Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.     

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k_cat/K_m = 10⁵-10⁷ m⁻¹ s⁻¹). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.     

Structural basis for dual nucleotide selectivity of aminoglycoside 2'-phosphotransferase IVa provides insight on determinants of nucleotide specificity of aminoglycoside kinases

Enzymatic phosphorylation through a family of enzymes called aminoglycoside O-phosphotransferases (APHs) is a major mechanism by which bacteria confer resistance to aminoglycoside antibiotics. Members of the APH(2″) subfamily are of particular clinical interest because of their prevalence in pathogenic strains and their broad substrate spectra. APH(2″) enzymes display differential preferences between ATP or GTP as the phosphate donor, with aminoglycoside 2″-phosphotransferase IVa (APH(2″)-IVa) being a member that utilizes both nucleotides at comparable efficiencies. We report here four crystal structures of APH(2″)-IVa, two of the wild type enzyme and two of single amino acid mutants, each in complex with either adenosine or guanosine. Together, these structures afford a detailed look at the nucleoside-binding site architecture for this enzyme and reveal key elements that confer dual nucleotide specificity, including a solvent network in the interior of the nucleoside-binding pocket and the conformation of an interdomain linker loop. Steady state kinetic studies, as well as sequence and structural comparisons with members of the APH(2″) subfamily and other aminoglycoside kinases, rationalize the different substrate preferences for these enzymes. Finally, despite poor overall sequence similarity and structural homology, analysis of the nucleoside-binding pocket of APH(2″)-IVa shows a striking resemblance to that of eukaryotic casein kinase 2 (CK2), which also exhibits dual nucleotide specificity. These results, in complement with the multitude of existing inhibitors against CK2, can serve as a structural basis for the design of nucleotide-competitive inhibitors against clinically relevant APH enzymes.     

Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2��-IVa

Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2″-IVa (APH(2″)-IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi-Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2″)-IVa enzyme phosphorylates various 4,6-disubstituted aminoglycoside antibiotics with catalytic efficiencies (k_cat/K_m) of 1.5 × 10³ to 1.2 × 10⁶ (M⁻¹ s⁻¹). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2″)-IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP-utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP-based enzyme inhibitors which would not be expected to interfere with crucial ATP-dependent enzymes.     

Crystal structures of two aminoglycoside kinases bound with a eukaryotic protein kinase inhibitor

Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3')-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3')-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eukaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.     

Aminoglycoside 2'-phosphotransferase IIIa (APH(2')-IIIa) prefers GTP over ATP: structural templates for nucleotide recognition in the bacterial aminoglycoside-2' kinases

Contrary to the accepted dogma that ATP is the canonical phosphate donor in aminoglycoside kinases and protein kinases, it was recently demonstrated that all members of the bacterial aminoglycoside 2'-phosphotransferase IIIa (APH(2')) aminoglycoside kinase family are unique in their ability to utilize GTP as a cofactor for antibiotic modification. Here we describe the structural determinants for GTP recognition in these enzymes. The crystal structure of the GTP-dependent APH(2')-IIIa shows that although this enzyme has templates for both ATP and GTP binding superimposed on a single nucleotide specificity motif, access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template. Similar GTP binding templates are conserved in other bacterial aminoglycoside kinases, whereas in the structurally related eukaryotic protein kinases this template is less conserved. The aminoglycoside kinases are important antibiotic resistance enzymes in bacteria, whose wide dissemination severely limits available therapeutic options, and the GTP binding templates could be exploited as new, previously unexplored targets for inhibitors of these clinically important enzymes.     

Crystal structures of antibiotic-bound complexes of aminoglycoside 2'-phosphotransferase IVa highlight the diversity in substrate binding modes among aminoglycoside kinases

Aminoglycoside 2'-phosphotransferase IVa [APH(2')-IVa] is a member of a family of bacterial enzymes responsible for medically relevant resistance to antibiotics. APH(2')-IVa confers high-level resistance against several clinically used aminoglycoside antibiotics in various pathogenic Enterococcus species by phosphorylating the drug, thereby preventing it from binding to its ribosomal target and producing a bactericidal effect. We describe here three crystal structures of APH(2')-IVa, one in its apo form and two in complex with a bound antibiotic, tobramycin and kanamycin A. The apo structure was refined to a resolution of 2.05 ?, and the APH(2')-IVa structures with tobramycin and kanamycin A bound were refined to resolutions of 1.80 and 2.15 ?, respectively. Comparison among the structures provides insight concerning the substrate selectivity of this enzyme. In particular, conformational changes upon substrate binding, involving rotational shifts of two distinct segments of the enzyme, are observed. These substrate-induced shifts may also rationalize the altered substrate preference of APH(2')-IVa in comparison to those of other members of the APH(2') subfamily, which are structurally closely related. Finally, analysis of the interactions between the enzyme and aminoglycoside reveals a distinct binding mode as compared to the intended ribosomal target. The differences in the pattern of interactions can be utilized as a structural basis for the development of improved aminoglycosides that are not susceptible to these resistance factors.     

