osmotically sensitive membrane, and maintaining cell morphology throughout the bacterial life cycle.⁴ It is composed of polysaccharide chains made up of alternating β-1,4-linked *N*-acetylglucosamine (GlcNAc) and *N*-acetylmuramic acid (MurNAc) units, which are cross-linked by stem peptides containing three to five amino acid residues. During the bacterial life cycle, PG is continuously remodeled and recycled, undergoing both degradation and biosynthesis simultaneously.⁵

This highly coordinated process involves several enzymes that act either on the glycan chains or on the stem peptides.⁶ Among them, lytic transglycosylases (LTs), penicillin-binding proteins (PBPs), and L,D-transpeptidases (Ldts) are extensively studied. LTs cleave the
glycosidic bond between MurNAc and GlcNAc residues, generating a 1,6-anhydro-*N*-acetyl-
muramic acid (anhMurNAc) unit, and contribute to morphogenesis⁷ and pathogenesis.⁸ PBPs,
which are closely associated with antibiotic resistance, catalyze glycan polymerization and
peptide cross-linking.⁹ Ldts also mediate peptide cross-linking¹⁰ and anchor PG to the outer
membrane in Gram-negative bacteria.¹¹

Regulatory proteins play a crucial role in the spatial and temporal coordination of this
enzymatic machinery. Among these, SPOR proteins¹² have recently attracted growing
attention. These PG-binding proteins, which are characterized by the presence of a
sporulation-related repeat (SPOR) domain from which their name is derived, participate in
cell division. SPOR proteins localize to the bacterial cell septum, where they guide septal PG
synthetases to their substrate by promoting interactions between the enzymes and regions of
peptidoglycan lacking stem peptides.¹³ *E. coli* harbors four members of this protein family:¹⁴
(i) FtsN, the only SPOR protein essential for septal PG synthesis; (ii) DamX and DedD,
which enhance the activity of PBP1A and PBP1B, with DedD also contributing to cell
constriction at division sites;¹⁵ and (iii) RlpA, which, unlike the other three, is anchored to the
outer membrane and has a homologue with lytic transglycosylase activity in *Pseudomonas
aeruginosa*.¹⁶ The structure of all four SPOR domains have been resolved (PDB ID 1UTA,
2LFV, 6ZTG, and 6I05 for FtsN, DamX, DedD, and *P. aeruginosa* RlpA, respectively). Each
binds 3–15 kDa soluble fragments of denuded PG from *Staphylococcus aureus*, albeit with
varying affinities, which likely modulate their functional roles in the cell.¹⁷ To date, only the
X-ray structures of the SPOR domain of *P. aeruginosa* RlpA have been reported, both in the
apo form and in complex with a tetrasaccharide ligand.¹⁶ The precise interaction patterns of
other SPOR domains with denuded glycans remains to be elucidated.

Although the enzymatic or binding activities of several PG-interacting proteins have been
demonstrated using intact murein, detailed mechanistic and atomic-resolution structural
studies remain scarce,¹⁸ hindered by the insolubility and heterogeneity of the PG polymer.

Another major obstacle is the lack of large synthetic PG oligomers that replicate the inherent complexity of the natural substrate. Producing soluble, well-defined PG oligomers in sufficient quantities for structural studies remains a significant challenge. To date, chemical synthesis has proven to be the most efficient method. Mobashery and coworkers developed a convergent, imidate-based strategy to synthesize PG oligomers, ranging from tetra-¹⁹,²⁰ to octasaccharides.²¹ They also reported a unique synthesis of a tetrasaccharide featuring a terminal anhMurNAc unit.²² Fukase et al. prepared PG octasaccharides using the same glycosylation approach but with a different protecting group strategy.²³,²⁴ Despite their excellent overall yields, these chemical syntheses suffer from a major drawback: the extensive number of steps required. Enzymatic pathways present a viable alternative to address this shortcoming. However, only one example of *in vitro* enzymatic synthesis of PG oligomers has been reported to date. Using the natural lipid II donor and a PG glycosyltransferase (SgtB) from S. aureus, Walker et al. prepared a mixture of compounds of varying chain lengths exclusively at an analytical scale.²⁵ Glycosynthases, as potent tools for glycosidic bond formation,²⁶,²⁷ may significantly aid in the synthesis of PG oligomers. Those derived from endo-glycosidases have been shown to be particularly effective at synthesizing long-chain oligosaccharides.²⁷,²⁸ Our group has previously reported the efficient synthesis of cellulose²⁸,²⁹ and xyloglucan³⁰,³¹ oligosaccharides using a cellulase-based glycosynthase, and more recently, chitin oligosaccharides with the D52S mutant of hen egg-white lysozyme (HEWL).³² The use of glycosyl donors that are incapable of undergoing self-glycosylation have enabled the precise synthesis of oligosaccharides with perfect control over their size.

