Carbohydrate recognition and lysis by bacterial peptidoglycan hydrolases

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ScienceDirect Carbohydrate recognition and lysis by bacterial peptidoglycan hydrolases Martı´n Alcorlo*, Siseth Martı´nez-Caballero*, Rafael Molina* and Juan A Hermoso The major component of bacterial cell wall is peptidoglycan (PG), a complex polymer formed by long glycan chains crosslinked by peptide stems. PG is in constant equilibrium requiring well-orchestrated coordination between synthesis and degradation. The resulting cell-wall fragments can be recycled, act as messengers for bacterial communication, as effector molecules in immune response or as signaling molecules triggering antibiotics resistance. Tailoring and recycling of PG requires the cleavage of different covalent bonds of the PG sacculi by a diverse set of specific enzymes whose activities are strictly regulated. Here, we review the molecular mechanisms that govern PG remodeling focusing on the structural information available for the bacterial lytic enzymes and the mechanisms by which they recognize their substrates.

core structure of the non-crosslinked stem attached is in Gram-negative bacteria (m-DAP for 2,6-diaminopimelic acid) and L-Ala-g-D-Glu-L-Lys-D-Ala-D-Ala in most Gram-positive bacteria [2]. This conserved core of the PG is constantly edited, by chemical modification (e.g., O-acetylation, N-deacetylation or amidation) or by incorporation of Gly residues or non-canonical D-amino acids at the peptide-stem, in response to different environmental stresses (reviewed in Ref. [3]). L-Ala-g-D-Glu-m-DAP-D-Ala-D-Ala

Introduction

Bacterial murein hydrolases contribute to this PG plasticity and form a vast and highly diverse group of enzymes capable of cleaving bonds in sacculi and/or its soluble fragments. The growing body of data generated by massive genome sequencing is progressively shedding more light on the number and variety of the enzymes involved in PG metabolism in different bacteria. However, identification and classification of novel PG hydrolases from genomic and metagenomic data is difficult due to their lack of homology with the previously well-characterized PG hydrolases. Efforts have been made in the development of computational tools to aid in the identification and classification of these novel PG hydrolases [4]. Fifty years of research work have led to the discovery of more than 35 PG hydrolases in Escherichia coli that have been classified into 12 families (reviewed in Ref. [5]). However, present knowledge on the regulation mechanism of these activities is still fragmentary and limited to a few examples. Hydrolases are involved in several critical functions [6], including PG maturation, turnover, recycling, autolysis, cleavage of the septum during cell division [7], and antibiotic resistance [8]. PG degradation products also act as critical activators of the mammalian immune system [9].

Peptidoglycan (murein) is an essential and specific component of the bacterial cell wall preventing cell lysis as a result of the cell’s high intracellular osmotic pressure. PG is intimately involved in the processes of cell growth and cell division, contributes to the maintenance of a defined shape and also serves as a scaffold for anchoring other cell envelope components such as proteins [1] and teichoic acids [2] (Figure 1). PG is composed by linear glycan strands cross-linked by short peptides. The glycan strands are made up of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues linked by b(1–4) bonds. The D-lactoyl group of each NAM residue is substituted by a peptide stem. Although several variations in the structure of the peptide stem are known, the

Hydrolases can cleave glycosidic (glycosidases) or amide bond (peptidases) in the PG network (Figure 1). Regarding glycosidases, three different subgroups, glucosaminidases, lysozymes and lytic transglycosylases, can be distinguished. While glucosaminidases hydrolytically cleave the b(1–4) glycosidic bond between NAG and NAM, lysozymes and lytic transglycosylases (LTs) cleave the b(1–4) glycosidic bond between sequential NAM and NAG (Figure 1). Differences between lysozymes and LTs arise from their specific catalytic mechanisms. While lysozymes are hydrolytic enzymes, the reaction catalyzed by LTs is the non-hydrolytic cleavage of the glycan strand of PG as first demonstrated by Holtje et al. in 1975 [10],

Address Department of Crystallography and Structural Biology, Inst. Quı´mica-Fı´sica “Rocasolano”, CSIC, Serrano 119, 28006 Madrid, Spain Corresponding author: Hermoso, Juan A ([email protected]) These authors equally contributed to this work.

*

Current Opinion in Structural Biology 2017, 44:87–100 This review comes from a themed issue on Carbohydrates Edited by Ute Krengel and Thilo Stehle

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88 Carbohydrates

Figure 1

(b)

(a) Gram-positive bacteria

(c)

Gram-negative bacteria

Murein hydrolases

Lipopolysaccharide Surface protein Lipoteichoic acid

Teichoic acids

exo-LTs

Lysozymes

Porin

endo-LTs NAG

NAM

anhNAM

Outer Membrane 20-35 nm

40-60 nm

35-40 nm

BLP

Periplasmic protein

PG amidases 2-8 nm

Peptidoglycan

Endopeptidases Glucosaminidase

Lipoprotein Inner Membrane Membrane proteins

Carboxypeptidases Permease

Current Opinion in Structural Biology

Gram-positive (a) and Gram-negative (b) cell wall organization scheme. The PG layer comprises long polymers of the repeating disaccharide Nacetylglucosamine-N-acetylmuramic acid (NAG–NAM, dark and pale green respectively) that are linked via peptide bridges. BLP refers to Braun’s lipoprotein (dark blue) attached to the outer membrane and covalently crosslinked with the PG. (c) Detailed scheme of the crosslinked PG and the different bacterial peptidoglycan hydrolases involved in the cell wall processing.

