View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Research Paper 747 Mechanism of aminoglycoside 3’-phosphotransferase type Illa: His188 is not a phosphate-accepting residue Paul R Thompson ‘, Donald W Hughes* and Gerard D Wright’ Background: The enzyme aminoglycoside 3’-phosphotransferase Type llla Addresses: Departments of ‘Biochemistry and (APH(3’)-llla), confers resistance to many aminoglycoside antibiotics by %hemistry, McMaster University, Hamilton, ON Canada LEN 325. regiospecific phosphorylation of their hydroxyl groups. The chemical mechanism of phosphoryl transfer is unknown. Based on sequence homology, it has been Correspondence: Gerard D Wright suggested that a conserved His residue, His1 88, could be phosphorylated by e-mail: [email protected] ATP, and this phospho-His would transfer the phosphate to the incoming Key words: aminoglycoside, antibiotic resistance, aminoglycoside. We have used chemical modification, site-directed mutagenesis mutagenesis, PIX and positional isotope exchange methods to probe the mechanism of phosphoryl transfer by APH(3’)-llla. Received: 31 Jul 1996 Revisions requested: 15 Aug 1996 Revisions received: 20 Aug 1996 Results: Chemical modification by diethylpyrocarbonate implicated His in Accepted: 23 Aug 1996 aminoglycoside phosphorylation by APH(3’)-llla. We prepared His + Ala mutants of all four His residues in APH(3’)-llla and found minimal effects of the Chemistry & Biology September 1996, mutations on the steady-state phosphorylation of several aminoglycosides. One 31747-755 of these mutants, His1 88Ala, was largely insoluble when compared to the wild- 0 Current Biology Ltd ISSN 1074-5521 type enzyme. Positional isotope exchange experiments using y-[lsO]-ATP did not support a double-displacement mechanism. Conclusions: His residues are not required for aminoglycoside phosphorylation by APH(3’)-llla. The conserved His1 88 is thus not a phosphate accepting residue but does seem to be important for proper enzyme folding. Positional isotope exchange experiments are consistent with direct attack of the aminoglycoside hydroxyl group on the y-phosphate of ATP Introduction both the Enterococcus and Staphylococcus genera can harbor Hospital-acquired infections have become a particularly specific aminoglycoside-modifying isozymes, which cir- serious problem because of the widespread dissemination cumvent the bactericidal effects of these antibiotics [9]. of antibiotic resistance genes. The Gram positive cocci of One of these enzymes, the 3’-aminoglycoside phospho- the genera Enterococcus and Staphyylococcus are among the transferase type IIIa (APH(3’)-IIIa), confers resistance to a more prevalent causes of these infections [l]. Entero- broad spectrum of aminoglycosides including the coccal infections are treated with a combination of clinically important drug amikacin [ 10,111. aminoglycoside and B-lactam antibiotics, or with the glycopeptide vancomycin [Z]. These infections have APH(3’)-IIIa catalyzes the transfer of the y-phosphate of become increasingly difficult to treat because of the ATP to the 3’ hydroxyl group of 4,6-disubstituted deoxy- selection of large numbers of antibiotic resistance deter- streptamine aminoglycosides and to both the 3’- and minants [3]. This increase in antibiotic-resistant organisms 5”-hydroxyls of 4,5-disubstituted deoxystreptamine has brought into question the future usefulness of aminoglycosides, such as neomycin, that possess available aminoglycosides and other antibiotics [4,5]. hydroxyl groups (Fig. 1) [1’2]. Six other classes of APH(3’)s have been identified and their protein sequences have In general, aminoglycoside antibiotics are composed of a significant homology in the carboxy-terminal region [8]. central Z-deoxystreptamine aminocyclitol ring which is Three conserved motifs have been identified (Fig. 2) and, linked to various aminosugars via glycosyl bonds (e.g., Fig. although their function is not known, Martin et a/.) [13] 1). Bacterial resistance to the aminoglycosides occurs have speculated that, based on homology searches of predominantly through a variety of enzyme-catalyzed protein databases, motif 2 is part of the nucleotide binding processes [6]. These mechanisms involve the covalent site, motif 3 is involved in aminoglycoside binding modification of aminoglycosides by 0-phosphorylation, and motif 1 is directly involved in catalysis. Specifically, N-acetylation or 0-adenylation. These modifications are they postulated that the strictly conserved histidine in catalyzed by a superfamily of transferases, comprising APH(3’) enzymes (His188 in APH(3’)-IIIa) may act as a greater than fifty individual enzymes [7,8]. Species of phosphate-accepting residue, mediating the transfer of 748 