Diversity in Fosfomycin Resistance Proteins$ Matthew K

Perspectives in Science (2015) 4,17–23

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REVIEW Diversity in fosfomycin resistance proteins$ Matthew K. Thompsona, Mary E. Keithlyb, Gary A. Sulikowskia,b, Richard N. Armstronga,b,c,n aDepartment of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA bDepartment of Chemistry, Vanderbilt University, Nashville, TN 37235, USA cCenter for Structural Biology, Vanderbilt University, Nashville, TN 37235, USA

Received 17 March 2014; accepted 1 December 2014 Available online 25 December 2014

KEYWORDS Abstract Antibiotic; Certain strains of the soil microorganism Streptomyces produce an antibiotic, fosfomycin [(1R,2S)- Fosfomycin resis- epoxypropylphosphonic acid], which is effective against both Gram-positive and Gram-negative tance; pathogens by inhibiting the first committed step in cell-wall biosynthesis. Fosfomycin resistance Phosphonate metabo- proteins are metallo-enzymes that are known to inactivate the antibiotic by the addition of lism; nucleophiles such as water, glutathione (GSH), L-cysteine and bacillithiol (BSH) to the oxirane ring Bacillithiol-S-transfer- of the molecule. Progress in the characterisation of FosB-type fosfomycin resistance proteins found ase; Diversification of in many Gram-positive organisms has been slow. This paper provides a brief description of the function diversity of fosfomycin resistance proteins in general and, more specifically, new data characteris- ing the substrate selectivity, structure, mechanism and metal-ion dependence of FosB enzymes from pathogenic strains of Staphylococcus and Bacillus. These new findings include the high- resolution X-ray diffraction structures of FosB enzymes from Staphylococcus aureus and Bacillus cereus in various liganded states and kinetic data that suggest that Mn(II) and BSH are the preferred divalent cation and thiol substrate for the reaction, respectively. The discovery of the inhibition of the enzyme by Zn(II) led to the determination of a ternary structure of the FosB Á Zn(II) Á fosfomycin Á L-Cys complex which reveals both substrates present in a pose prior to reaction. & 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Contents

Introduction...... 18 FosA ...... 18 FosX ...... 18 FosB ...... 19

☆This article is part of an special issue entitled, "Proceedings of the Beilstein ESCEC Symposium 2013 – Celebrating the 100th Anniversary of Michaelis-Menten Kinetics''. Copyright by Beilstein-Institut www.beilstein-institut.de. nCorresponding author. E-mail address: [email protected] (R.N. Armstrong). http://dx.doi.org/10.1016/j.pisc.2014.12.004 2213-0209/& 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 18 M.K. Thompson et al.

Mechanism of fosfomycin inactivation...... 19 Moving forward...... 22 Variety is the price of life ...... 22 Conﬂict of interest ...... 22 Acknowlegdgements...... 22 References ...... 22

