Figure 1. (a) Outline of the synthesis of the phosphinate analogue of tetraglycine. Reagents and conditions: (a) hexamethyldisilazane, 90–100 °C, 2 h; (b) hexamethyldisilazane, 80–90 °C, 3 h; (c) MeOH, yield 85% starting from 1; (d) 33% HBr/AcOH, room temperature, 2 h; (e) Cbz-Gly-OH, isobutyl chloroformate, N-methylmorpholine, anhydrous tetrahydrofuran (THF), 0 °C→room temperature, overnight; (f) bromotrimethylsilane, anhydrous CH₂Cl₂, 3d, room temperature, (g) water, then conc. HCl, then HPLC, yield 15% starting from 3. (b) Effect of the tetraglycine phosphinate analogue on LytM_185-316 activity. (c) Effect of glycine hydroxamate on LytM_185-316 activity. The assays in (b) and (c) were done with 10 μM enzyme and 0.5 mM Gly4-ethylenediamine-Dnp, which is below the K_m for this substrate, so that apparent and actual inhibition constants are related by a correction factor close to 1.

Figure 2. LytM_185-316 activity in TLC assay as a function of the pH. The assay was done with 100 μM enzyme and 4 mM Gly4-ethylenediamine-Dnp, which is above the K_m for this substrate at neutral pH. The lower spots correspond to the Gly4-ethylenediamine-Dnp substrate, the upper to the Gly2-ethylenediamine-Dnp product.

Figure 3. Effects of divalent metal ions on LytM_185-316 activity. (a) Metal reactivation of Zn²⁺-depleted LytM_185-316 was tested in a TLC-based activity assay after 30 min preincubation with the indicated metal ions at 37 °C. Spots corresponding to the substrate (lower) and the product (upper) were visualized by exposure to UV light. (b) HPLC-based assay demonstrating the quantitative measurement of Zn²⁺-depleted LytM_185-316 reactivation by selected divalent cations. The initial velocity was determined for an apo-LytM_185-316 as control, and for Zn²⁺-depleted LytM_185-316 preincubated with appropriate metal ions: Zn²⁺, Co²⁺, Mn²⁺, Cu²⁺. (c) The influence of different metal ions on Zn²⁺-containing LytM_185-316 was tested by the TLC-based activity assay.

Figure 4. Superposition of the six models of LytM_185-316 with full-length LytM (PDB accession code 1QWY). The full-length LytM is shown in grey, the models for the P3(2)21 form are in yellow (molecule A) and brown (molecule B), the models for the P2₁2₁2₁ form are in blue (molecule A) and red (molecule B), and the models for the P4₁ form are in orange (molecule A) and green (molecule B). (a) Side view to illustrate the groove, and (b) top view to show the flexibility of the groove walls. (c) Molecule B of the P4₁ form in surface representation. The triglycine of a loop of the neighbouring molecule A is shown in CPK representation. (d) Molecule A of the P3₂21 form in surface representation. The triglycine of a loop of a crystallographic symmetry mate of molecule A is shown in CPK representation. For (d), one of the two conformations of the loop and the compatible receptor conformation were chosen. The arrows in (c) and (d) indicate the direction of the polypeptide chain in the triglycine loop.

Figure 5. Binding mode comparison. (a) Stereo diagram of the interaction of the glycine-rich loop of monomer B (shown with omit map density, contoured at 1.5 σ) with the active site region of monomer A in the P4₁ crystal form. Residues of monomer B and water molecules that come closer than 3.5 Å to the three glycine residues are presented in ball-and-stick representation in standard colour-coding and without density. The black ball represents Zn²⁺. For orientation, the tartrate molecule in the active site of this crystal form has been included in the drawing. Hydrogen bonds to or from the three loop glycine residues are indicated by broken lines. All interactions in (a) result from the packing of the P4₁ crystal form and are not present in the other crystal forms. (b) Stereo diagram of the interaction of the N-terminal part of full-length LytM (shown with omit map density, contoured at 2.0 σ) with the active site region of the latent enzyme. For comparison, the same residues of the active LytM region as in (a) are drawn, irrespective of whether they interact with the N-terminal part.

Figure 6. Ball-and-stick representation of the selected residues of the active site of LytM_185-316. The electron density covering these residues is shown. The densities are 2F_o−F_c omit maps, calculated omitting all residues and ligands shown in the Figure, contoured at 1σ. The Zn²⁺ is shown as a black ball, and water molecules are shown as red balls. All Zn²⁺-coordinating interactions are represented by green, dotted lines. Hydrogen bonds are indicated by black, dotted lines. (a) The trigonal form with the monohydrogen phosphate anion (PO₄). (b) The orthorhombic form with the cacodylate anion (CAC). (c) The tetragonal form with the tartrate molecule (TAR) interacting with Zn²⁺ and other active site residues shown in stereo.

Figure 7. Distortion of the active site in the P3₂21 form. The active site Zn²⁺-coordinating residues of molecules A of the P3₂21 form (yellow) and of the P2₁2₁2₁ form (blue) were superimposed. The Figure shows selected fragments of the LytM_185-316 structure in the proximity of H260 and H291, and the loop 281–290, located above them. The hydrogen bonds that have changed upon the rearrangement are shown as black, broken lines. The distances between the centroids of H260 and H291 are indicated by broken-line-arrows. Note that H260 in the trigonal form is flipped around the C^β–C^γ bond in comparison with H260 in the orthorhombic form, which is representative of all crystal structures obtained at pH around neutral.

Data collection and refinement statistics