Molecular mechanism of aminoglycoside antibiotic kinase APH(3')-IIIa: roles of conserved active site residues

The aminoglycoside antibiotic kinases (APHs) constitute a clinically important group of antibiotic resistance enzymes. APHs share structural and functional homology with Ser/Thr and Tyr kinases, yet only five amino acids are invariant between the two groups of enzymes and these residues are all located within the nucleotide binding regions of the proteins. We have performed site-directed mutagenesis on all five conserved residues in the aminoglycoside kinase APH(3')-IIIa: Lys(44) and Glu(60) involved in ATP capture, a putative active site base required for deprotonating the incoming aminoglycoside hydroxyl group Asp(190), and the Mg(2+) ligands Asn(195) and Glu(208), which coordinate two Mg(2+) ions, Mg1 and Mg2. Previous structural and mutagenesis evidence have demonstrated that Lys(44) interacts directly with the phosphate groups of ATP; mutagenesis of invariant Glu(60), which forms a salt bridge with the epsilon-amino group of Lys(44), demonstrated that this residue does not play a critical role in ATP recognition or catalysis. Results of mutagenesis of Asp(190) were consistent with a role in proper positioning of the aminoglycoside hydroxyl during phosphoryl transfer but not as a general base. The Mg1 and Mg2 ligand Asp(208) was found to be absolutely required for enzyme activity and the Mg2 ligand Asn(195) is important for Mg.ATP recognition. The mutagenesis results together with solvent isotope, solvent viscosity, and divalent cation requirements are consistent with a dissociative mechanism of phosphoryl transfer where initial substrate deprotonation is not essential for phosphate transfer and where Mg2 and Asp(208) likely play a critical role in stabilization of a metaphosphate-like transition state. These results lay the foundation for the synthesis of transition state mimics that could reverse aminoglycoside antibiotic resistance in vivo.     

Biochemical studies and ligand-bound structures of biphenyl dehydrogenase from Pandoraea pnomenusa strain B-356 reveal a basis for broad specificity of the enzyme

Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphB_B-356), the crystal structures of the apo-enzyme, the binary complex with NAD⁺, and the ternary complexes with NAD⁺-2,3-dihydroxybiphenyl and NAD⁺-4,4′-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphB_B-356 converts the dihydrodiol metabolites of 3,3′-dichlorobiphenyl, 2,4,4′-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphB_B-356 elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls.     

Global Conformational Change Associated with the Two-step Reaction Catalyzed by Escherichia coli Lipoate-Protein Ligase A

Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645–33651). Here, we report two new LplA structures, LplA·lipoyl-5′-AMP and LplA·octyl-5′-AMP·apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5′-AMP reaction intermediate and rotation of the C-terminal domain by about 180°. These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys¹³³ residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.     

Structural Basis of Human p70 Ribosomal S6 Kinase-1 Regulation by Activation Loop Phosphorylation

p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3′-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3′-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.     

The Crystal Structures of Substrate and Nucleotide Complexes of Enterococcus faecium Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily

