Mohammed Terrak

The penicillin‐binding proteins: structure and role in peptidoglycan biosynthesis

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.

Structural basis of protein phosphatase 1 regulation

The coordinated and reciprocal action of serine/threonine (Ser/Thr) protein kinases and phosphatases produces transient phosphorylation, a fundamental regulatory mechanism for many biological processes. The human genome encodes a far greater number of Ser/Thr protein kinases than of phosphatases. Protein phosphatase 1 (PP1), in particular, is ubiquitously distributed and regulates a broad range of cellular functions, including glycogen metabolism, cell-cycle progression and muscle relaxation. PP1 has evolved effective catalytic machinery but lacks substrate specificity. Substrate specificity is conferred upon PP1 through interactions with a large number of regulatory subunits. The regulatory subunits are generally unrelated, but most possess the RVxF motif, a canonical PP1-binding sequence. Here we reveal the crystal structure at 2.7 Å resolution of the complex between PP1 and a 34-kDa N-terminal domain of the myosin phosphatase targeting subunit MYPT1. MYPT1 is the protein that regulates PP1 function in smooth muscle relaxation. Structural elements amino- and carboxy-terminal to the RVxF motif of MYPT1 are positioned in a way that leads to a pronounced reshaping of the catalytic cleft of PP1, contributing to the increased myosin specificity of this complex. The structure has general implications for the control of PP1 activity by other regulatory subunits.

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan‐polymerizing penicillin‐binding protein 1b of Escherichia coli

The penicillin‐binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid‐transported N‐acetyl glucosaminyl‐β‐1,4‐N‐acetylmuramoyl‐l‐alanyl‐γ‐d‐glutamyl‐(l)‐meso‐diaminopimelyl‐(l)‐d‐alanyl‐d‐alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46‐N844) PBP1bγ. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46‐N844) PBP1bγ possesses an amino‐terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an ≅ 100‐amino‐acid insert, to a D198‐G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of ≅ 39 000 M−1 s−1. Glu‐233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu‐233 γ‐COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4‐OH group of a nucleophile N‐acetylglucosamine. Asp‐234 of motif 1 or Glu‐290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4‐OH group of the N‐acetylglucosamine. In turn, Tyr‐310 of motif 4 is an important component of the amino acid sequence‐folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447‐N844 acyl transferase module, which possesses the catalytic centre‐defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active‐site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser‐510‐assisted double‐proton shuttle are glycan strands substituted by cross‐linked tetrapeptide–pentapeptide and tetrapeptide–tetrapeptide dimers and uncross‐linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on d‐alanyl‐d‐alanine‐terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross‐linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.

The essential cell division protein FtsN interacts with the murein (peptidoglycan) synthase PBP1B in Escherichia coli

Bacterial cell division requires the coordinated action of cell division proteins and murein (peptidoglycan) synthases. Interactions involving the essential cell division protein FtsN and murein synthases were studied by affinity chromatography with membrane fraction. The murein synthases PBP1A, PBP1B, and PBP3 had an affinity to immobilized FtsN. FtsN and PBP3, but not PBP1A, showed an affinity to immobilized PBP1B. The direct interaction between FtsN and PBP1B was confirmed by pulldown experiments and surface plasmon resonance. The interaction was also detected by bacterial two-hybrid analysis. FtsN and PBP1B could be cross-linked in intact cells of the wild type and in cells depleted of PBP3 or FtsW. FtsN stimulated the in vitro murein synthesis activities of PBP1B. Thus, FtsN could have a role in controlling or modulating the activity of PBP1B during cell division in Escherichia coli.

Two distinct myosin light chain structures are induced by specific variations within the bound IQ motifs—functional implications

IQ motifs are widespread in nature. Mlc1p is a calmodulin‐like myosin light chain that binds to IQ motifs of a class V myosin, Myo2p, and an IQGAP‐related protein, Iqg1p, playing a role in polarized growth and cytokinesis in Saccharomyces cerevisiae. The crystal structures of Mlc1p bound to IQ2 and IQ4 of Myo2p differ dramatically. When bound to IQ2, Mlc1p adopts a compact conformation in which both the N‐ and C‐lobes interact with the IQ motif. However, in the complex with IQ4, the N‐lobe no longer interacts with the IQ motif, resulting in an extended conformation of Mlc1p. The two light chain structures relate to two distinct subfamilies of IQ motifs, one of which does not interact with the N‐lobes of calmodulin‐like light chains. The correlation between light chain structure and IQ sequence is demonstrated further by sedimentation velocity analysis of complexes of Mlc1p with IQ motifs from Myo2p and Iqg1p. The resulting ‘free’ N‐lobes of myosin light chains in the extended conformation could mediate the formation of ternary complexes during protein localization and/or partner recruitment.