with formation of the non-reducing N-acetyl 1,6-anhydromuramic and the N-acetylglucosamine as the two termini (Figure 1). LTs can be classified as exolytic, when the cleavage is performed at the end of the glycan strands, or endolytic if the cleavage is carried out in the middle of the PG chain. Recent analysis of the reactions products of all LTs from E. coli have revealed that almost all LTs are able to exert, in different degree, both exolytic and endolytic activities, and even, although to a lesser extent, muramidase activity [11]. Regarding peptidases, two different groups, PG amidases and peptidases, can be distinguished depending on the nature and localization of amide bond to be hydrolyzed (Figure 1). While PG amidases are NAM-L-Ala amidases, which cleave the amide bond between L-Ala residue of the stem peptide and NAM, peptidases cleave the amide bonds within the stem peptide itself. Depending on the position of the amide bond to be cleaved, peptidases are subdivided into carboxypeptidases that remove C-terminal residues, and endopeptidases that cleave within the peptide, with prefixes DD-, LD- or DL referring to the stereochemistry of the two amino acid residues constituting the cleavage site. Recent results on the structural and functional characterization of the different PG-hydrolysing enzymes are summarized below. Relevant databases for PG hydrolases classification

Different databases are now available providing updated information on both the enzymes processing PG and different carbohydrate-binding modules. The PG hydrolases can be analyzed based on sequence similarity and Current Opinion in Structural Biology 2017, 44:87–100

structure motifs through three different databases (see Table 1). (i) The Carbohydrate-Active enZymes (CAZy) database (http://www.cazy.org/) and its extension CAZypedia (http://www.cazypedia.org/) for glycosidic hydrolases; this is an encyclopedic resource on structurallyrelated enzymes that degrade, modify, or create glycosidic bonds. These carbohydrate-active enzymes are classified into different glycoside hydrolase (GH) families according to their catalytic mechanism, enzyme active site residues or three-dimensional structure. For each GH family, a description of its known activity and catalytic mechanism is provided, in addition to the GenBank, Uniprot and PDB accession codes of its members. (ii) MEROPS is an on-line database (http://merops.sanger.ac. uk/) for peptidases (also known as proteases, proteinases or proteolytic enzymes) and their inhibitors. The summary page describing a given peptidase can be reached by using an index under its name, a MEROPS identifier or the source organism. The MEROPS database uses a hierarchical, structure-based classification of peptidases. Each peptidase is assigned to a family on the basis of statistically significant similarities in the amino acid sequence, and families that are thought to be homologous are grouped together in a Clan. The summary page for each family provides links to supplementary pages with further information. (iii) The Pfam is a database containing a large collection of protein families (http://www. xfam.org/), each represented by multiple sequence alignments and hidden Markov probabilistic models (HMM) used for the statistical inference of homology sequences. A high-quality seed alignment provides the basis for the position-specific amino-acid frequencies, gap and length www.sciencedirect.com

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Table 1 Classification according CAZy (glycoside hydrolases), MEROPS (peptidases) and Pfam (amidases) databases for various types of enzymes involved in the bacterial peptidoglycan metabolism described in this work Enzyme type Glucosaminidases (CAZy) Lysozymes (CAZy) Lytic transglycosylases (CAZy) LT family 1

Family

Activities

Mechanism

Catalytic residues

3D structure

Retaining

Asp, Glu

NagZ (2OXN)

V. cholerae

[14]

GH73 GH25

endo-b-N-Acetylglucosaminidase Lysozyme

Not known Retaining

Glu Asp, Glu

LytB (4Q2W) LytC (2WWD)

S. pneumoniae S. pneumoniae

[17] [19]

SleM (5JIP)

C. perfringes

[71]

MltE (4HJY) MltF (4P0G), (5A5X) Slt70 (1QSA) EtgA (4XP8) MltC (4CFP) RpfE (4CGE) RpfB (4KPM)

E. coli P. aeruginosa

[27] [33] a

E. coli E. coli E. coli M. tuberculosis M. tuberculosis

[72] [28] [30] [37] [36]

E. coli P. aeruginosa Pseudomonas bacteriophage C. difficile P. aeruginosa

[74] [32] [76] [40] [50]

P. aeruginosa

[51]

E. coli E. coli

[49] [73]

AmiA (4KNL) LytA (5CTV)

S. aureus S. pneumoniae

[47] [46]

DacB (4OXD) Van XYc (4MUT)

S. pneumoniae E. gallinarum

[56] [59]

GH23

Lytic transglycosylase

Retaining

Glu

Lytic transglycosylase Lytic transglycosylase Lytic transglycosylase

Retaining Retaining Retaining

Asp Glu Glu

MltA (2PI8) SltB3 (5AO8) Phi KZ (3BKV)

LT family 5 Peptidoglycan amidases (Pfam)

– PF01510 (amidase 2)

Lytic transglycosylase N-Acetylmuramoyl-L-alanine amidase

– Zinc amidases

Glu Glu, His

SpoIID (5I1T) AmpDh2 (4BOL, 4BPA) AmpDh3 (4BXD, 4BXE) AmiD (3D2Z) AmiC (4BIN)

PF01520 (amidase 3)

M15 M15

N-Acetylmuramoyl-L-alanine amidase

Zinc D-Ala-D-Ala carboxypeptidase Peptidase

Redden E, Thunnissen AMWH, to be published.