Aminoglycoside-2′′-phosphotransferase-IIa [APH(2′′)-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2′′)-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2′′)-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5′-(β,γ-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2′′) family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2′′)-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R_free, 0.271) at a resolution of 2.5 Å. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2′′)-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2′′) subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.The emergence of bacteria resistant to several important classes of antibiotics has become a major clinical problem over the last few years. Almost every antibacterial compound in clinical use today has associated examples of resistant bacterial isolates (39), including life-threatening strains of Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and various enterococci. The latter are among the most common antibiotic-resistance bacteria isolated from patients with nosocomial infections in the United States today. The synergistic use of either ampicillin or vancomycin with an aminoglycoside, such as kanamycin or gentamicin, has long been the optimal therapy for serious enterococcal infections; however, many previously susceptible enterococcal strains have since acquired resistance to the aminoglycosides. The mechanisms of resistance are many and varied, although only three are readily understood: (i) mutation of the ribosomal target, (ii) reduced permeability and/or increased efflux of the drug, and (iii) enzymatic deactivation of the drug. Resistance to the aminoglycosides through enzymatic deactivation, although seemingly straightforward, is in reality a complex problem involving three different classes of enzyme. These enzyme classes are the ATP-dependent phosphotransferases (APH) and adenyltransferases (ANT), and the acetyl coenzyme A-dependent N-acetyltransferases (AAC). This area of research has been extensively reviewed in the past few years (2, 4, 13, 29, 39, 47, 52, 53).Originally isolated from soil bacteria, including various species of Streptomyces and Micromonospora (20), the aminoglycosides are a family of potent, broad-spectrum antibiotics that includes clinically relevant drugs such as gentamicin, neomycin, amikacin, kanamycin, and streptomycin. The structures of these compounds, with the exception of that of streptomycin, are all similar, consisting of a central aminocyclitol ring (the B ring) with two or three substituted aminoglycan rings (A, C, and in some cases, D) attached at either the 4 and 5 positions (the 4,5-disubstituted aminoglycosides, which include neomycin and lividomycin) or the 4 and 6 positions (the 4,6-disubstituted aminoglycosides, such as gentamicin and kanamycin). Streptomycin, a competitive inhibitor of aminoglycoside-2′′-phosphotransferase-IIa [APH(2′′)-IIa] (45), is an atypical aminoglycoside that does not fall into either the 4,5-disubstituted or 4,6-disubstituted classes. It has a modified ribose (ring B) attached to position 4 on a 1,3-diguanidinium-substituted aminocyclitol ring (ring A) with no substituent at the 5 or 6 position. The structures of gentamicin, kanamycin, neomycin, and streptomycin are shown in Fig. ​Fig.1.1. The aminoglycosides are targeted to the 16S rRNA of the bacterial 30S ribosomal subunit, where they selectively bind to the decoding aminoacyl (A) site (31, 51) and stabilize the conformation of the tRNA bound to a cognate mRNA codon. This decreases the dissociation rate of aminoacyl-tRNA and promotes miscoding (28). The structures of a number of the aminoglycosides with either the 30S subunit or oligonucleotides containing minimal A sites are known (51).Open in a separate windowFIG. 1.Structures of gentamicin, kanamycin, streptomycin, and neomycin. Gentamicin and kanamycin are classified as 4,6-disubstituted aminoglycosides, whereas neomycin is an example of a 4,5-disubstituted compound. The three structural variants which comprise gentamicin C are indicated. Amikacin is similar to kanamycin, although the substituent on the N1 amine is a 4-amino-2-hydroxy-1-oxobutyl group. Taken together, the A and B rings of aminoglycosides, such as gentamicin, kanamycin, and neomycin, are commonly known as the neamine moiety.The enzymes which deactivate the aminoglycosides are named according to the reaction they catalyze and the site on the aminoglycoside at which they act. The APH(2′′) enzymes, which give rise to high-level resistance to gentamicin in enterococci, phosphorylate gentamicin and kanamycin at the 2′′-hydroxyl group of the C ring (Fig. ​(Fig.1).1). The APH(3′) enzymes, another major subfamily of the phosphotransferases, phosphorylate kanamycin and neomycin at the 3′-hydroxyl on the A ring but cannot deactivate gentamicin, since it has no corresponding 3′-hydroxyl. The individual members of each family can normally bind only a subset of the available drugs, and this difference in drug specificity is known as the resistance profile, designated with a roman numeral and, in some cases, a letter identifying a specific gene. The first APH(2′′) enzyme discovered for enterococci was the bifunctional AAC(6′)-Ie-APH(2′′)-Ia enzyme, which possesses both 6′-acetylating and 2′′-phosphorylating activities (17, 33). Enterococci with the corresponding gene show resistance to almost all clinically relevant aminoglycosides (38). Four additional APH(2′′) enzymes have since been isolated for Enterococcus spp.; they are designated APH(2′′)-Ib (27), APH(2′′)-Ic (11), APH(2′′)-Id (46), and APH(2′′)-Ie (10) and were initially classified as genetic variants of an APH(2′′)-I-type enzyme. Recently, APH(2′′)-Ib, APH(2′′)-Ic, and APH(2′′)-Id have been reclassified as distinct enzymes with different resistance profiles and, more importantly, different nucleotide specificities, such that they are now named APH(2′′)-IIa, APH(2′′)-IIIa, and APH(2′′)-IVa, respectively (44). APH(2′′)-Ie was not included in the latter study, but based upon the very high sequence similarity with APH(2′′)-IVa (93%) (see Table S1 in the supplemental material), it is possible that it is a genetic variant of APH(2′′)-IVa.Structural details are currently known for only two members of the APH(3′) family, APH(3′)-IIIa (5, 18, 23) and APH(3′)-IIa (37). These enzymes share a two-domain structure similar to the catalytic domains of the eukaryotic Ser/Thr and Tyr protein kinases. Moreover, the phosphotransferases and kinases share several important sequence motifs related to nucleotide binding and phosphoryl transfer, most notably the catalytic loop (HXDXXXXN) and the activation segment (GXIDXG), where X is any amino acid. Not surprisingly, the catalytic mechanisms of the phosphotransferases and the kinases are identical, involving the nucleophilic attack by the target hydroxyl on the γ phosphate of ATP, facilitated by a conserved aspartate residue from the catalytic loop (29, 54). A comparison of the known APH(2′′) and APH(3′) sequences shows that the two families of phosphotransferases share these kinase-like motifs, and there appears to be some partial conservation of acidic residues in the substrate binding region. It has been suggested that their structures may be similar (37). Here, we report the first structure of an APH(2′′) enzyme, APH(2′′)-IIa as the binary complex with the preferred substrate gentamicin and the ternary complex with the nonhydrolyzable ATP analog adenosine-5′-(β,γ-methylene)triphosphate (AMPPCP) and the competitive inhibitor streptomycin.     

