Abstract
In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of approximately 100 m(-1) cm(-1), which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M(S) = |+/-1/2), E/D = 0.14, g(real(x,y)) = 2.37, and g(real(z)) = 2.03. These parameters lead to the prediction that Co(II) in the enzyme is five-coordinate and that there may be at least one solvent-derived ligand. Single scattering fits of EXAFS data indicate that the metal ions in both native Zn(II)-containing and Co(II)-substituted VanX have the same coordination number and that the metal ions are coordinated by 5 nitrogen/oxygen ligands at approximately 2.0 angstroms. These data demonstrate that Co(II) (and Zn(II) from EXAFS studies) is five-coordinate in VanX in contrast to previous crystallographic studies (Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T., and Park, C. H. (1998) Mol. Cell 2, 75-84). These spectroscopic studies also demonstrate that the metal ion in Co(II)-substituted VanX when complexed with a phosphinate analog of substrate D-Ala-D-Ala is also five-coordinate.
Highlights
Vancomycin is a cup-shaped glycopeptide that has been called the “antibiotic of last resort” [1, 2] because it is often used for antibiotic-resistant bacterial infections and in patients who are allergic to penicillins
This depsipeptide is added to a sugar-linked tripeptide by MurF, producing a modified bacterial cell wall precursor that can be incorporated into the growing peptidoglycan layer by the normal bacterial enzymes [3, 5, 12]
Since VanX is essential for vancomycin resistance in bacteria, the enzyme has been targeted for drug/inhibitor design studies in hopes that an inhibitor can be given in combination with vancomycin as a treatment for vancomycin-resistant bacterial infections
Summary
Vancomycin is a cup-shaped glycopeptide that has been called the “antibiotic of last resort” [1, 2] because it is often used for antibiotic-resistant bacterial infections and in patients who are allergic to penicillins. VanA is a ligase that uses the D-lactate produced by VanH and generates a D-Ala-Dlactate depsipeptide [11, 13] This depsipeptide is added to a sugar-linked tripeptide by MurF, producing a modified bacterial cell wall precursor that can be incorporated into the growing peptidoglycan layer by the normal bacterial enzymes [3, 5, 12]. Phosphinate and phosphonate analogs of DAla-D-Ala have been shown to be inhibitors of VanX, suggesting that the enzyme utilizes a nucleophile-driven hydrolysis reaction that may proceed via a tetrahedral transition state intermediate [17, 22, 23] These structurally similar compounds exhibit Ki values varying over several orders of magnitude, and the crystal structure of VanX complexed with a phosphonate and a phosphinate demonstrated two distinct binding interactions [1]. Wu and Walsh [24] reported several
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