Zinc amidases

Metallo Metallo

Glu

Glu, His, Asp Glu His Asp

Structure and function of bacterial peptidoglycan hydrolases Alcorlo et al. 89

Current Opinion in Structural Biology 2017, 44:87–100

b-N-Acetylhexosaminidase

GH102 GH103 GH23

a

References

GH3

LT family 2 LT family 3 LT family 4

Carboxypeptidases (MEROPS)

Organism

90 Carbohydrates

Figure 2

(a)

Glucosaminidases

(b) Lysozymes

(c)

Lytic Transglycosilases

NagZ (V. cholerae) LytC (S. pneumoniae)

LytBcat (S. pneumoniae)

PsmCat (phiSM101 phage)

MltF (P. aeruginosa)

MltE (E. coli)

SpollD (B. anthracis)

SltB3 (P. aeruginosa) Current Opinion in Structural Biology

Overall structures of glycosidases involved in PG hydrolysis. Their different folds and modular nature is shown. Protein structures are displayed in ribbon representation and ligands as sticks. (a) Crystal structure of glucosaminidase NagZ from V. cholerae in complex with PUGNAc (PDB code 2OXN, [14]), and the catalytic domain of LytB from S. pneumoniae (PDB code 4Q2W, [17]). Catalytic domains are displayed in green while WW and SH3b domains in LytB are colored in purple and blue, respectively. (b) Crystal structures of lysozyme LytC from S. pneumoniae in complex with a PG fragment (PDB code 2WWD, [19]), and the catalytic domain of Psm from phiSM101 phage in complex with NAG (PDB code 4KRU, [21]). Catalytic and binding domains are coloured in red and blue, respectively. (c) Crystal structures of lytic transglycosylases. Catalytic domains are colored in orange; MltF in complex with bulgecin and muropeptide (PDB code 4P0G, Redden E, Thunnissen AMWH, to be published), MltE (PDB code 4HJZ, [28]) is shown with bound chitopentaose (dark green), SpoIID (PDB code 5I1T, [40]) in complex with triacetylchitotriose and SltB3 in complex with NAG-NAM pentapeptide (PDB code 5AO8, [32]). anhNAM residues are depicted in red sticks and NAG residues in dark green. All ligands are represented in sticks with peptides in yellow and glycan chains in dark green, light green or salmon corresponding to NAG, NAM or anhNAM, respectively. White sticks depict other ligand moieties.

parameters in the profile HMM. In Pfam, the profile HMM is searched against a large sequence collection, based on UniProt Knowledgebase (UniProtKB), to find all members of the family. Glucosaminidases

cleave not only its natural substrate (NAG-anhNAMpeptide) but also the NAG-NAM-peptide [15,16]. The requirement of NagZ for AmpC b-lactamase induction and, more specifically, its role in production of the anhNAM-peptide signal molecule(s), makes NagZ a key target for inhibitor development.

NagZ, the only cytoplasmic N-acetylglucosaminidase identified up to now, is involved in cell wall recycling in Gram-negative bacteria [12]. NagZ is predicted to use a two-step, double displacement catalytic mechanism involving a covalent glycosyl-enzyme intermediate and highly dissociative oxocarbenium ion-like transition state [13]. The crystal structure of NagZ from Vibrio cholerae (VcNagZ) in complex with PUGNAc (an inhibitor that mimics the mentioned oxocarbenium ion-like transition state) has been reported [14] (Figure 2a). VcNagZ adopts the common TIM-barrel fold found in many glycoside hydrolases and features a large open pocket containing the active site. Regarding substrate specificity, NagZ can

In Streptococcus pneumoniae, the glucosaminidase LytB plays a critical role in cell division. LytB is a cholinebinding protein (CBP, a pneumococcal family of modular surface-exposed proteins) composed by a choline-binding module, recognizing the choline-containing teichoic acids, followed by a C-terminal catalytic domain. The crystal structure of the catalytic domain of LytB [17] shows three structurally independent modules (SH3b, WW, and GH73) (Figure 2a). The active site is at the interface of the three modules; their intersection creates creating a groove to accommodate the PG substrate. It has been recently reported that LytB hydrolysis occurs at

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Structure and function of bacterial peptidoglycan hydrolases Alcorlo et al. 91

sites with fully acetylated NAG moieties, suggesting a substrate-assisted mechanism with anchimeric assistance of the acetamido group of NAG, and with preference for non-crosslinked muropeptides [18]. Lysozymes

Lysozymes cleave the bond between sequential NAM (located at 1 position of the active site) and NAG (located at +1 position) saccharide subunits in the PG backbone. Some structures of bacterial lysozymes such as autolysin LytC from S. pneumoniae [19] or phage lysozymes as SPN1S [20] and Psm [21] (Figure 2b) have been reported. LytC is a CBP involved in the fratricide phenomena, a competence-programmed mechanism of predation of noncompetent sister cells [22]. Its crystal structure in complex with muropeptide and choline revealed an unusual hook-shaped conformation (Figure 2b) of the multimodular protein (choline-binding module and catalytic module) that restricts LytC hydrolysis to non-crosslinked PG chains [19]. The observed structure would explain why LytC is activated by CbpD in fratricide and also how bacteria regulates lytic activity of this autolysin. Lytic transglycosylases

LTs exist as either soluble (named as Slts) or membranebound forms (named Mlts) (Figure 2c). LTs have important roles in bacterial fitness such as growth, division, insertion of pili or cell-wall recycling [8,23] and their activities are is also directly related with the lethal effect of b-lactam antibiotics [24]. LTs were organized into four families, however the recent discovery of new LTs has provoked an updated classification scheme in which an additional subfamily (1F) in family 1, as well as new families 5 and 6 have been defined [25]. Despite their low sequence identity, the catalytic module is, in most cases, highly similar to the goose-type lysozyme (GEWL). In addition to their catalytic module, most LTs comprise several other modules with functions that are often poorly understood. Considering that LTs are inactive against chitin or chitooligosaccharides, it has been proposed that the presence of the peptide side chains in PG is a requirement for LT’s activity. Family 2 LTs would show a different behavior being active on naked glycan chains. In addition, new studies on the reaction products of all E. coli LTs have revealed information on their preferences for non-crosslinked vs. crosslinked PG chains [11]. The three-dimensional structures of different LTs have recently been reported (Table 1) providing clues about their catalytic activity and regulation. MltE is a membrane-bound LT displaying endolytic activity that has been shown to be required for the www.sciencedirect.com

