Structure of Escherichia coli Lytic Transglycosylase MltA with Bound Chitohexaose

Abstract

Crystal structures of an inactive mutant (D308A) of the lytic transglycosylase MltA from Escherichia coli have been determined in two different apo-forms, as well as in complex with the substrate analogue chitohexaose. The chitohexaose binds with all six saccharide residues in the active site groove, with an intact glycosidic bond at the bond cleavage center. Its binding induces a large reorientation of the two structural domains in MltA, narrowing the active site groove and allowing tight interactions of the oligosaccharide with residues from both domains. The structures identify residues in MltA with key roles in the binding and recognition of peptidoglycan and confirm that Asp-308 is the single catalytic residue, acting as a general acid/base. Moreover, the structures suggest that catalysis involves a high energy conformation of the scissile glycosidic linkage and that the putative oxocarbenium ion intermediate is stabilized by the dipole moment of a nearby ␣-helix. Lytic transglycosylases (LTs)4 are bacterial muramidases that cleave the bacterial cell wall heteropolymer peptidoglycan (murein) for turnover and recycling to facilitate cell growth and division, as well as to allow local cell wall opening without loss of integrity (1, 2). The lytic activity of these enzymes is directed toward the ␤-1,4-glycosidic bonds between N-acetylmuramic acid (MurNAc) and GlcNAc residues, the two sugar units that make up the glycan strands of peptidoglycan. Concomitant to bond cleavage, they catalyze an intramolecular transglycosylation reaction resulting in the formation of muropeptides terminated with a non-reducing 1,6-anhydromuramic acid residue.LTs are found in a wide variety of bacteria, as well as in a few bacteriophages, and are often present as several distinct enzymes within the same species. For instance, in Escherichia coli, at least six different LTs have been identified: one soluble (Slt70) and five that are outer membrane-anchored (MltAMltD, EmtA) (1, 3, 4). A comparison of genes encoding LTs allowed their classification into four families based on the identification of different sets of consensus motifs (5). LT family 1 includes proteins with amino acid sequence similarity to Slt70, MltC, MltD, and EmtA, whereas MltA and MltB are representatives of families 2 and 3, respectively. The fourth LT family comprises mostly enzymes encoded by bacteriophages.Studies of the LTs have focused on understanding their precise cellular roles, as well as on explaining their reaction mechanism. Concerning the latter interest, important insights have been obtained from the study of crystal structures of these enzymes, both in unliganded forms and in complex with various murein-derived compounds (6 -11). These investigations showed that members from LT families 1, 3, and 4 have a catalytic domain that resembles the fold of goose-type lysozyme (12). The reaction catalyzed by these enzymes most likely takes place via a general acid/base mechanism that requires participation of a single catalytic residue, i.e. an invariant glutam...

Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization

“…For MltA, a 2-step mechanism is proposed for peptidoglycan cleavage, with a single Asp residue playing the central role in both steps (21). In the first step, it acts as an acid, attacking and cleaving the glycosidic bond; and in the second step, it acts as a base, activating the hydroxyl group of MurNAc to induce the formation of the 1,6-anhydroMurNAc product.…”

“…Superposition of EXLX1 D1 with the EcMltA-chitohexaose complex (21) shows that this chitin fragment would fit very well in the shallow groove of EXLX1 D1 (Fig. 1E), and most of the interactions between chitohexaose and D1 of EcMltA are potentially conserved in EXLX1 (Fig.…”

The Dynamics of Lysozyme from Bacteriophage Lambda in Solution Probed by NMR and MD Simulations

“…In the closed form, these regions of secondary structure are lost and the loop regions extend across the active-site cleft. The experimental chemical shift, [9] RDC, 3 J HNHa and NOE data suggest that both the b-strands in the lower lip and the initial helical region at the start of helix a6, seen in the open form, are retained in solution. However, the 15 N relaxation and hydrogen exchange [12] data show that these regions display a significant degree of mobility in solution.…”

“…Similarly for Leu59 TALOS+ predicts a b-sheet conformation with a f angle of À130 AE 258 and a y angle of +121 AE 218 while in molecules A and B in the crystal structure the f, y torsion angles are À1238, +1098 and À928, À68, respectively. 3 J HNHa coupling constants have been measured for 104 residues in l lysozyme and have been compared with values predicted from the f torsion angles in molecules A and B from the 1AM7 crystal structure. Overall, better agreement is seen between the experimental coupling constants and those pre- dicted from the structure of molecule A (correlation coefficient 0.860) compared to those predicted from the structure of molecule B (correlation coefficient 0.788).…”

“…[2] In this respect l lysozyme resembles the lytic transglycosylases. [3] Similar to all lysozymes, the structure of l lysozyme consists of two domains (Figure 1). [4] Domain I contains two b-sheets formed from six b-strands and one a-helix (a2), while domain II contains four a-helices (a1, a4, a5, a6).…”