Introduction There are currently three distinct classes of fosfomycin resistance enzymes (FosA, FosX, and FosB) all of which Since the introduction of antibiotic compounds for the belong to the Vicinal Oxygen Chelate (VOC) superfamily of treatment of infectious diseases, mankind has enjoyed a metalloenzymes. They are structurally characterised by a βαβββ historically unprecedented increase in life expectancy and 3D domain-swapped arrangement of tandem motifs in overall quality of life. However, the pathogens that cause which both subunits of the homodimer participate in these infectious diseases adapt quickly, and as a result, coordination of a metal ion and formation of the active 2 + + many of the antibiotic compounds once used to treat sites (Figure 1). FosA enzymes are Mn and K dependent infections are quickly becoming ineffective. Currently, glutathione-S-transferases that catalyse nucleophilic addi- there exist numerous pathogenic bacteria that are resistant tion of glutathione (GSH) to carbon-1 of fosfomycin, opening fi to multiple drugs. While the exact mechanism of antibiotic the epoxide ring of the antibiotic and resulting in a modi ed resistance can vary, the primary means of bacterial survival compound with no bactericidal properties (Arca et al., are: (1) decreased cell permeability for the antibiotic, (2) 1990, 1988; Bernat et al., 1997, 1999). Genes encoding fi active efflux pumping of the antibiotic out of the cell, and FosA have been identi ed in several Gram-negative bacter- (3) destruction/inactivation of the compound via antibiotic ial species including the opportunistic human pathogen modifying enzymes (Arias and Murray, 2009). Antibiotic Pseudomonas aeruginosa (Rife et al., 2002). FosX enzymes, 2+ modifying enzymes represent the most common mode of also from Gram-negative bacterial species, are Mn depen- microbial resistance and are, therefore, obvious targets for dent hydrolases that catalyse the hydration of fosfomycin at the development of new therapeutic agents. carbon-1 forming a vicinal diol and inactivating the anti- Fosfomycin, or (1R,2S)-epoxypropylphosphonic acid, is a biotic (Fillgrove et al., 2007, 2003). FosB enzymes were broad-spectrum antibiotic produced by certain strains of the later discovered in Gram-positive organisms such as Strep- soil microorganism Streptomyces (Falagasetal.,2008; Patel tomyces aureus, Bacillus cereus, and Bacillus subtilis (Cao et al., 1997). It is effective against both Gram-negative and et al., 2001; Rigsby et al., 2005; Thompson et al., 2013) and 2+ Gram-positive bacteria, including numerous human pathogens. catalyse the Mn dependent addition of L-cysteine (L-Cys) Fosfomycin inhibits cell wall biogenesis by inactivating the or bacillithiol (BSH) to carbon-1 of fosfomycin (Thompson enzyme UDP-N-acetylglucosamine-3-enolpyruvyltransferase, et al., 2013; Lamers et al., 2012). alsoknownasMurA.TheMurAenzymecatalysesthefirst committed step in peptidoglycan biosynthesis, namely the FosA ligation of phosphoenolpyruvate to the 3'-hydroxy group of UDP-N-acetylglucosamine. This pyruvate moiety is further FosA was initially discovered as a plasmid-borne resistance reduced by UDP-N-acetylenolpyruvoylglucosamine reductase gene isolated from clinical samples of Serratia marcescens (MurB) in the presence of NADPH and ultimately provides the (García-Lobo and Ortiz 1982). This gene encodes a 16 kDa linker that bridges the glycan and peptide portions of pepti- protein that adds glutathione to carbon-1 of fosfomycin, doglycan. Fosfomycin is a phosphoenolpyruvate analogue that opening the epoxide ring as indicated in Figure 1. This irreversibly inhibits MurA by alkylating an active site cysteine modification of the antibiotic results in a compound with no residuethatactsasageneralbaseduringcatalysis(Eschenburg bactericidal properties, as it is no longer a suitable electro- et al., 2003, 2005; Krekel et al., 2000). phile for the active site cysteine residue of MurA. Thus, FosA Fosfomycin was initially characterised in 1969 (Hendlin + 2 enzymes are Mn -dependent glutathione-S-tranferases of et al., 1969; Stapley et al., 1969) and is used in the United the VOC superfamily. Their activity is significantly enhanced States under the trade name Monurol. Taken within appro- + in the presence of K or other monovalent cations (Beharry priate dosing recommendations, the drug is well tolerated, and Palzkill 2005). Genes encoding FosA enzymes have been has a low incidence of harmful side effects and is eliminated identified in other Gram-negative bacterial species includ- in its unmetabolised, active form (Patel et al., 1997). It is ing the opportunistic human pathogens Pseudomonas aeru- most often prescribed as a single three gram oral dose for ginosa (Rigsby et al., 2007), Klebsiella pneumoniae and the treatment of certain urinary tract infections. The large Acinetobacter baumannii. dose results in very high concentrations of the active drug in the urine and thus circumvents two of the primary bacterial resistance mechanisms: decreased cell permeability and FosX active efflux pumping. When combined with other antibacterial drugs, it has been effective against various antibiotic- A probe of nucleotide sequence databases revealed another resistant strains. Shortly after the introduction of fosfomy- class of fosfomycin resistance enzymes, termed FosX cin, however, bacterial strains containing fosfomycin mod- (Fillgrove et al., 2007). The FosX enzymes share 30–35% ifying enzymes began to emerge. nucleotide sequence identity with the FosA enzymes, but Diversity in fosfomycin resistance proteins 19

