Crystal Structure of a Genomically Encoded Fosfomycin Resistance Protein (FosA) at 1.19 Å Resolution by MAD Phasing Off the L-III Edge of Tl⁺

Abstract

The fosfomycin resistance protein (FosA) catalyzes the Mn(II)- and K+-dependent addition of glutathione to the oxirane of the antibiotic fosfomycin. The crystal structure of FosA from Pseudomonas aeruginosa was solved at a resolution of 1.19 A by multiwavelength anomalous diffraction at the L-III edge of a Tl+ derivative. The structure solution took advantage of the ability of Tl+ to substitute for K+. The existence of multiple Tl sites in the asymmetric unit suggests that this may be a generally useful technique for phasing protein crystal structures. A 1.35 A resolution structure with phosphate bound in the active site shows that the Mn(II) center has a rare four-coordinate geometry. The structure of the fosfomycin complex at 1.19 A resolution indicates that the Mn(II) center is close to five-coordinate with trigonal bipyramidal geometry and a ligand set consisting of two histidines (H7 and H64) and one phosphonate oxygen occupying the equatorial sites and the carboxylate of E110 at one of the apical sites. The oxirane oxygen of the substrate is located at the other apical site but is 0.2 A beyond the average Mn-O distance for five-coordinate Mn(II). The Mn(II) center is proposed to stabilize the alkoxide in the transition state, while the nearby hydroxyl group of T9 acts as a proton donor in the reaction. The K+ ion located 6.5 A from the Mn(II) appears to help orient the substrate for nucleophilic attack.

Determinants of Regioselectivity and Chemoselectivity in Fosfomycin Resistance Protein FosA from QM/MM Calculations

“…The initial geometry was built from the X-ray structure of FosA from P. aeruginosa complexed with fosfomycin (PDB code: 1LQP, resolution 1.19 Å). 13 The dimeric form was used, and an active region for the setup was selected containing residues within 30 Å of the Mn atom of chain A. The protonation states of the titratable residues (His, Asp, and Glu) were determined on the basis of their pK a values obtained via the PROPKA 51 program (see Supporting Information for details) and verified by visual inspection of the hydrogen-bonding environment of the residues assuming pH = 8 (the working pH of FosA).…”

“…As mentioned in the Introduction, the crystal structure of FosA in complex with fosfomycin has a water-accessible open pocket. 13 However, water addition is not catalyzed by FosA, but by another enzyme (FosX). 15−17 Theoretical calculations gave very similar barriers for alcohol and thiol attack on amide, 74 and for thiolysis and alcoholysis of phosphate monoester, 75 so that one might expect similar reactivity for GSH and water attack on fosfomycin.…”

“…In the structure of FosA in complex with fosfomycin, Mn 2+ is ligated to His7 of chain A, His64 and Glu110 of chain B, oxirane oxygen, and one phosphonate oxygen of the substrate. 13 The metal is pentacoordinated in a trigonal-bipyramidal fashion, with the glutamate oxygen and the oxirane oxygen in the axial positions. EPR studies of FosA showed that the Mn 2+ ion (3d 5 ) is in the high-spin sextet state.…”

Functional Analysis of Active Site Residues of the Fosfomycin Resistance Enzyme FosA from Pseudomonas aeruginosa

“…Furthermore, there is no significant sequence similarity between FosA and the cytosolic (members of the alpha, pi, mu, sigma, theta, zeta, or kappa classes) GSTs. However, the three-dimensional structure of FosA from Pseudomonas aeruginosa exhibits similarity to members of the vicinal oxygen chelate family such as glyoxalase I and the extradiol dioxygenases (19), which are characterized by the ability to bind a metal cofactor with available sites for substrate coordination (20 -22).…”

“…The crystal structure of substrate-bound FosA showed that, like other members of the vicinal oxygen chelate family, fosfomycin binds at the active site Mn 2ϩ with distances of 2.0 Å between Mn 2ϩ and one of the phosphonate oxygens and 2.4 Å between Mn 2ϩ and the oxirane oxygen of fosfomycin (Scheme 1) (19). The crystal structure also revealed a number of amino acids (Thr ) that interact with the bound fosfomycin at the active site (Fig.…”