assembly of the type VI secretion system (T6SS) in enteroaggregative E. coli [26]. The structure of MltE from E. coli showed a single-domain enzyme with an extended binding groove (by inclusion of an extra a-helix) that can accommodate up to eight sugars (from 4 to +4) (vs. the six sites observed in most LTs) [27,28] (Figure 2c). Because of the active-site conformation and the absence of other structural domains, MltE can cleave in the middle of the polymeric substrate. A catalytic model was proposed to explain the experimental observation of tetrasaccharides and hexasaccharides as products [27]. The catalytic core of EtgA from enteropathogenic E. coli [29] showed features in common with both LTs and lysozymes and still awaits precise classification. A new single-domain LT from Rhodobacter sphaeroides, named SltF, involved in flagella assembly was recently reported [30] and it has been classified as a new subfamily, 1F. SltF, like MltE, was demonstrated to be an endolytic enzyme. The crystal structure of MltC from E. coli [31] revealed a bimodular enzyme featuring a novel N-terminal module (Pfam DUF3393) and a catalytic module showing a long PG-binding site (23 A˚) that is further elongated (up to 30 A˚) by the N-terminal domain enabling the accommodation of up to nine saccharides [31]. The structure not only provides an explanation for the main exolytic activity of MltC, but also for the residual endolytic activity experimentally observed [7]. The structure of SltB3 from Pseudomonas aeruginosa has been reported in its apo conformation and in complex with the substrate and the reaction products [32]. SltB3 has four domains arranged in a unique annular conformation with the polymeric linear peptidoglycan substrate threading through the opening of the annulus of the enzyme [32] (Figure 2c). Analysis of the reaction products, together with the three-dimensional structure of the complexes, provided indications about the exolytic character of the reaction and the structural plasticity of the active-site groove during catalysis [32]. The first case of allosteric regulation in LTs has recently been described. MltF from P. aeruginosa is a membranebound LT comprising two modules: an ABC-transporterlike regulatory module and a catalytic module connected by a linker region [33] (Figure 2c). The apo structure shows an inactive conformation with the active site blocked by the regulatory module and the linker region. Occupancy of the regulatory module by peptidoglycanderived peptide molecules triggers allosteric activation by a dramatic and long distance (36 A˚) conformational change. Why this regulation is required in MltF is presently unknown, however the fact that the loss of LTs MltF and Slt in P. aeruginosa increases b-lactam Current Opinion in Structural Biology 2017, 44:87–100

92 Carbohydrates

susceptibility [34] points to specific physiological role for these two proteins.

residue (F217 in Slt35 and W199 in SltB3) that would further contribute in clamping the substrate.

While LTs have been extensively described in Gramnegative bacteria, some cases have been also described for Gram-positive bacteria involved in peptidoglycan remodeling during Bacillus spore germination (SleB protein, [35]), in Staphylococcus aureus septation (IsaA and SceD proteins [36]) or in resuscitation of dormant Mycobacterium tuberculosis bacteria (Rfps proteins [37–39]). SpoIID, a new LT from a Gram-positive organisms was recently reported [40]. Its structure from Bacillus anthracis and Clostridium difficile revealed a bilobal protein (Figure 2c) with a deep groove that is able to accommodate up to six sugar residues (Figure 4f). SpoIID does not show sequence similarity to any of the characterized murein hydrolases and thus has been proposed to represent the founding member of a new LT family, the so-called SpoIID-family or family 5. Several structural studies have elucidated the structure of the catalytic core from resuscitation promoting factors B and E (RpfB, RpfE) [37–39]. According to CAZy classification, these enzymes belong to GH23 family. Their catalytic core is a reduced version of the common architecture found in family 1, and also presents differences in the residues involved in substrate recognition (Figure 4d).

Similar to families 1 and 3, family 4 (belonging to GH23 family, Table 1) displays the catalytic residue (E115 in Gp144) located at the end of an a-helix (Figure 4e) and forms a H-bond with Tyr residue (Y197). A histidine residue (H200) is also making a H-bond with the catalytic residue and mimicks the stabilization of the sugar acetamide group at position 2 found in families 1 and 3. It is worth mentioning that very few polar interactions are observed with the glycan chain (Figure 4e). LTs belonging to family 5 have a groove able to accommodate at least seven saccharides (Figure 4f). A tyrosine residue (Y121 in SpoIID), together with a histidine (H318 in SpoIID), contributes to the stabilization of the sugar moiety at position 2. In the vicinity of position 2, there is Zn+2 ion that presumably plays a structural role [40].

