structural and mechanistic observations of the fosx class

Laura Busenlehner and the rest of the Armstrong lab for teaching me how to be a good scientist. Fosfomycin, (1R-2S)-epoxypropylphosphonic acid, was first isolated from cultures of Streptomyces in 1969 as a broad-spectrum antibiotic against Gram-positive and Gram-negative bacteria (Figure 2) (3,4). The compound was found to interfere with the first step of cell wall biosynthesis by inhibiting the reaction between UDP-GlcNAc) and PEP catalyzed by the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA). Members of the VOC group consist of paired βαβββ motifs arranged in different orientations to form a metal ion binding site ( Table 2 ) ( 19 ).

Although each of the classes confers resistance to fosfomycin, they do so with different substrates and metal ion dependencies (Figure 5). Hydrogen/deuterium exchange mass spectrometry (HXMS) is a powerful technique that uses solvent accessibility of amide hydrogen atoms along a protein backbone to predict the solution structure of the protein. Linderstrøm-Lang first conceptualized the idea that the rate of amide hydrogen exchange with solvent molecules is a reflection of protein stiffness.

The fastest exchange is called "EX2" and occurs when protein refolding occurs faster than the intrinsic rate of exchange of hydrogen for deuterium. Pepsin digestions with a ratio of 1:1.5 FosX:pepsin w/w were performed under the quenching conditions of the HXMS experiment. Peptide identities were determined using ExPASy-PeptideMass software (35) and confirmed by MS/MS analysis of individual peptide sequencing against theoretical fragmentation patterns generated by the ProteinProspector MS-Product program (36).

The pepsin map covers 85% of the protein, Aspergillus protease XIII covers 78%, and Rhizopus protease XVIII covers 69%.

Table 1. Antibiotic classes and their mechanisms of action. [Adapted from (2)].

It makes sense that this peptide near a metal-binding residue would experience a structural perturbation to accommodate the approach of the metal cation to the active site. A crystal structure of Listeria monocytogenes FosX shows that residue E126 is within coordination distance of the divalent Mn2+ cation. However, from Figure 19 and Table 7 we can clearly state that the exchange is very fast in the region around E126 - about 80-85% for Co2+ and Zn2+, 100% for Mn2+ (the program could not fit the data because the exchange was completed at 15s ), and 50% for apoenzyme (Although the best-fit lines look quite similar for the four species, the few lows at the beginning of the time course for native enzymes have been quite reproducible and should not be considered outliers).

Although we often attach little importance to regions of a protein that are not part of the catalytically active site, the data presented here suggest that these residues do in fact play a role in catalysis, despite their location several angstroms away from the center of activity. This peptide contains no metal binding or substrate recognition sites and is located furthest from the metal center than any other part of the protein. Constants for peptide 11-22 are also shown to demonstrate the reproducibility of the results from this technique.

This peptide covers the last few residues of the α-helix spanned by the aforementioned peptide 11-21, and the beginning of a large loop whose exact length is unknown due to missing electron density in the crystal structure. As is the case with 11-21, this peptide contains no functionally significant residues and is removed from the active site, yet the same exchange trend is observed.

Figure 17. HXMS backbone amide kinetic profile for peptide 117-124.

The amide protons that make up this peptide exchange significantly throughout the time course in the intermediate and slow phases, suggesting a conformational change rather than increased solvent accessibility. Several peptides spanning this region were isolated from the peptide mapping experiment, and the HXMS results from each are shown to convey reproducibility in Table 9. The exchange results for this peptide are quite dramatic; rates and amplitudes for the native enzyme, Co2+, and Zn2+ are virtually identical (they differ by <5%), whereas Mn2+-bound FosX exchange is >20% higher.

This peptide is also interesting because the only species that experiences complete exchange of both deuterons is Mn2+-bound FosX; the other three species appear to exchange only one hydrogen for deuterium. The HXMS data shown in Figure 24 and Table 12 indicate that a metal ion-dependent conformational change is occurring in this peptide.

Fast exchange with bound Mn2+ is 50% greater than with native, Co2+ or Zn2+ bound enzyme (rates of the three differ by <5%). Color-coded bars showing percentages of fast exchange for the native, Mn2+-bound, Co2+-bound, and Zn2+-bound enzyme are shown below in Figure 25. Inspection of these structures highlights that the act of Mn2+ binding increases solvent accessibility throughout the protein, as shown by abundance of red segments (indicating >80% rapid exchange).

The Co2+ structure can be considered an exchange medium, lying between the abundant fast exchange of the Mn2+ structure and the minimal fast exchange of the Zn2+ and apoenzyme structures. Since only very small percentages separate the exchange rates of Zn2+ and apoenzyme, these structures appear.

As the catalytic activity is reduced by binding to less preferred metals, the exchange rates begin to more closely resemble those of the natural enzyme. NMR spectra reveal that deuterium incorporation increases by several deuterons at both regulatory Ca2+ binding sites. Most of the protein exhibits a faster exchange upon metal binding, whereas the first ∼30 residues maintain the same level of exchange as native TnC ( 41 ).

To explain their findings, the authors theorize that the binding of Cu2+ leads to a destabilization of the protein's native state, which exhibits reduced stability. Perhaps before FosX binds to metal, hydrogen bonding forces within the protein and interactions with ordered water surrounding the protein are strong, reducing the overall flexibility of the structure. From this experiment we would be able to compare the folding of the protein/metal complex with the protein/metal/fosfomycin complex.

The following spectroscopic experiments were designed to clarify the binding saturation of FosX with its metal ion cofactors so that we could later achieve the best signal to noise possible in HXMS while still ensuring that we captured a realistic picture of the dynamics of the protein/metal structure. The fluorescence plot shown in Figure 30 reveals that although the data points appear to level off around 1-1.5 equivalents of [Mn2+]:[FosX], the shape of the curves is very different from those originating from Co2+. The reason for the sigmoid shape of the Mn2+ titration curve is unknown at this time.

A possible explanation is that the cuvette contained a competing metal species, either from inadequate acid soaking of the cuvette to remove contaminants or from a metal other than Mn2+ that was present in the Mn2+ stock used in the titration. Incubation times greater than ten minutes may lead to increased linearity of the data points. The abundance and size of colonies expressing Pseudomonas FosX were reproducible in several experiments.

To determine whether this high optimal pH is also characteristic of the Listeria enzyme, NMR experiments were performed according to the same procedure. The NMR data show that the FosX activity is nominal, and since the only peaks in the spectra are fosfomycin and its hydrolyzed product, this implies that the only change of the fosfomycin molecule is the conversion to diol. This activity represents the only known example of a C-P cleavage enzyme that is not under the control of the pho operon ( 46 ).

Figure 25. Ribbon diagrams illustrating fast exchange percentages for certain peptides selected to maximize protein coverage (clockwise from top left: native, Mn 2+ -bound, Zn 2+ -bound, Co 2+ -bound).

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