In this work, we applied the D52S HEWL glycosynthase to the synthesis of PG
oligosaccharides (Figure 1). A non-polymerizable donor, Gal-β-(1→4)-GlcNAc-β-(1→4)-
MurNAc-α-fluoride, was prepared from a PG recycling product of E. coli through a
combination of bacterial metabolic engineering and chemical modifications. PG oligosaccharides were further synthesized by an iterative process consisting of glycosylation-deglycosylation sequences from two disaccharide acceptors. Finally, these oligosaccharides were used to study the SPOR domain of *E. coli* DedD by liquid-state NMR and NMR-restraint-driven molecular dynamics.

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**Figure 1.** General strategy for the chemo-enzymatic synthesis of PG oligosaccharides. Metabolically engineered *E. coli* cells produce a trisaccharide precursor which is chemically modified into a fluorinated donor. Afterwards, an enzymatic cascade using a glycosynthase and a *β*-galactosidase provides the PG oligosaccharides of defined length.

## RESULTS AND DISCUSSION

### Synthesis of the glycosyl fluoride donor
The trisaccharide Gal-β-(1→4)-GlcNAc-β-(1→4)-anhMurNAc **1** (Figure 2) was designed as a key precursor in the synthesis of the non-polymerizable glycosyl fluoride donor. It has indeed been reported that HEWL can accommodate a galactosyl group in the -3 (B) subsite of its active site,³³ but not in its +1 (E) subsite.³⁴ To circumvent the complexity of a multistep chemical synthesis of **1**, we devised a bioinspired enzymatic route. Bacteria recycle about 50% of their PG every generation.³⁵ During this process, the disaccharide GlcNAc-β-(1→4)-anhMurNAc is released from the cell wall and transported into the cytoplasm to be recycled.³⁶ We recently reported a *nagZ*-deficient *E. coli* mutant in which the disaccharide accumulates within its cytoplasm at a concentration of 110 mg L⁻¹ culture medium.³⁷ Here, the *E. coli* Δ*nagZ* strain was further engineered to produce **1**. The *Neisseria meningitidis* LgtB galactosyltransferase has previously been expressed in *E. coli* to synthesize human milk oligosaccharides³⁸ and chitin oligosaccharide derivatives,³⁹ utilizing the host's endogenous pool of UDP-galactose. Thus, the Δ*nagZ* strain was transformed with the *N. meningitidis* *lgtB* gene. The chromosomal *lacZ* gene, coding for the *E. coli* β-galactosidase, was inactivated to avoid the degradation of the reaction product. The *E. coli* Δ*nagZ*Δ*lacZ*-lgtB strain grew well in M10 minimal medium and the accumulation of **1** in the cytoplasm was confirmed using mass-spectrometry analysis. Then, the trisaccharide was produced in a bioreactor in minimal medium under the conditions previously optimized for the production of GlcNAc-β-(1→4)-anhMurNAc.³⁷ After purification using anion-exchange chromatography followed by flash chromatography, **1** was isolated with a yield of 125 mg L⁻¹ culture medium and characterized using NMR (Figures S1-S5). Finally, an average yield of 500 mg of trisaccharide was obtained from 4 L of culture medium in bioreactors.
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Production of Gal-β-(1→4)-GlcNAc-β-(1→4)-anhMurNAc **1** in *E. coli* Δ*nagZ*
Δ*lacZ-lgtB*</figcaption></figure>

