A step-by-step in crystallo guide to bond cleavage and 1,6-anhydro-sugar product synthesis by a peptidoglycan-degrading lytic transglycosylase.

Lytic transglycosylases (LTs) are a class of enzymes important for the recycling and metabolism of peptidoglycan (PG). LTs cleave the ß-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and GlcNAc in the PG glycan strand, resulting in the concomitant formation of 1,6-anhydro-N-acetylmuramic acid and GlcNAc. No LTs reported to date have utilized chitins as substrates, despite the fact that chitins are GlcNAc polymers linked via ß-1,4-glycosidic bonds, which are the known site of chemical activity for LTs. Here, we demonstrate enzymatically that LtgA, a non-canonical, substrate-permissive LT from Neisseria meningitidis utilizes chitopentaose ((GlcNAc)5) as a substrate to produce three newly identified sugars: 1,6-anhydro-chitobiose, 1,6-anhydro-chitotriose, and 1,6-anhydro-chitotetraose. Although LTs have been widely studied, their complex reactions have not previously been visualized in the crystalline state because macromolecular PG is insoluble. Here, we visualized the cleavage of the glycosidic bond and the liberation of GlcNAc-derived residues by LtgA, followed by the synthesis of atypical 1,6-anhydro-GlcNAc derivatives. In addition to the newly identified anhydro-chitin products, we identified trapped intermediates, unpredicted substrate rearrangements, sugar distortions, and a conserved crystallographic water molecule bound to the catalytic glutamate of a high-resolution native LT. This study enabled us to propose a revised alternative mechanism for LtgA that could also be applicable to other LTs. Our work contributes to the understanding of the mechanisms of LTs in bacterial cell wall biology.

Atg8 transfer from Atg7 to Atg3: a distinctive E1-E2 architecture and mechanism in the autophagy pathway.

Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7(NTD)) recruits a unique "flexible region" from Atg3 (Atg3(FR)). The structure of an Atg7(NTD)-Atg3(FR) complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7~Atg8-Atg3)(2) complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1~UBL-E2 architecture for enzymes mediating autophagy.

Visualization of a substrate-induced productive conformation of the catalytic triad of the Neisseria meningitidis peptidoglycan O-acetylesterase reveals mechanistic conservation in SGNH esterase family members.

Peptidoglycan O-acetylesterase (Ape1), which is required for host survival in Neisseria sp., belongs to the diverse SGNH hydrolase superfamily, which includes important viral and bacterial virulence factors. Here, multi-domain crystal structures of Ape1 with an SGNH catalytic domain and a newly identified putative peptidoglycan-detection module are reported. Enzyme catalysis was performed in Ape1 crystals and key catalytic intermediates along the SGNH esterase hydrolysis reaction pathway were visualized, revealing a substrate-induced productive conformation of the catalytic triad, a mechanistic detail that has not previously been observed. This substrate-induced productive conformation of the catalytic triad shifts the established dogma on these enzymes, generating valuable insight into the structure-based design of drugs targeting the SGNH esterase superfamily.

De-O-acetylation of peptidoglycan regulates glycan chain extension and affects in vivo survival of Neisseria meningitidis.

Peptidoglycan O-acetylation is a modification found in many bacteria. In Gram-positive pathogens, it contributes to virulence by conferring resistance to host lysozyme. However, in Gram-negative pathogens, its contribution to physiology and virulence is unknown. We examined the contribution of patA, patB and ape1 to peptidoglycan O-acetylation in the major human pathogen Neisseria meningitidis (Nm). Using genetic expression of all possible combinations of the three genes in Escherichia coli and Nm, we confirmed that PatA and PatB were required for PG O-acetylation, while ApeI removed the O-acetyl group. ApeI was active on all O-acetylated muropeptides produced by PatA and PatB during heterologous expression in E. coli and was also active on several PG structures in vitro. Interestingly, in Nm, ApeI was found to preferentially de-O-acetylate muropeptides with tripeptide stems (GM3), suggesting that its activity is highly regulated. Accordingly, de-O-acetylation of GM3 regulated glycan chain elongation and cell size. Additionally, the virulence of Nm lacking ApeI was drastically reduced suggesting that regulation of glycan chain length by O-acetylation contributes to bacterial fitness in the host. Altogether, our results suggest that ApeI represents an attractive target for new drug development.

Acetylation regulates the oligomerization state and activity of RNase J, the Helicobacter pylori major ribonuclease.

In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.

The mechanistic landscape of Lytic transglycosylase as targets for antibacterial therapy.

Lytic transglycosylases (Ltgs) are glycan strand cleaving enzymes whose role is poorly understood in the genesis of the bacterial envelope. They play multiple roles in all stages of a bacterial life cycle, by creating holes in the peptidoglycan that is necessary for cell division and separation. Here, we review recent advances in understanding the suitability of Ltgs as antibacterial drug targets. We specifically highlight a known inhibitor bulgecin A that is able to inhibit the function of structurally diverse Ltgs, as well as synergize with beta-lactams to improve its efficacy in antibiotic insensitive strains. Discovery of new antibiotics or new targets has been challenging. These studies could provide a viable path toward designing broad-spectrum inhibitors that targets Ltgs.