Figure 1 Top: Overall structures of FosA (PDB 1LQP) (Rife et al., 2002), FosB (PDB 4JH6) (Thompson et al., 2013), and FosX (PDB 1R9C) (Fillgrove et al., 2003). Bottom: Reactions catalysed by fosfomycin resistance enzymes. catalyse the hydration of fosfomycin at carbon-1, as shown aureus and Deinococcus radiodurans (Figure 2)(Newton in Figure 1. The hydrolase reaction opens the epoxide ring et al., 2009). Similar to the function of mycothiol in and forms a vicinal diol with no bactericidal properties in mycobacteria, BSH serves as a substitute for glutathione the same manner as FosA. FosX enzymes do not require in Gram-positive organisms (Gaballa et al., 2010). Recent monovalent cations for optimal activity. Genes encoding kinetic analyses of the FosB enzymes from multiple Gram- FosX have been found in numerous Gram-negative bacterial positive organisms have indicated that BSH is the preferred species including Listeria monocytogenes, Mesorhizobium thiol substrate over L-Cys when activated by Mn(II) (Figure loti, and Clostridium botulinum. 2)(Thompson et al., 2013; Lamers et al., 2012; Roberts The FosX enzyme from M. loti (FosXMl) demonstrates et al., 2013; Thompson et al., 2014), catalysing addition of catalytic promiscuity and is able to perform both fosfomycin the cysteinyl-moiety of BSH to carbon-1 of fosfomycin glutathione transferase (FosA) and fosfomycin hydrolase similar to FosA addition of GSH. In addition, Zn2 + is a (FosX) reactions as might be expected given the relationship potent inhibitor of FosB for either BSH or L-Cys transferase to Gram-negative bacteria. However, the enzyme performs activity. Both BSH and FosB knockout/null cells exhibit a both of these reactions with markedly reduced catalytic significant increase in their sensitivity to fosfomycin (Figure efficiencies, and as a result, FosXMl does not confer 2; Thompson et al., 2014). These results have led to the appreciable fosfomycin resistance. For this reason it is FosB enzymes being characterised as bacillithiol-S- likely that FosXMl actually functions in phosphonate meta- transferases. bolism and is an evolutionary precursor to true fosfomycin resistance enzymes (Fillgrove et al., 2003; O'Brien and Mechanism of fosfomycin inactivation Herschlag, 1999). The structural data for FosB in particular is extensive and FosB has provided the most insight into the mechanism of action of the VOC superfamily of fosfomycin resistance enzymes. A third, distinct fosfomycin resistance factor, FosB, was There are a total of nine deposited structures for FosB from later discovered in Gram-positive organisms such as S. B. cereus (FosBBc)(Thompson et al., 2013) and five for FosB aureus, B. cereus and B. subtilis (Cao et al., 2001; Rigsby from S. aureus (Thompson et al., 2014). Together, they et al., 2005; Thompson et al., 2013). These are genomically provide detailed information about how the FosB enzymes encoded fosfomycin resistance enzymes that are also mem- function. bers of the VOC metalloenzyme superfamily. However, FosB For FosBBc, the three most informative structures were enzymes are not glutathione-S-transferases like FosA or the FosBBc Á Mn(II) Á Fosfomycin structure (PDB 4JH6), the Bc hydrolases like FosX. Rather, FosB enzymes catalyse the FosB Á Mn(II) Á L-Cys-Fosfomycin product structure (PDB + 2 Bc Mn -dependent addition of bacillithiol (BSH) or L-cysteine 4JH7), and the FosB Á Zn(II) Á Fosfomycin and L-Cys ternary (L-Cys) to carbon-1 of fosfomycin (Figure 1)(Cao et al., structure (PDB 4JH8) (Thompson et al., 2013). Comparison Bc Bc 2001). The end result is a similar, chemically modified of the FosB Á Mn(II) Á Fosfomycin and FosB Á Mn(II) Á L-Cys- inactive form of the antibiotic. Glutathione is not found in Fosfomycin product structures revealed a unique trigonal Gram-positive bacteria and thus explains why the FosB bipyramidal metal-ligand coordination geometry (Figure 3). enzymes have not evolved to be glutathione-transferases. Formation of the product relieves strain on the atomic Instead, BSH is the abundant low-molecular-weight thiol orbitals of the metal and ring strain in the epoxide of the found in nearly equal concentrations as L-cysteine. BSH is antibiotic. In addition, the unique coordination geometry the α-anomeric glycoside of L-cysteinyl-D-glucosaminyl-L- explains why the VOC metalloenzymes are activated by Mn malate. It was first isolated and identified in 2009 from S. (II) and inhibited by Zn(II). The highest symmetry axis places 20 M.K. Thompson et al.