Transformation of **1** into the glycosyl fluoride **5** required a few chemical modifications (Erreur ! Source du renvoi introuvable.A). First, the carboxylic acid group was protected as a methyl ester using an H⁺ cation-exchange resin, affording **2** at a 90% yield (Figures S6-S10). Attempts to functionalize the anomeric position of **2** via simultaneous ring opening of the 1,6-anhydro bridge were unsuccessful. Consequently, a slightly longer yet more reliable approach was adopted. Acetolysis of the 1,6-anhydro-bridge provided an α/β mixture of peracetylated trisaccharides that was directly subjected to selective anomeric deacetylation with hydrazine acetate.⁴⁰ After purification, **3** was isolated with a yield of 43% (over two
steps) as a mixture of α/β anomers (55:45) as determined by ¹H NMR (Figures S11-S15). The yield was limited due to the formation of a significant amount of a 1,2-oxazoline derivative formed by the attack of the acetamido group onto the anomeric carbon during acetolysis. Fluorination of **3** with (dimethylamino)sulfur trifluoride (Me-DAST) under standard conditions (-30°C in dichloromethane) produced a mixture of α/β glycosyl fluorides. Again, a significant amount of oxazoline was formed during the reaction. Solvent and temperature were modified to favor the formation of the α-glycosyl fluoride, the required anomer to serve as a donor for the HEWL glycosynthase. The reaction was carried out at different temperatures (from -75°C to 20°C) in different solvents (dichloromethane, toluene or THF). The highest yield (52%) in pure α-fluoride **4** (Figures S16-S20) was obtained when the reaction was conducted in dichloromethane at 20°C. The use of other fluorination reagents such as diethylaminodifluorosulfinium tetrafluoroborate (XtalFluor-E)⁴¹ and 2-pyridinesulfonyl fluoride (PyFluor)⁴² did not result in any improvement. Finally, Zemplén deacetylation afforded the expected donor **5** with a quantitative yield.
<figure><img src="image_5.png" /><figcaption>**Scheme 1.** A) Chemical synthesis of glycosyl fluoride donor 5; B) Stepwise synthesis of PG oligosaccharides from donor 5.</figcaption></figure>

i) Amberlyst 15 (H⁺) resin, MeOH, 90%; ii) Ac₂O:AcOH:H₂SO₄ (7:3:0.05 v/v/v); iii) NH₂-NH₂.AcOH, DMF, 43% over 2 steps; iv) Me-DAST, CH₂Cl₂, 52%; v) MeONa in MeOH, quantitative yield; vi) Acceptor (6a or 6b or 8a or 8b or 10a or 10b), 5 (1.5 equiv.), D52S HEWL (0.005 equiv.), 60 mM potassium phosphate buffer pH 6.5, 37°C, overnight with 67%, 73%, 62%, 66%, 54%, and 42% yield for 7a, 7b, 9a, 9b, 11a, and 11b, respectively; vii) Aspergillus oryzae β-galactosidase (0.007 equiv.), 100 mM potassium phosphate buffer pH 5.8, 37°C, 7h, with 77%, 81%, 83%, 83%, 78%, and 82% for 8a*, 8b*, 10a*, 10b*, 12a, and 12b, respectively (* When conducted in one vessel, the overall yield of glycosylation and degalactosylation reached 69%, 70%, 57% and 63% yield for 8a, 8b, 10a and 10b, respectively instead of 51%, 59%, 51% and 55%); viii) LiOH (10 equiv.), THF:1,4-dioxane:H₂O (0.8:0.4:0.13 v/v/v), quantitative yield.