An important body of structural information is now available for LTs of families 1 and 3 (less abundant for the other families) in complex with different substrate analogues (Figure 4a–f) allowing the identification of some conserved features. Whereas most LTs would require peptide stems for catalysis, very few structural data are available for peptide-stem recognition in LTs. In contrast, glycan recognition is better understood. Families 1 and 3 show a substrate-binding site lined by several conserved Tyr residues (Y120, Y146 and Y192 in MltE) playing an essential role in stabilization of the substrate: Y120 establishes polar interactions through its OH group with NAM( 3) and NAG( 2) sugars, while Y146 provides van der Waals interactions stabilizing the acetamide group of NAG( 2) and allowing proper orientation of the NAM( 1). The Y192 residue makes polar interactions with the catalytic residue (E64 in MltE) or with NAM ( 1) and NAG(+1) depending on the specific complex, thus pointing to its dynamic behavior during catalysis. An extra Tyr residue is also observed in family 3 (Y234 in Slt35) that could play a role in the stabilization of the glycan chain through polar interactions with sugar moieties at positions 3 and 4 (Figure 4b). The glycan chain is further stabilized by different polar and acidic residues of the protein (Figure 4). It is worth mentioning the presence of a structurally conserved loop (residues 66– 85 in MltE), which exhibits a certain degree of mobility, to clamp the substrate at the reaction site (positions 1 and +1). In family 3, the top of the loop has an aromatic Current Opinion in Structural Biology 2017, 44:87–100

Family 2 LT (exemplified by E. coli MltA) exhibits a completely different fold and binding site. The catalytic domain folds as a double-c b-barrel similarly to the catalytic domain of the family GH45 endoglucanase V from Humicola insolens, an aspartate as the catalytic residue (D308, Figure 4c) and a strong preference for naked glycan strands. The sugar moeity at the 2 position is stabilized by stacking interaction with a tyrosine (Y180 in MltA) and other aromatic and polar residues contribute to substrate stabilization (Figure 4c). Peptidoglycan amidases

While PG amidases usually perform similar functions, they exhibit rather low amino acid conservation. This sequence diversity could be related to differential recognition of substrates and/or different interaction mechanisms with additional domains [41]. They play important roles in cell division (AmiA, AmiB and AmiC of E. coli, AmiE in Staphylococcus epidermidis) [42,43], in antibiotic resistance (i.e., AmpDh2 and AmpDh3 of P. aeruginosa) [44], in PG recycling (i.e., AmpD of Gram-negative bacteria) [45], or in bacterial autolysis and fratricidal lysis (i.e., LytA from S. pneumoniae) [46]. According to the Pfam classification, the catalytic domains of AmiA (S. aureus), LytA (S. pneumoniae), AmiD (E. coli), AmpD (Citrobacter freundii), AmpDh2 (P. aeruginosa) and AmpDh3 (P. aeruginosa) belong to the amidase 2 family (PF01510), while AmiA, AmiB and AmiC from E. coli belong to the amidase 3 family (PF01520) (Table 1). All of them are zinc-ion-dependent amidases showing the catalytic Zn+2 cation located at the intersection of the glycanbinding site and the peptide-binding site. Recent structural work revealed important structural features dealing with the specificity and regulation of these enzymes. The crystal structure of catalytic domain of S. aureus AmiA in complex with a PG ligand (NAM-tetrapeptide) www.sciencedirect.com

Structure and function of bacterial peptidoglycan hydrolases Alcorlo et al. 93

Figure 3

(a)

Amidases

(c)

Carboxypeptidases

VanXYc (E. gallinarum)

AmpD (C. freundii)

LytAcat (S. pneumoniae) AmpDh2 (P. aeruginosa)

(b)

Endopeptidases

VanXYg (E. faecalis)

AmiA (S. aureus)

Csd6 (H. pylori)

AmpDh3 (P. aeruginosa)

AmiC (E. coli)

PcsB (S. pneumoniae)

DacB (S. pneumoniae)

Current Opinion in Structural Biology

Overall structures of peptidases involved in PG hydrolysis. (a) Crystal structure of PG amidases AmiA in complex with a PG-derived ligand (PDB code: 4KNL, [47]); AmiC (PDB code 4BIN, [73]); AmpD in complex with anhNAM pentapeptide (PDB code 2Y2B, [45]); AmpDh2 dimer (functional state) in complex with (NAG-NAM)2 pentapeptide (combined structures of AmpDh2: (NAG-NAM)2 complex PDB code 4BPA ([50]) and AmpDh2: pentapeptide complex PDB code 4BOL, [50]); AmpDh3-tetramer (functional state) in complex with (NAG-NAM(pentapeptide))2 (PDB code 4BXD, [51]) and catalytic domain of LytA in complex with (NAG-NAM-pentapeptide)2 (PDB code 5CTV, [48]). Catalytic domains colored in light brown and remaining domains in blue. (b) Crystal structure of endopeptidase PcsB (PDB code 4CGK, [62]). (c) Crystal structure of carboxypeptidases VanXYc in complex with D-Ala (PDB code 4MUT, [60]), VanXYg in complex with D-Ala-D-Ala (PDB code 4OAK, [60]), Csd6 in complex with D-Ala (PDB code: 4Y4V, [61]) and DacB in complex with NAM-tetrapeptide (PDB code 4OXD, [57]). Color code as in Figure 2.

has been recently reported [47], revealing that ligand recognition is performed mostly by the peptide and also showing the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond. The crystal structure of the closely-related pneumococcal catalytic domain of LytA in complex with a synthetic cellwall based PG fragment (NAG-NAM-pentapeptide)2 (Figure 3a) revealed a similar binding site like in AmiA with strong interaction with the four saccharide units [48]. Indeed, structural superimposition of LytA with AmiA (Figure 4g) reveals that both enzymes have a Y-shaped substrate-binding crevice (allowing peptide cleavage at NAM in position 2) and conserved glycaninteracting residues (T28, E38, Y41 and H147; LytA numbering). The glycan chain is stabilized through hydrogen bond, van der Waals and CH-p interactions [48]. Most of these glycan-interacting residues have been reported as being pivotal for enzymatic activity in AmiA from S. aureus [47] and AmiE from S. epidermidis www.sciencedirect.com