Chemistry & Biology 1996, Vol3 No 9 Figure 1 APH(3’)-Ma catalyzes the regiospecific transfer of the y-phosphate of ATP to an aminoglycoside hydroxyl group. Two examples are shown. HO ATP ADP Neomycin B HO *-ospo 7 ATP ADP Kanamycin A the y-phosphate of ATP to an aminoglycoside hydroxyl residues, such as Ser and Tyr, or Leu [18], that are group via a phospho-enzyme intermediate (Fig. 3a) [13]. potential phosphate acceptors (but very poor phosphate We have not found any steady-state kinetic evidence for a donors), or by Gln [19] demonstrated the importance of phospho-enzyme intermediate with APH(3’)-IIIa [14,15]. this residue for conferring aminoglycoside resistance; for Thus, an equally plausible mechanism of phosphate cells harboring a mutant enzyme, a dramatic decrease in transfer would be by the direct in-line transfer of the minimal inhibitory concentrations for a number of terminal phosphate of ATP to a specific aminoglycoside aminoglycosides was observed. Assays of crude cell-free hydroxyl group (Fig. 3b) [ 16,171. A dissociative mecha- extracts on the Hisl88Gln mutant revealed a two-fold nism in which a hyper-electrophilic metaphosphate anion increase in the K, for ATP and a 35fold decrease in is generated is theoretically possible, but would be relative activity, but the enzyme was not purified [19]. unprecedented. All known enzymatic phosphoryl transfers occur by associative mechanisms [ 171. To understand the mechanism of phosphoryl transfer for the rational design of inhibitors and/or new antibiotics, we Site-directed mutagenesis of His188 has been reported for have undertaken to delineate the chemical mechanism of the APH(3’)-II from Tn.5. Substitution of His188 with phosphoryl transfer. The question of whether phosphate transfer occurs through a phospho-enzyme intermediate or Figure 2 by a direct in-line attack is complicated by the fact that the kinetic mechanism of APH(3’)IIIa is Theorell-Chance [14]. In this version of an ordered BiBi mechanism, ATP binds to the enzyme first and the aminoglycoside second; this is followed by release of the phospho-aminoglycoside and ADP. In such a mechanism the ternary complex is transparent in the steady state because product release is completely rate limiting, and as such the existence of a phospho-enzyme intermediate cannot be discounted. To discern between double-displacement (phospho-enzyme) Conserved motifs (1,2,3) in APH(3’) sequences. His1 88 is found in and direct-attack associative mechanisms, and to eluci- motif 1. Lys44 can be labeled with p-fluorosulfonylbenzoyl adenosine 1381, and thus contacts ATP The single-letter code for amino acids is used. date the possible role of His188 in catalysis, we have approached the problem in two ways. First, we examined Research Paper Mechanism of kanamycin kinase Thompson et al. 749 Figure 3 Possible mechanisms of phosphate transfer catalyzed by APH(3’Wa. The reaction may a) r( ON NH; proceed (a) by a double-displacement mechanism in which a phospho-His ADp.o-p-o-: NvNH intermediate is formed or(b) by direct in-line v’l 0. transfer of the terminal phosphate of ATP to a specific aminoglycoside hydroxyl group. 11 the roles of the His residues in APH(3’)-IIIa by chemical this agent [ZZ]. Rates of inactivation were best fit to a modification and site-directed mutagenesis. Second, we double exponential decay model, suggesting that more addressed the question of the route of phosphoryl transfer than one residue was modified and that these (Fig. 3a versus 3b) by the positional isotope exchange modifications had different effects on the enzymatic rate (PIX) technique of Midlefort and Rose [20,21]. These (Table 2). This was confirmed by stoichiometric experiments provide evidence that neither His188 nor any measurements, which indicated that approximately three other His residue in the enzyme is required for catalysis His residues were modified (Table 2). and that the most likely mechanism is direct attack of the aminoglycoside hydroxyl group on the -y-phosphate Table 1 of ATP Chemical modification of APH(3’)-llla. Results Reagent Target Concentration Activity Inactivation of APH(3’)-llla by chemical modifying agents hM) Wo) We initially explored the roles of several different amino acid residues in catalysis by a survey of the susceptibility of Control, pH 7.5 100 APH(3’)-IIIa to various chemical modifying agents N-Ethylmaleimide CYS 0.5 47 (Table 1). The enzyme showed broad sensitivity to com- p-Chloromercuribenzoic acid CYS 0.5 3 pounds modifying carboxyl and thiol groups as well as Tyr Diethylpyrocarbonate His, Tyr, Cys and Arg residues. The susceptibility to diethylpyro- 10 2 carbonate (DEPC) suggested a possible role for His 1 -Ethyl-3- C-term COO-, 2.5 23 residues in catalysis [Z].