## Size-controlled synthesis of PG oligomers

The synthesis of PG oligosaccharides with the D52S HEWL glycosynthase was initiated from donor 5 and two disaccharide acceptors 6a and 6b using an iterative approach (Scheme 1B). Acceptor 6a containing an anhMurNAc unit was used to produce oligosaccharides mimicking PG chains having just been cleaved by a lytic transglycosylase, and 6b mimics an intact PG
chain. Disaccharide **6a** was obtained by methyl-esterification of GlcNAc-β-(1→4)-anhMurNAc,³⁷ with a yield of 85% (Figures S21-S25). GlcNAc-β-(1→4)-MurNAc-β-O-Bn **2b** was prepared by chemical modification of chitinbiose.⁴³ A first glycosylation step with the D52S HEWL glycosynthase afforded pentasaccharides **7a** and **7b** with yields of 67% and 73%, respectively, after purification using size-exclusion chromatography. Both pentasaccharides were then deprotected with a commercial *Aspergillus oryzae* β-galactosidase, and PG tetrasaccharides **8a** and **8b** were isolated with yields of 77% and 81%, respectively, after purification using the same technique.

A second glycosylation-deprotection cycle performed between donor **5** and the tetrasaccharides **8a** and **8b** as acceptors under similar conditions afforded the heptasaccharides **9a** and **9b** with yields of 62% and 66%, respectively, and then hexasaccharides **10a** and **10b** with an 83% yield for both molecules. Finally, the nonasaccharides **11a** and **11b** were isolated with slightly lower yields (54% and 42%, respectively), before the final deprotection to obtain the octasaccharides **12a** and **12b** (78% and 82%, respectively).

To minimize purification steps and increase overall yield, we investigated the feasibility of conducting glycosylation and degalactosylation in single reaction vessel without intermediate isolation. Tetrasaccharides **8a** and **8b** were obtained in improved yields of 69% and 70%, respectively, compared to the previously obtained yields of 51% and 59%. Hexasaccharides **10a** and **10b** were prepared with yields of 57% and 63%, respectively, instead of 51 and 55%. Although the octasaccharides were not synthesized in one vessel, the suppression of a chromatographic step increased the overall yield by around 10 to 15%.

The penta- and tetrasaccharides **7a**, **7b** and **8a**, **8b** respectively were fully characterized using ¹H and ¹³C NMR (Figures S26-S45). In particular, heteronuclear multiple bond correlation
(HMBC) experiments confirmed the regioselectivity of the newly formed glycosidic linkages.
For example, in tetrasaccharide **8a**, the hydrogen covalently attached to carbon 1 of the third
MurNAc unit (hereafter referred to as H-1<sup>III</sup>) appeared as a doublet at 4.57 ppm in the ¹H
NMR spectrum, with a ³J<sub>HH</sub> scalar coupling constant of 8.3 Hz, characteristic of a 1,2-trans
glycosidic linkage (Figure S36). In the HMBC spectrum, this hydrogen showed coupling with
the carbon 4 (³J<sub>HC</sub>) of the second GlcNAc unit (C-4<sup>II</sup>) at 79.10 ppm, confirming the β-(1→4)
selectivity of the glycosylation (Figure S40). Quantitative removal of the galactosyl protecting
group from **7a** was highlighted by the disappearance of the H-1<sup>V</sup> and C-1<sup>V</sup> signals at 4.45
ppm and 102.97 ppm, respectively, on the spectrum of **8a** (Figures S36-S37). The
characterization of **7b** (Figures S31-S35) and **8b** (Figures S41-S45) led to similar conclusions.
Deprotection of the esters with LiOH afforded tetra-, hexa- and octasaccharides **13a**, **13b**,
**14a**, **14b**, **15a**, **15b** with quantitative yields.