The Monofunctional Glycosyltransferase of Escherichia coli Localizes to the Cell Division Site and Interacts with Penicillin-Binding Protein 3, FtsW, and FtsN

The monofunctional peptidoglycan glycosyltransferase (MtgA) catalyzes glycan chain elongation of the bacterial cell wall. Here we show that MtgA localizes at the division site of Escherichia coli cells that are deficient in PBP1b and produce a thermosensitive PBP1a and is able to interact with three constituents of the divisome, PBP3, FtsW, and FtsN, suggesting that MtgA may play a role in peptidoglycan assembly during the cell cycle in collaboration with other proteins.

Kinetic characterization of the monofunctional glycosyltransferase from Staphylococcus aureus

The glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs (MGTs) belong to the GT51 family in the sequence-based classification of GTs. They both possess five conserved motifs and use lipid II precursor (undecaprenyl-pyrophosphate-N-acetylglucosaminyl-N-acetylmuramoyl- pentapeptide) to synthesize the glycan chain of the bacterial wall peptidoglycan. MGTs appear to be dispensable for growth of some bacteria in vitro. However, new evidence shows that they may be essential for the infection process and development of pathogenic bacteria in their hosts. Only a small number of class A PBPs have been characterized so far, and no kinetic data are available on MGTs. In this study, we present the principal enzymatic properties of the Staphylococcus aureus MGT. The enzyme catalyzes glycan chain polymerization with an efficiency of approximately 5,800 M(-1) s(-1) and has a pH optimum of 7.5, and its activity requires metal ions with a maximum observed in the presence of Mn2+. The properties of S. aureus MGT are distinct from those of S. aureus PBP2 and Escherichia coli MGT, but they are similar to those of E. coli PBP1b. We examined the role of the conserved Glu100 of S. aureus MGT (equivalent to the proposed catalytic Glu233 of E. coli PBP1b) by site-directed mutagenesis. The Glu100Gln mutation results in a drastic loss of GT activity. This shows that Glu100 is also critical for catalysis in S. aureus MGT and confirms that the conserved glutamate of the first motif EDXXFXX(H/N)X(G/A) is likely the key catalytic residue in the GT51 active site.

Monofunctional transglycosylases are not essential for Staphylococcus aureus cell wall synthesis.

The polymerization of peptidoglycan is the result of two types of enzymatic activities: transglycosylation, the formation of linear glycan chains, and transpeptidation, the formation of peptide cross-bridges between the glycan strands. Staphylococcus aureus has four penicillin binding proteins (PBP1 to PBP4) with transpeptidation activity, one of which, PBP2, is a bifunctional enzyme that is also capable of catalyzing transglycosylation reactions. Additionally, two monofunctional transglycosylases have been reported in S. aureus: MGT, which has been shown to have in vitro transglycosylase activity, and a second putative transglycosylase, SgtA, identified only by sequence analysis. We have now shown that purified SgtA has in vitro transglycosylase activity and that both MGT and SgtA are not essential in S. aureus. However, in the absence of PBP2 transglycosylase activity, MGT but not SgtA becomes essential for cell viability. This indicates that S. aureus cells require one transglycosylase for survival, either PBP2 or MGT, both of which can act as the sole synthetic transglycosylase for cell wall synthesis. We have also shown that both MGT and SgtA interact with PBP2 and other enzymes involved in cell wall synthesis in a bacterial two-hybrid assay, suggesting that these enzymes may work in collaboration as part of a larger, as-yet-uncharacterized cell wall-synthetic complex.

Characterization of the Bifunctional Glycosyltransferase/Acyltransferase Penicillin-Binding Protein 4 of Listeria monocytogenes

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.

Importance of the Conserved Residues in the Peptidoglycan Glycosyltransferase Module of the Class A Penicillin-binding Protein 1b of Escherichia coli

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage λ lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237 and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to maintain the structure of the active site and the positioning of the catalytic Glu233.