Figure 2 Upper: Structure of bacillithiol (Left). Time course of FosBSa catalysed-addition of BSH or L-Cys to fosfomycin in the presence of Mn2 + or Zn2 + (Right). Reactions were carried out at 25 1C in 20 mM HEPES, pH 7.0 with 4 mM fosfomycin and 0.50 mM enzyme in the presence of ( ) 1.6 mM BSH and 10 mMMn2 + ,( ) 1.6 mM BSH and 10 mMZn2 +,( ) 1.6 mM BSH, 10 mM Mn2+, 2 + 2 + 2 + and 100 mMZn ,(■) 1.8 mM L-Cys and 10 mMMn ,( ) 1.8 mM L-Cys and 10 mMZn or ( ) 1.8 mM L-Cys, 10 mM Mn2+, and 100 mMZn2 + . Botton: Fosfomycin disk diffusion assays for MRSA (JE2). The amount off fosfomycin on each disk is shown in red. (Left) Wild type MRSA. (Center) BSH knockout MRSA. (Right) FosB knockout MRSA. The zone of clearing for the knockout strains has doubled. (Figures adapted from (Choi et al., 2012)).

Figure 3 (Left) Comparison of the FosBBc Á Mn(II) Á Fosfomycin Á (PDB 4JH6) and (Center) FosBBc Á Mn(II) Á L-Cys-Fosfomycin Á (PDB

4JH7) structures. If the Z-axis is deﬁned along E115Oε–Mn(II)–FosfomycinO(Oxirane), there is a distorted trigonal bipyramidal geometry about the Mn(II) center. When the product is formed, the equatorial distances lengthen, and the metal-oxygen(oxirane) bond shortens, effectively creating a more symmetric trigonal bipyramidal geometry around Mn(II) and relieving strain on both the atomic orbitals of the metal and the epoxide ring of the antibiotic (Thompson et al., 2013). (Right) Activation of fosfomycin (FosBBc Á Mn(II) Á 2 Fosfomycin, PDB 4JH6) occurs through σ donation of the epoxide lone pair into the dz orbital of Mn(II), increasing the polarity of the + carbon-1-O(Oxirane) bond and resulting in a partial positive charge (δ ) on carbon-1. Furthermore, the inherent electron withdrawing nature of the phosphonate group will further enhance δ + on carbon-1 through polarisation of the carbon-1-P bond. The arrows denote electron charge redistribution away from carbon-1 to both the phosphonate and the epoxide oxygen. This prepares the antibiotic for nucleophilic attack by the incoming thiolate. (Figure adapted from (Thompson et al., 2013)).

2 a lone pair of the oxirane oxygen in the position to form a σ- electron density from the lone pair into the dZ orbital. Zn(II) 2 10 2 type interaction with the dZ orbital of the active site metal is a d transition metal species and therefore has a full dZ ion. Mn(II) is a d5 transition metal species and can accept orbital that cannot accept electron density from the Diversity in fosfomycin resistance proteins 21

Bc Bc Figure 4 Superposition of the FosB Á Mn(II) Á L-Cys-Fosfomycin product structure (PDB 4JH7) and the FosB Á Zn(II) Á Fosfomycin and L-Cys ternary structure (PDB 4JH8). The positions of all other amino acids stay the same, but Asn50 in the ternary complex is rotated approximately 90 degrees relative to the product complex. Rotation of Asn50 about the Cβ–Cγ bond is the only signiﬁcant difference observed in the reported structures of FosBBc.