**Interaction between the SPOR domain of DedD and PG oligosaccharides**

According to Hermoso *et al.*,¹⁶ SPOR domains are expected to interact with the denuded
glycan chain of PG, but the interaction may be influenced by the presence of an 1,6-anhydro
group at the reducing end of the oligosaccharide. To address this question regarding the
modulation of the interaction, a solution of ¹³C,¹⁵N-labeled DedD was titrated with
octasaccharides **12a**, **12b**, **15a** and **15b** to evaluate the impact (i) of the presence of a charge
(carboxylate) at the lactoyl group of MurNAc and (ii) of the presence of an anhydro group at
the reducing end of the oligosaccharide. NMR (and more specifically [¹H,¹⁵N]-correlation
resonances of protein amides) was used to directly monitor the interaction in the same
conditions for all four oligosaccharides as alternatives to mass spectrometry and X-ray
crystallography, two monitoring methods that may contain pitfalls. Excerpts of the ¹⁵N-
BEST-TROSY spectra of ¹³C,¹⁵N-labeled DedD throughout the titration with each of the four
octasaccharides are shown in Figure 3. DedD interacted with octasaccharides **15a** and **15b** bearing lactoyl extremities, independently of the presence of an anhydro or benzyl group at the reducing end (Figures 3A and 3C), but it did not interact (up to a 50 molar equivalent ratio) with octasaccharides **12a** and **12b** bearing a lactoyl group protected with a methyl ester (Figures 3B and 3D). This discrepancy between octasaccharides suggests that the presence of an anhydro group at the reducing end of the oligosaccharide does not impact SPOR domain binding, whereas the presence of a negative charge (carboxylate) at the lactoyl group of MurNAc is critical for the interaction with PG.

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**Figure 3.** Titration of ¹³C,¹⁵N-labeled DedD with octasaccharides (A) **15a**, (B) **12a**, (C) **15b**, and (D) **12b**, monitored by ¹⁵N-BEST-TROSY HSQC of the protein. Protein-to-ligand molar ratios are indicated by a color key at the bottom, namely 0 (black), 0.5 (dark blue), 1.1 (cyan), 2 (green), 5 (orange), 10 (red). Residues close to the interaction site show significant chemical shift perturbations. The presence of the anhydro group at the reducing end has no impact in
comparison to the benzyl group, but the presence of the charge at the lactoyl group is
necessary for a significant interaction.

We then addressed the impact of oligosaccharide chain length on the interaction with DedD.
Figure 4 shows excerpts of the NMR titration experiments of DedD with tetra- and
octasaccharides **8a**, **12a**, **13a** and **15a** within the oligosaccharide series presenting an anhydro
group at the reducing end (Figures S62, S63, S64, and S65 show the entire <sup>15</sup>N-BEST-
TROSY spectra for oligosaccharides **13a**, **14a**, **15a** and **8a/12a**, respectively). Again, the
presence of a charge (carboxylate) at the lactoyl group of MurNAc proved to be key to the
interaction, regardless of oligosaccharide size (Figures 4B and 4D). In contrast, ligand length
influenced interaction strength within the charged lactoyl oligosaccharide series, as shown by
the greater chemical shift perturbations in the spectrum of **15a** (Figures 4C and 4F) compared
to **13a** (Figures 4A and 4E) during the titration of DedD with increasing molar ratios of the
oligosaccharide ligand.
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<figure><img src="image_16.png" /><figcaption>**Figure 4.** Titration of ¹³C,¹⁵N-labeled DedD with octasaccharides (A) **13a**, (B) **8a**, (C) **15a**, and (D) **12a**, monitored by ¹⁵N-BEST-TROSY HSQC of the protein at 25°C in 150 mM HEPES buffer, 300 mM NaCl at pH 8.0. Ligand-to-protein molar ratios are indicated by a color key at the top of the excerpt of the ¹⁵N-BEST-TROSY HSQC. Only oligosaccharides with a charged lactoyl group show evidence of an interaction. For these oligosaccharides, significant chemical shift perturbations were fitted to determine the dissociation constant</figcaption></figure>
upon DedD interaction. Results of these fits are presented in panels (E) and (F) for
tetrasaccharide **13a** and octasaccharide **15a**, respectively, in the case of residue Gly209.
Tetrasaccharide **13a** interacted weakly with an estimated Kd value of 6.4 ± 0.3 mM.
Octasaccharide **15a** interacted more strongly with a Kd value of 72.7 ± 4.5 µM.