[43]. Peptide-stem recognition is also mainly conserved in AmiA and LytA (Figure 4g). The crystal structures of some members of the amidase 2 family of G( ) bacteria AmiD (E. coli) [49], AmpDh2 (P. aeruginosa) [50] and AmpDh3 (P. aeruginosa) [51] have been reported in their apo form and in complex with synthetic turnover products (Figure 3a). They show a flexible N-terminal extension involved in membrane anchoring (in AmiD and AmpDh2) and in oligomerization (in the three enzymes), and a catalytic domain and a Cterminal domain involved in PG binding (PG_binding_1, Pfam PF01471). Structural superposition reveals a conserved active site consisting of a L-shaped cavity (allowing recognition of, at least, four sugar units with peptide cleavage at NAM in position 4) with a segment for the glycan binding, another for the peptide-stem binding and the catalytic Zn+2 ion located at the junction of the two (Figure 4h). Conserved residues line the grooves and Current Opinion in Structural Biology 2017, 44:87–100

94 Carbohydrates

Figure 4

Lytic transglycosylases Family 1 (a)

Family 3 (b)

RfpB (d)

Family 2 (c)

Family 4 (e)

Family 5 (f)

PG Amidases Periplasmic G(–) PG amidases

G(+) PG amidases (g)

(h)

Cytosolic G(–) PG amidases (i)

Current Opinion in Structural Biology

PG recognition in LTs and PG amidases. Conserved key residues in PG recognition are depicted in sticks. Color code for the ligand is the same as in Figures 1–3. Zn2+ cation is represented as spheres. (a) Structural superposition for family 1 LTs. The catalytic domain of Slt70 (purple) in complex with 1,6 anhydromuropeptide and bulgecin (PDB code 1QSA, [74]), MltC (salmon) in complex with tetrasaccharide (PDB code 4CFP, [31]), MltE (green) in complex with chitopentaose (PDB code 4HJI, [75]) and MltF (gray) in complex with bulgecin and muropeptide (PDB code 4P0G, Redden E, Thunnissen AMWH, to be published). For clarity reasons, only chitopentaose is shown. Residue numbering stands for MltE. (b) Structural superposition for family 3 LTs. Catalytic module of SltB3 (blue) in complex with NAG-NAM pentapeptide (PDB code 5AO8, [32]) and Slt35 (orange) in complex with two murodipeptides (PDB code 1D0K, [76]). NAG-NAM-L-Ala-D-Glu murodipeptide in complex with Slt35 is shown in sticks. Residue numbering for Slt35. Chitopentaose ligand from MltE complex (PDB code 4HJI, [75]) superimposed to indicate 3 and 4 positions. (c) MltA protein in complex with chitohexaose (PDB code 2PI8, [77]). (d) Structural superposition between RfpE (pink) in complex with triacetyl chitotriose (PDB code 4KDM, [78]) and MltE (green) (PDB code 4HJI, [75]). Triacetyl chitotriose as green sticks. Residue numbering for RfpB. (e) PG-binding site of Gp144 (yellow) in complex with chitotetraose, in green (PDB code 3BKV, [79]). (f). Structural superposition of SpoIIA from C. difficile (yellow) in complex with triacetyl chitotriose (green sticks) (PDB code 5I1T, [40]) and SpoIIA from B. anthracis (brown) (PDB code 4RWR, [40]). (g) Substrate recognition in G(+) PG amidases. Structural superimposition of LytA (molecular surface and ribbon in wheat) in complex with (NAG-NAM(pentapeptide))2 (PDB code 5CTV, [48]) and AmiA (slate ribbon) in complex with Mur-tetrapeptide (PDB code 4KNL, [47]). NAGNAM(pentapeptide)2 and conserved residues depicted as sticks. (h) Substrate recognition in periplasmic G( ) PG amidases. AmiD from E. coli

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Structure and function of bacterial peptidoglycan hydrolases Alcorlo et al. 95

participate in peptide-binding (W85, R160, E106; AmpDh2 numbering) and glycan-binding (Y43, Y112, W121, F241, and K161; AmpDh2 numbering). A b hairpin containing aromatic residues (Y112 and W121 in AmpDh2) (Figure 4h) is involved in clamping the carbohydrate chain [50]. Interestingly, the oligomeric arrangement is also playing an essential role in their activities as their active sites are extended in the oligomer. Thus, dimeric membrane-bound AmpDh2 would allow hydrolysis of external cross- and noncross-linked PG chains, and tetrameric soluble AmpDh3, would allow multivalent anchoring onto the cell wall and further hydrolysis of inner cross- and non-crosslinked PG chains [50,51]. The solution and crystal structures of cytosolic AmpD from C. freundii [52,45] (Figure 3a), also from the amidase 2 family, revealed the existence of a closed and open conformation for the enzyme. AmpD activation involves large conformational rearrangements (up to 17 A˚) and provokes both the formation of a substrate-binding site in which the peptide is strongly stabilized by three saltbridge interactions (by R71, R107 and R161) and some polar and hydrophobic contacts with the backbone of the peptide-stem (especially through W95) (Figure 4i). Upon activation AmpD builds a small glycan-binding site (compared with periplasmic amidases) in which Y63 is playing a critical role in anhNAM specificity (Figure 4i) [45]. Enzymes processing bacterial peptide stems: carboxypeptidases and endopeptidases

Carboxypeptidases have a crucial role in bacterial cell wall biosynthesis and recycling. Importantly, the re-use of peptides in the murein synthesis pathway is limited to tripeptides and actually, an L,D-carboxypeptidase activity is required for the re-using the cell wall components [53]. Several L,D-carboxypeptidase genes have been identified recently, including DacB (Figure 3c) from Lactococcus lactis and S. pneumoniae [54,55]. DacB from S. pneumoniae is a lipoprotein with an N-terminal region composed of a transmembrane helix, a linker region and a globular catalytic domain that contains the Zn2+-dependent catalytic machinery (Figure 3c). The crystal structures of DacB alone and in complex with a muropeptide have been reported providing details about key amino acids to substrate recognition and a potential groove through which crosslinked PG peptides may pass during proteolysis [56,57]. Vancomycin resistance in Gram-positive bacteria is caused by the D,D-peptidases VanX [58] and VanY [59] that hydrolyses dipeptide (D-Ala-D-Ala) and