Structural basis for the sugar nucleotide and acyl-chain selectivity of Leptospira interrogans LpxA.

The first step of lipid A biosynthesis is catalyzed by LpxA in Escherichia coli (EcLpxA), an acyltransferase selective for UDP-GlcNAc and R-3-hydroxymyristoyl-acyl carrier protein (ACP). Leptospira interrogans LpxA (LiLpxA) is extremely selective for R-3-hydroxylauroyl-ACP and an analogue of UDP-GlcNAc, designated UDP-GlcNAc3N, in which NH(2) replaces the GlcNAc 3-OH group. EcLpxA does not discriminate between UDP-GlcNAc and UDP-GlcNAc3N; however, E. coli does not make UDP-GlcNAc3N. With LiLpxA, R-3-hydroxylauroyl-methylphosphopantetheine efficiently substitutes for R-3-hydroxylauroyl-ACP. We now present crystal structures of free LiLpxA and its complexes with its product UDP-3-N-(R-3-hydroxylauroyl)-GlcNAc3N and with its substrate R-3-hydroxylauroyl-methylphosphopantetheine. The positions of the acyl chains of the R-3-hydroxylauroyl-methylphosphopantetheine and the UDP-3-N-(R-3-hydroxylauroyl)-GlcNAc3N are almost identical and are similar to that of the acyl chain in the EcLpxA/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc complex. The selectivity of LiLpxA for UDP-GlcNAc3N may be explained by the orientation of the backbone carbonyl group of Q68, which differs by approximately 82 degrees from the corresponding Q73 carbonyl group in EcLpxA. This arrangement provides an extra hydrogen-bond acceptor for the 3-NH(2) group of UDP-GlcNAc3N in LiLpxA. The R-3-hydroxylauroyl selectivity of LiLpxA is explained by the position of the K171 side chain, which limits the length of the acyl-chain-binding groove. Our results support the role of LiLpxA H120 (which corresponds to EcLpxA H125) as the catalytic base and provide the first structural information about the orientation of the phosphopantetheine moiety during LpxA catalysis.

Structure guided design of an antibacterial peptide that targets UDP-N-acetylglucosamine acyltransferase.

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of an R-3-hydroxyacyl chain from its acyl carrier protein (ACP) to the 3-OH group of UDP-GlcNAc. Essential in the growth of Gram-negative bacteria, LpxA is a logical target for antibiotics design. A pentadecapeptide (Peptide 920) with high affinity towards LpxA was previously identified in a phage display library. Here we created a small library of systematically designed peptides with the length of four to thirteen amino acids using Peptide 920 as a scaffold. The concentrations of these peptides at which 50% of LpxA is inhibited (IC50) range from 50 nM to >100 µM. We determined the crystal structure of E. coli LpxA in a complex with a potent inhibitor. LpxA-inhibitor interaction, solvent model and all contributing factors to inhibitor efficacy were well resolved. The peptide primarily occludes the ACP binding site of LpxA. Interactions between LpxA and the inhibitor are different from those in the structure of Peptide 920. The inhibitory peptide library and the crystal structure of inhibitor-bound LpxA described here may further assist in the rational design of inhibitors with antimicrobial activity that target LpxA and potentially other acyltransferases.

The cryo-electron microscopy supramolecular structure of the bacterial stressosome unveils its mechanism of activation.

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria.

Author Correction: N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes.

This Article contains a URL for a publically available whole-genome browser ( http://nterm.listeriomics.pasteur.fr ). However, due to technical constraint, this website has been replaced with an alternative ( https://listeriomics.pasteur.fr ).

Bulgecin A: The Key to a Broad-Spectrum Inhibitor That Targets Lytic Transglycosylases.

Lytic transglycosylases (Lts) are involved in recycling, cell division, and metabolism of the peptidoglycan. They have been understudied for their usefulness as potential antibacterial targets due to their high redundancy in Gram-negative bacteria. Bulgecin A is an O-sulphonated glycopeptide that targets primarily soluble lytic tranglycosylases (Slt). It has been shown that bulgecin A increases the efficacy of ß-lactams that target penicillin bindings proteins (PBPs). Here, we present the high-resolution crystal structure of LtgA from Neisseria meningitidis strain MC58, a membrane bound homolog of Escherichia coli Slt, in complex with bulgecin A. The LtgA-bulgecin A complex reveals the mechanism of inhibition by bulgecin A at near atomic resolution. We further demonstrate that bulgecin A is not only a potent inhibitor of LtgA, but most importantly, it restores the efficacy of ß-lactam antibiotics in strains of N. meningitidis and Neisseria gonorrhoeae that have reduced susceptibility to ß-lactams. This is particularly relevant for N. gonorrhoeae where no vaccines are available. This work illustrates how best to target dangerous pathogens using a multiple drug target approach, a new and alternative approach to fighting antibiotic resistance.

N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes.