Following these preliminary conclusions, chemical shift perturbations (CSPs) of DedD
residues in the <sup>15</sup>N-BEST-TROSY spectra upon titration with ligands **13a** (Figure S62), **14a**
(Figure S63), and **15a** (Figure S64) were systematically investigated as a function of the
ligand-to-protein molar ratios (Table S1). CSPs at higher ratios were plotted over the protein
sequence to determine average and standard deviation values to establish a list of significantly
perturbed protein residues for titrations with each of the oligosaccharide ligands: **13a** (Figure
S62B), **14a** (Figure S63B), and **15a** (Figure S64B). The CSPs of these residues were
subsequently fitted as a function of ligand concentration to extract a unique dissociation
constant (Kd) for each of the ligands (Table S2). Kd values of 6.4 ± 0.3 mM, 200 ± 10 µM,
and 73 ± 5 µM were determined for the tetrasaccharide **13a** (Figure S66), hexasaccharide **14a**
(Figure S67), and octasaccharide **15a** (Figure S68), respectively. These results are consistent
with previous interaction studies between SPOR domain proteins and soluble glycan
fragments from *S. aureus* containing a mixture of 6 to 30 saccharides, with an estimated Kd
value of 3.7 ± 1.5 µM from ITC experiments in the case of DedD.<sup>17</sup> Affinities of denuded
charged glycans clearly increased with oligosaccharide chain length. The interaction was
extremely weak with the tetrasaccharide and only became significant for the hexasaccharide.
Adding two more saccharides only increased the affinity by a factor of 2 to 3. These results
suggest that the DedD protein can only efficiently participate in cell constriction when
sufficiently long chains of denuded peptidoglycan have been produced, with a minimal
requirement of three GlcNAc-β-(1→4)-MurNAc units.

Determining the atomic scale DedD recognition pattern of PG oligosaccharides

DedD residues affected by the addition of oligosaccharides **13a**, **14a**, and **15a** as deduced from NMR titration experiments were mapped on the previously determined DedD NMR structure (PDB ID 6ZTG)¹⁴ (Figures S62C, S63C, and S64C, respectively). Because none of the residues of the N-terminal intrinsically disordered tail interacted with oligosaccharides, only the SPOR domain of the DedD protein (residues 140 to 220) was considered. In parallel, residues above the previously determined thresholds for Kd determination were entered as ambiguous restraints for the molecular docking between the oligosaccharides and the DedD SPOR domain. The lower energy models for these complexes are depicted in Figure 5. The X-ray structure of the *P. aeruginosa* RlpA:13a complex is also shown in Figure 4B for comparison.
A)

<figure><img src="image_17.png" /></figure>

B)

<figure><img src="image_18.png" /></figure>

C)

<figure><img src="image_19.png" /></figure>

D)

<figure><img src="image_20.png" /></figure>

**Figure 5.** Structural models of SPOR domains with denuded charged glycans. (A) DedD:13a HADDOCK model of lower energy deduced from NMR data in the present work; (B) RlpA:13a X-ray structure 6I0N solved by Hermoso *et al.*¹⁶; (C) DedD:14a HADDOCK model of lower energy deduced from NMR data in the present study; (D) DedD:15a HADDOCK model of lower energy deduced from NMR data in the present study. SPOR domain orientations have been superimposed for these representations. This figure only shows lower-energy structural models, but for longer oligosaccharide chains, alternate orientation of the reducing end is also possible.

Tetrasaccharides in the NMR-derived HADDOCK DedD model (Figure 5A) and in the X-ray structure with RlpA (Figure 5B) adopt a similar orientation with respect to the *β*-sheet and
helix-1. In both structures, the largest number of interactions are observed for anhMurNAc 1 and GlcNAc 2. Nevertheless, interaction between RlpA and **13a** is mediated by two hydrogen bonding interactions between the carboxylate moiety of anhMurNAc 1 and MurNAc 3 with Arg 302 side chain on the one side and between MurNAc 3 and Ala 275/Asn276 backbone amide (Figure S68) on the other side. Only the first hydrogen bonding interaction persists in the DedD model interaction between the carboxylate moiety of anhMurNAc 1 and Gln 147 side chain depicted in Figure S64. An alternative orientation of ligand **13a** that is parallel to the SPOR domain *β*-sheet and approximately perpendicular to the orientation in Figure 5A is also proposed by HADDOCK (Figures S70-S73 and Table S3). This alternative conformation for the DedD:**13a** complex, albeit with a slightly higher HADDOCK score (worse energetic model in comparison to the model in Figure 5A), relies on a higher electrostatic contribution and accordingly shows interaction between the charge of the lactoyl group of MurNAc 3 with the side chain amino group of Lys 156 (Table S4). This orientation furthermore matches the mapping of higher CSPs on the DedD structure (Figure S62B) and lower contribution of E<sub>air</sub>, the energy penalty arising because some of the ambiguous NMR restraints in Table S3 are not fulfilled by the model. Therefore, DedD seems to be able to accommodate two orientations for the tetrasaccharide, the lower energy one driven by van der Waals interactions, similar to the orientation found in crystals in the case of RlpA, and the higher energy one that is driven by electrostatic interactions. These two competing interactions and the low number of contacts may explain the low affinity for the tetrasaccharide.