positive charge generated on carbon-1 of the antibiotic and the negatively charged incoming thiolate. Finally, three of the five deposited structures of FosBSa (PDB entries 4NB0, 4NB1, and 4NB2) show a highly conserved active site loop region of the FosB enzymes in an open conformation relative to all of the deposited FosBBc structures. The loop region is now believed to function as a door that can open and close to allow fosfomycin to enter the active cavity of the enzyme (Figure 5). Closure of the door surrounds the polar phosphonate end of fosfomycin with multiple hydrogen bonding interactions and locks the antibiotic in a position at the bottom of a solvent channel where the thiolate enters to attack carbon-1. In addition, closure of the door forms the final side of the BSH binding cavity on the surface of the enzyme. Interaction of the glucosaminyl-L-malate domain of BSH with the amino acid Figure 5 (Left) Apo FosBSa (PDB 4NB2) with the binding loop residues of the binding cavity is important for substrate in the open conformation. (Right) FosBBc Á Mn(II) Á Fosfomycin recognition and is a thermodynamic driving force in the (PDB 4JH6) with the binding loop in the closed conformation. preference of BSH over L-Cys for the FosB enzymes Closure of the loop surrounds the polar phosphonate end of (Thompson et al., 2013; Roberts et al., 2013). That fosfo- fosfomycin with multiple hydrogen bonding interactions and mycin must bind for the door to close and form the BSH locks the antibiotic in a position at the bottom of a solvent binding cavity is consistent with published “order of addi- channel where the thiolate enters to attack carbon-1. Closure tion” kinetic experiments for FosB (Roberts et al., 2013). of the loop also forms the final side of the BSH binding cavity on In summary, the mechanism of fosfomycin inactivation for the surface of the enzyme. the VOC fosfomycin resistance enzymes can be characterised as acid-catalysed nucleophilic epoxide ring opening. During attack of carbon-1 of fosfomycin, Mn(II) serves as the coordinated antibiotic. Sigma donation of electron density Lewis acid to facilitate the partial positive charge build up 2 from the oxirane oxygen of the antibiotic into the dZ atomic on carbon-1. In FosX, a glutamate residue (Glu44) acts as a orbital of the metal plays a large role in generating the general base in the direct addition of water to the partial positive charge (δ + ) of carbon-1 of fosfomycin, antibiotic, whereas in FosA and FosB, Tyr39 is proposed to activating the antibiotic for nucleophilic attack by the be in the tyrosinate form stabilised by an adjacent arginine incoming L-Cys or BSH molecule (Figure 3). residue. The tyrosinate form of Tyr39 can then deprotonate Bc Examination of the FosB Á Zn(II) Á Fosfomycin and L-Cys the incoming respective thiol substrate. In FosB, Asn50 is in ternary structure revealed the only amino acid that under- an excellent position to stabilise the negatively charged goes any significant change in any of the deposited FosBBc incoming thiolate and the positive charge on the antibiotic Bc structures. Asn50 in the the FosB Á Zn(II) Á Fosfomycin and L- just before nucleophilic addition occurs (Figure 3). The Cys ternary structure is rotated 90 degrees relative to the ternary structure of FosB with both co-substrates in the Bc Bc FosB Á Mn(II) Á Fosfomycin or FosB Á Mn(II) Á L-Cys-Fosfomy- active site (PDB 4JH8) clearly demonstrates this mechanistic cin product structures indicating that Asn50 likely has an arrangement. In FosA, Ser50 probably serves the function of important functional role in the mechanism of the FosB stabilising the partial positive charge on the antibiotic, but enzymes (Figure 4). Asn50 is a conserved amino acid among lack of a FosA structure with bound glutathione makes it the FosB enzymes and is believed to stabilise both the difficult to infer which residues may play a role in 22 M.K. Thompson et al.

Variety is the price of life

Diversiﬁcation of function in the VOC superfamily of fosfomycin resistance enzymes indicates that this particular resistance mechanism to the antibiotic had to have devel- oped prior to the evolutionary split of Gram-positive, Gram- negative, and Mycobacterium species. The variety of mechanisms is then a result of the availability of the native nucleophile for the individual organisms. Inactivation of fosfomycin still occurs via the same regioselective acid- catalysed, SN2-type epoxide ring opening reaction mechanism that depends on the native nucleophile in each organ- ism. Thus, with respect to the evolution of microbial resistance to fosfomycin, variety is not just the price of life; it is the requirement for survival. 31 Figure 6 P NMR can be used to detect fosfomycin or the L- Cys-fosfomycin product. Conﬂict of interest

The authors declare that there is no conﬂict of interest. stabilisation of the incoming thiolate. In any case, inversion of conﬁguration of carbon-1 of the antibiotic indicates that the reaction proceeds via direct SN2 addition of the Acknowlegdgements nucleophile for all of the VOC superfamily fosfomycin resistance enzymes. Supported by Grants R01 GM030910 to R.N.A. and T32 ES007028 to M.K.T. and M.E.K. from the National Institutes of Health.

Moving forward References

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