To adapt to changing environments, bacteria have evolved numerous pathways that activate stress response genes. In Gram-positive bacteria, the stressosome, a cytoplasmic complex, relays external cues and activates the sigma B regulon. The stressosome is structurally well-characterized in Bacillus, but how it senses stress remains elusive. Here, we report a genome-wide N-terminomic approach in Listeria that strikingly led to the discovery of 19 internal translation initiation sites and 6 miniproteins, among which one, Prli42, is conserved in Firmicutes. Prli42 is membrane-anchored and interacts with orthologues of Bacillus stressosome components. We reconstituted the Listeria stressosome in vitro and visualized its supramolecular structure by electron microscopy. Analysis of a series of Prli42 mutants demonstrated that Prli42 is important for sigma B activation, bacterial growth following oxidative stress and for survival in macrophages. Taken together, our N-terminonic approach unveiled Prli42 as a long-sought link between stress and the stressosome.

Structural basis for the acyl chain selectivity and mechanism of UDP-N-acetylglucosamine acyltransferase.

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the reversible transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein to the glucosamine 3-OH group of UDP-GlcNAc. Escherichia coli LpxA is highly selective for R-3-hydroxymyristate. The crystal structure of the E. coli LpxA homotrimer, determined previously in the absence of lipid substrates or products, revealed that LpxA contains an unusual, left-handed parallel beta-helix fold. We have now solved the crystal structures of E. coli LpxA with the bound product UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc at a resolution of 1.74 A and with bound UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc at 1.85 A. The structures of these complexes are consistent with the catalytic mechanism deduced by mutagenesis and with a recent 3.0-A structure of LpxA with bound UDP-GlcNAc. Our structures show how LpxA selects for 14-carbon R-3-hydroxyacyl chains and reveal two modes of UDP binding.

Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide.

UDP-GlcNAc acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein (ACP) to the glucosamine 3-OH group of UDP-GlcNAc. LpxA is essential for the growth of Escherichia coli and related Gram-negative bacteria. The crystal structure of the E. coli LpxA homotrimer, determined previously at 2.6 A in the absence of substrates or inhibitors, revealed that LpxA contains an unusual, left-handed parallel beta-helix fold. We now present the crystal structure at 1.8 A resolution of E. coli LpxA in a complex with a pentadecapeptide, peptide 920. Three peptides, each of which adopts a beta-hairpin conformation, are bound per LpxA trimer. The peptides are located at the interfaces of adjacent subunits in the vicinity of the three active sites. Each peptide interacts with residues from both adjacent subunits. Peptide 920 is a potent inhibitor of E. coli LpxA (Ki = 50 nM). It is competitive with respect to acyl-ACP but not UDP-GlcNAc. The compact beta-turn structure of peptide 920 bound to LpxA may open previously uncharacterized approaches to the rational design of LpxA inhibitors with antibiotic activity.

Enzymatic synthesis of lipid A molecules with four amide-linked acyl chains. LpxA acyltransferases selective for an analog of UDP-N-acetylglucosamine in which an amine replaces the 3"-hydroxyl group.

LpxA of Escherichia coli catalyzes the acylation of the glucosamine 3-OH group of UDP-GlcNAc, using R-3-hydroxymyristoyl-acyl carrier protein (ACP) as the donor substrate. We now demonstrate that LpxA in cell extracts of Mesorhizobium loti and Leptospira interrogans, which synthesize lipid A molecules containing 2,3-diamino-2,3-dideoxy-d-glucopyranose (GlcN3N) units in place of glucosamine, do not acylate UDP-GlcNAc. Instead, these LpxA acyltransferases require a UDP-Glc-NAc derivative (designated UDP 2-acetamido-3-amino-2,3-dideoxy-alpha-d-glucopyranose or UDP-GlcNAc3N), characterized in the preceding paper, in which an amine replaces the glucosamine 3-OH group. L. interrogans LpxA furthermore displays absolute selectivity for 3-hydroxylauroyl-ACP as the donor, whereas M. loti LpxA functions almost equally well with 10-, 12-, and 14-carbon 3-hydroxyacyl-ACPs. The substrate selectivity of L. interrogans LpxA is consistent with the structure of L. interrogans lipid A. The mechanism of L. interrogans LpxA appears to be similar to that of E. coli LpxA, given that the essential His(125) residue of E. coli LpxA is conserved and is also required for acyltransferase activity in L. interrogans. Acidithiobacillus ferrooxidans (an organism that makes lipid A molecules containing both GlcN and GlcN3N) has an ortholog of LpxA that is selective for UDP-GlcNAc3N, but the enzyme also catalyzes the acylation of UDP-GlcNAc at a slow rate. E. coli LpxA acylates UDP-GlcNAc and UDP-GlcNAc3N at comparable rates in vitro. However, UDP-GlcNAc3N is not synthesized in vivo, because E. coli lacks gnnA and gnnB. When the latter are supplied together with A. ferrooxidans lpxA, E. coli incorporates a significant amount of GlcN3N into its lipid A.