When the length of the oligosaccharide is increased to six (Figures 5C and S74-S75) or eight (Figure 5D and S76-S77) saccharide units, the alignment of the oligosaccharide scaffold along the SPOR domain *β*-sheet structure seems to dominate. The higher affinity detected by NMR in these cases is consistent with the higher electrostatic energetic contribution in the HADDOCK models of lower energy (Table S5). Nevertheless, no unique persistent contact
(Table S4) or oligosaccharide orientation can be established with high confidence between the hexasaccharide **14a** (Figures S74 and S75) and the octasaccharide **15a** (Figures S76 and S77). Furthermore, no specific pattern seems to recognize the anhydro-MurNAc 1 in comparison to other MurNAc saccharides of the chain. This versatility may reflect the fact that the DedD protein must adapt to the heterogeneity of denuded glycan strands for cell constriction. However, electrostatic interactions appear to be the driving force of the interaction of the DedD SPOR domain with the denuded glycan chain. The hexasaccharide appears to offer the optimal number of saccharide units needed for interaction with DedD in terms of structural overlap with the SPOR domain size and to be the minimal required length for an appreciable affinity.

Because DedD shows structural similarity to the four other SPOR domains in *E. coli* and because interaction with denuded hexasaccharides with charged lactoyl moieties is mainly directed by electrostatic interactions, we studied whether the electrostatic surface is conserved among the SPOR domains. Figure 6 depicts the SPOR domains of DedD, *P. aeruginosa* RlpA, DamX, and FtsN (Figure 6, panels A–D). All SPOR domains present a positively charged face, ready to interact with the negative charges of the carboxylate moieties of the denuded glycans. However, the distribution of positive patches with respect to the orientation of the core SPOR β-sheet differed from one SPOR domain to the other. In DedD, we showed that the preferred orientation of the glycan chain is relatively parallel to the β-sheet (Figure 6A). Carboxylate moieties of different MurNAc residues can be accommodated by each of the two positive patches on the left and right of the structure depicted in the second row. The perpendicular orientation of the positive RlpA patch is consistent with the orientation of the glycan chain in the X-ray determined structure (Figure 6B). Based on these conclusions, we suggest that the glycan chain in interaction with DamX takes on a different orientation, as shown in Figure 6C (second row). The interaction pattern of the glycan chain with FtsN
(Figure 6D) is more difficult to predict, the overall structure of the SPOR domain and electrostatic surface being highly different from the other three domains. These differences in the relative orientation of the glycan chain with respect to the β-sheet of the SPOR domain may be the basis for the functional differences among the SPOR-domain proteins.

<figure><img src="image_21.png" /></figure>

**Figure 6.** Comparison of the structure of the four SPOR domain proteins (A) DedD, (B) *P. aeruginosa* RlpA, (C) DamX and (D) FtsN. All structures are presented in the same orientation (superimposition with the alignment plugin of Pymol). The first two top rows depict the cartoon and APBS<sup>44</sup> electrostatic surface represented in the exact same orientation. The third row depicts the APBS electrostatic structure after a 90° rotation along the vertical axis with respect to the second row. The fourth row depicts the APS electrostatic structure
after a 90° rotation along the horizontal axis with respect to the second row. Blue and red
emphasize the location of negatively and positively charged residues on the APBS surfaces as
indicated in the color scale at the bottom of the figure. Red arrows indicate the proposed
overall orientation of the glycan chain in interaction with the SPOR domain.