NAM-pentapeptide (N-acetyl-muramy-L-Ala-D-g-Glu-LLys-D-Ala-D-Ala) respectively. VanXYc is a Zn2+-dependent D,D-carboxypeptidase (Figure 3c), which hydrolyses both dipeptides and pentapeptides [60 ]. The structure of VanXYc reveals the molecular basis of their specificity towards vancomycine-susceptible precursors and explains the dual function of VanXYc [60] (Figure 3c). The crystal structure of Csd6, one of the cell shape-determining proteins in Helicobacter pylori, is comprised of three domains, and was proposed to constitute a new family of L,D-carboxypeptidases (Figure 3c) [61]. The PG endopeptidases are involved in multiple processes considered essential for the bacteria including cell growth and division [62]. Some of these proteins are members of the so-called CHAP superfamily that includes CHAP and NlpC/p60 families of peptidases. Data from the literature indicate that NlpC/p60 and CHAP proteins cleave distinct peptidoglycan bonds [63]. CHAP and NlpC/p60 domains are similar in size (110–140 residues), show a papain-like fold with a catalytic cysteine residue involved in a nucleophilic-attack mechanism [64–66]. There are several available structures corresponding to CHAP and NlpC/p60 domains (for recent examples see [67–71]). Recently, three putative NlpC/P60 cell wall hydrolases – containing SH3 domains – were biochemically and structurally characterized. These enzymes all have g-D-Glu-m-DAP cysteine amidase (or DL-endopeptidase) activities but with different substrate specificities. Structure analysis showed the involvement of SH3b domains in the modulation of substrate specificity [70]. Of particular interest is PcsB from S. pneumoniae (Figure 3b), a modular protein involved in cell division which contains a CHAP domain and a coiled-coil domain [62]. The protein adopts a dimeric state in which the V-shaped coiled-coil domain of each monomer acts as a pair of molecular tweezers locking the catalytic CHAP domain of each partner in an inactive configuration (Figure 3b). The release of the catalytic domains would require an ATP-driven conformational change in the transmembrane FtsEX complex, ensuring that the enzyme digests the cell wall only at the right time in the right place. Common principles of PG recognition and regulatory mechanisms

Whereas principles of PG recognition and regulatory mechanisms of the enzymes involved in its metabolism are still largely unknown, the growing available structural and biochemical information is starting to shed light on how these processes may occur. Some key aspects on PG recognition for LTs (families 1 and 3) and PG amidases

(Figure 4 Legend Continued) (pink) in complex with NAM-L-Ala-D-g-Glu-L-Lys (PDB code 3D2Z, [49]), AmpDh2 from P. aeruginosa (molecular surface and ribbon in wheat) in complex with (NAG-NAM)2 (PDB code 4BPA, [50]) and with pentapeptide (PDB code 4BOL, [50]) both represented in sticks, and AmpDh3 (blue ribbon) from P. aeruginosa. (i) PG recognition in cytosolic G( ) PG amidases. Active site of AmpD from C. freundii in complex with reaction products 1,6-anhydro-N-acetylmuramyl and L-Ala-D-g-Glu-mDAP (PDB code 2Y2B, [45]). www.sciencedirect.com

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96 Carbohydrates

Figure 5

(a)

Lytic Transglycosylases

Catalytic domain

Membrane-bound vs soluble • Access to exposed vs inner glycan chains

S-acylation

Additional domains • 6 subsites at active site • Tyr involved in glycan recognition and catalysis

S

• Extension of the active site

O

• Allosteric regulation of lytic activity

• Clamping loop

• In vivo substrate specificity (crosslinked vs non-crosslinked)

• Additional structural elements (α-helik, Ca2+-binding loop...) –4

–3

–2

–1

+1

• Interaction with protein partners

+2

• PG-binding • Other

(b)

PG Amidases Membrane-bound vs soluble • Access to exposed vs inner PG chains

Catalytic Domain

Oligomerization

• Zn2+-dependent activity • Activation mechanism (AmpD)

S-acylation S

O

S

• Recognition anhNAM (AmpD)

O

• In vivo substrate specificity (crosslinked vs non-crosslinked)

• Extra elements: Clamping loop.. • Multi punctual PG attachment

• Active site plasticity Zn2+

Sp-LytA, Sa-AmiA

Cf-AmpD Ec-AmiD, Pa-AmpDh2 Pa-AmpDh3

Additional Domain • Extension of the glycan-binding site

Tyr residue Catalytic residue Clamping loop Catalytic Zn2+ Current Opinion in Structural Biology

Key aspects for PG recognition by LTs (families 1 and 3) and PG amidases (amidase 2 family). (a) Schematic representation of the domain composition of a prototypic LT. (b) Elements modulating PG binding and hydrolysis in PG amidases. Relevant features involved in PG recognition and in the regulation of the enzyme activity have been highlighted with arrows.