CONCLUSION

In this study, we implemented a straightforward chemo-enzymatic synthesis of peptidoglycan
oligosaccharides, combining bacterial metabolic engineering and glycosynthase-mediated
catalysis. Glycosylation of Gal-β-(1→4)-GlcNAc-β-(1→4)-MurNAc-α-fluoride on PG
acceptors with the D52S HEWL glycosynthase followed by enzymatic degalactosylation led
to PG oligosaccharides bearing either a benzyl β-MurNAc or an 1,6-anhydro-MurNAc
extremity in high yields. These synthetic ligands were then used to study their interaction with
the SPOR domain of the E. coli cell division protein DedD. We showed that hexa- and
octasaccharides bearing denuded charged lactoyl groups interact with the protein with a
dissociation constant in the ~100 μM range, but tetrasaccharides have poor affinity. This
affinity allows DedD to localize at the septum between daughter cells during cell division and
to activate PBP1A and PBP1B,¹⁴ which incorporate new peptidoglycan chains into the
ingrowing septum and potentially facilitate the gliding of the saccharide chain along the core
protein β-sheet. The negligible impact of the terminal unit of the denuded charged hexa- and
octasaccharides on DedD binding suggests that the protein could bind such fragments as part
of a longer polysaccharide chain in which the other stem peptides have not been cleaved by
amidases. The orientation of the oligosaccharide with respect to the SPOR domain appears to
be mediated by electrostatic interactions. The distribution of the positive charges on the
surface of the different SPOR domains may then direct the glycan chain orientation and
provide the basis for functional differences. The synthesis methodology developed here paves
the way for rapid access to real PG fragments after conjugation of stem peptides onto
MurNAc units. This synthesis approach thus offers the possibility to study a wider range of PG-binding proteins with soluble synthesis mimetics, work that is already underway in our laboratory.

## ASSOCIATED CONTENT

Detailed experimental procedures, characterization data for all new compounds (NMR spectra), protein-oligosaccharide interaction data (NMR titration, determination of dissociation constants, HADDOCK models).

This material is available free of charge via the Internet at http://pubs.acs.org.

## AUTHOR INFORMATION

### Corresponding authors

Sébastien Fort : Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France ; https://orcid.org/0000-0002-6133-9900 ; sebastien.fort@cermav.cnrs.fr

Catherine Bougault : Univ. Grenoble Alpes, CNRS, CEA, IBS, 38044 Grenoble Cedex 9, France ; catherine.bougault@ibs.fr

### Authors

Antoine Rousseau : Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France

Dindet Steve-Evanes Koffi Teki : Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France

Célia Boyat : Univ. Grenoble Alpes, CNRS, CEA, IBS, 38044 Grenoble Cedex 9, France

Emeline Richard : Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France
Stéphanie Pradeau : Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France

Louis Brigandat : Univ. Grenoble Alpes, CNRS, CEA, IBS, 38044 Grenoble Cedex 9, France

Isabel Ayala : Univ. Grenoble Alpes, CNRS, CEA, IBS, 38044 Grenoble Cedex 9, France

Jean-Pierre Simorre : Univ. Grenoble Alpes, CNRS, CEA, IBS, 38044 Grenoble Cedex 9, France

Sylvie Armand : Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France

Sylvain Cottaz : Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France

## FUNDING SOURCES

This work was supported by the Agence Nationale de la Recherche (ANR) under grant number ANR-20-CE07-0012-01 (Glyco_SWIM).

## ACKNOWLEDGEMENTS

This work was supported by the French National Research Agency (ANR) through Glyco_SWIM (ANR-20-CE07-0012-01), Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003), Glyco@Alps (ANR-15-IDEX-02), Carnot PolyNat (CARN-025-01). The NanoBio ICMG (UAR 2607) is acknowledged for providing facilities for mass spectrometry and oligosaccharide NMR analyses. The protein interaction work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRSCEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003).
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For Table of Content Only

<figure><img src="image_22.png" /></figure>