(amidase 2 family) for which more information is now available, are summarized in Figure 5. As PG hydrolases must be directed to the appropriate subcellular localization to ensure proper function, a general characteristic of these proteins is the presence of one or more of the cell wall-binding domains recognizing a specific fragment of the cell wall. Such is the case of SPOR (PF05036), LysM (PF01476), SH3b (PF08460), PG-binding domain type 1 (PF01471), DUF3393 (PF11873) or WxL (PF13731) domains. In many cases, the specific target of these domains has not been identified yet, although the SH3b domain specifically binds to the penta-glycine Current Opinion in Structural Biology 2017, 44:87–100

cross-bridge in S. aureus PG and LysM domain recognizes the NAG-X-NAG motif of PG [72]. Another factor affecting the subcellular localization pattern in bacteria is the covalent attachment of fatty acids to a protein (acylation) that serves to anchor it to the outer membrane. In LTs (Figure 5a), membrane anchoring restricts the substrate to glycan chains that are more exposed in the PG mesh, while soluble LTs can reach inner regions in the PG. The catalytic module adopts the goose-type lysozyme fold in most of LTs (families 1, 3 and 4); for instance, eight out of eleven LTs in P. aeruginosa exhibit www.sciencedirect.com

Structure and function of bacterial peptidoglycan hydrolases Alcorlo et al. 97

such a fold. The catalytic site is a long groove in which six sugar residues are stabilized (from 4 to +2 positions) (Figures 4 and 5a). The saccharides are recognized by some conserved Tyr residues (Figure 4) and further stabilized through polar interactions. It is worth mentioning the presence of a loop clamping the substrate at the reaction site (positions 1 and +1) whose flexibility would be important to accommodate at the 1 position both the NAM ring or the bulky anhNAM during the course of the reaction. Whereas the peptide-stem is required for activity in many LTs, no clear structural data are currently available regarding the specific peptide-binding sites decorating the active site. Additional structural elements are observed in the catalytic modules, such as an a-helix (e.g., MltE) which extends of the active site groove or a Ca2+-binding loop (e.g., family 3) which is likely involved in protein-protein interactions. The presence of additional domains modulate/expand the functionalities of LTs. In fact, most of LTs exhibit one or more additional domains (e.g., one or two in family 1, and two or three in family 3) playing important roles in substrate specificity, for instance endolytic vs. exolytic cleavage (e.g., Slt70) or discrimination between crosslinked or non-crosslinked substrate (e.g., SltB3). Additional domains may also play roles in extending the active site groove (e.g., MltC), in the interaction with other protein partners as part of macromolecular complexes regulating PG remodeling (e.g., Slt70) or even in the allosteric regulation of their lytic activity (e.g., MltF). PG amidases also provide a very interesting demonstration of the plasticity of the PG hydrolases (Figure 5). Amidase 2 family of PG amidases are distributed in both Gram-negative and Gram-positive bacteria (Figure 4). While all share a common fold, a catalytic Zn2+-dependent machinery and a quite well defined peptide-binding site, they possess very different specificity and regulation mechanisms. The anchorage to the membrane (e.g., AmpDh2) or the soluble state (e.g., AmpDh3) determines whether the enzyme degrades exposed or inner PG chains respectively. The catalytic domain shows a great plasticity at the active site groove, for instance while both G(+)-exposed and G( )-periplasmic PG amidases recognize a tetrasaccharide, the G(+) enzymes exhibit a Y-shaped active site that serves to hydrolyze peptidestem at position 2, and G( ) enzymes possess an L-shaped groove cleaving the peptide stem at position 4 (Figures 4 and 5). Interestingly, the cytosolic paralogue in G( ), AmpD, presents additional structural elements which afford a conformational change from an inactive to an active state. This activation mechanism would shelter the cytoplasm molecules from adventitious proteolytic activities of AmpD. An additional domain has also been found for G( ) periplasmic enzymes that acts to extend the glycan-binding site. This additional domain (PF01471) is also found in LTs from families 3 and 4. Oligomerization is another factor that plays a key role www.sciencedirect.com

in the activity of G( ) periplasmic enzymes allowing multi punctual PG attachment and expanding degradation to both crosslinked and non-crosslinked PG chains.

Conclusions Recent studies on the structural biology of PG hydrolases are revealing a great diversity in the way these enzymes, critical for bacterial survival, are regulated and perform their catalytic activities. It is worth noting that despite the strict conservation of bacterial PG carbohydrate composition, bacterial glycosidases have evolved to possess structural elements (loops, domains) that afford regulation and carbohydrate recognition at specific positions of the PG chain or presenting specific chemical modifications or temporal regulation. In the same sense, PG amidases provide a plethora of tactics (variation in the shape of the active-site groove, differences in oligomerization state, presence of activation mechanism, specific recognition of the peptide-stem or the glycan chain) in order to perform their different functions. The redundancy of the PG hydrolases and subtle differences in substrate specificities between the enzymes of each family reflect a large variety of as-yet-unidentified specific physiological functions. The elucidation of the regulatory mechanisms that rule the different PG hydrolases functionalities (i.e., by complexing with other proteins of the biosynthetic machinery, by subcellular localization dependency on modification of the PG substrate or by interacting with specific proteinaceous regulators), is one of the future challenges. Considering the relevance of bacterial cell wall as a major target for antibiotics, the indepth knowledge of the molecular mechanisms orchestrating cell-wall remodeling processes will be essential in the fight against the increasing problem of multidrug resistance. In addition, the identification and classification of novel PG hydrolases from complete genomic or metagenomic ORFs will provide new candidates with the potential to act as antibacterial agents. Future three-dimensional structure determination of PG glycosyl hydrolases in complex with different ligands and regulatory partners will provide valuable information for the elucidation of the molecular mechanisms underlying substrate recognition.

Acknowledgements We thank Douglas Laurents for critical reading of the manuscript. We apologize to all the researchers whose papers could not be cited in this review because of the limited space. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU201459389-P) and from Community of Madrid (S2010/BMD-2457).

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