The use of neamine as a molecular template: Inactivation of bacterial antibiotic resistance enzyme aminoglycoside 3′-phosphotransferase type IIa

Aminoglycoside 3′-phosphotransferase type IIa [APH(3′)-IIa] is a member of the family of bacterial aminoglycoside-modifying enzymes. Bacteria that harbor these enzymes are resistant to aminoglycoside antibiotics. Four aminoglycoside-based affinity inactivators were synthesized and were shown to be both substrates and inactivators for APH(3′)-IIa. These affinity inactivators are N-bromoacetylated derivatives of neamine, an aminoglycoside antibiotic, where the bromoacetyl moiety in each was introduced regiospecifically at a different amine of the parent compound.     

Mechanism of aminoglycoside antibiotic kinase APH(3')-IIIa: role of the nucleotide positioning loop

The aminoglycoside antibiotic resistance kinases (APHs) and the Ser/Thr/Tyr protein kinases share structural and functional homology but very little primary sequence conservation (<5%). A region of structural, but not amino acid sequence, homology is the nucleotide positioning loop (NPL) that closes down on the enzyme active site upon binding of ATP. This loop region has been implicated in facilitating phosphoryl transfer in protein kinases; however, there is no primary sequence conservation between APHs and protein kinases in the NPL. There is an invariant Ser residue in all APH NPL regions, however. This residue in APH(3')-IIIa (Ser27), an enzyme widespread in aminoglycoside-resistant Enterococci, Streptococci, and Staphylococci, directly interacts with the beta-phosphate of ATP through the Ser hydroxymethyl group and the amide hydrogen in the 3D structure of the enzyme. Mutagenesis of this residue to Ala and Pro supported a role for the Ser amide hydrogen in nucleotide capture and phosphoryl transfer. A molecular model of the proposed dissociative transition state, which is consistent with all of the available mechanistic data, suggested a role for the amide of the adjacent Met26 in phosphoryl transfer. Mutagenesis studies confirmed the importance of the amide hydrogen and suggest a mechanism where Ser27 anchors the ATP beta-phosphate facilitating bond breakage with the gamma-phosphate during formation of the metaphosphate-like transition, which is stabilized by interaction with the amide hydrogen of Met26. The APH NPL therefore acts as a lever, promoting phosphoryl transfer to the aminoglycoside substrate, with the biological outcome of clinically relevant antibiotic resistance.     

Hydrolysis of ATP by aminoglycoside 3'-phosphotransferases: an unexpected cost to bacteria for harboring an antibiotic resistance enzyme

Aminoglycoside 3'-phosphotransferases (APH(3')s) are common bacterial resistance enzymes to aminoglycoside antibiotics. These enzymes transfer the gamma-phosphoryl group of ATP to the 3'-hydroxyl of the antibiotics, whereby the biological activity of the drugs is lost. Pre-steady-state and steady-state kinetics with two of these enzymes from Gram-negative bacteria, APH(3')-Ia and APH(3')-IIa, were performed. It is demonstrated that these enzymes in both ternary and binary complexes facilitate an ATP hydrolase activity (ATPase), which is competitive with the transfer of phosphate to the antibiotics. Because these enzymes are expressed constitutively in resistant bacteria, the turnover of ATP is continuous during the lifetime of the organism both in the absence and the presence of aminoglycosides. Concentrations of the enzyme in vivo were determined, and it was estimated that in a single generation of bacterial growth there exists the potential that this activity would consume as much as severalfold of the total existing ATP. Studies with bacteria harboring the aph(3')-Ia gene revealed that bacteria are able to absorb the cost of this ATP turnover, as ATP is recycled. However, the cost burden of this adventitious activity manifests a selection pressure against maintenance of the plasmids that harbor the aph(3')-Ia gene, such that approximately 50% of the plasmid is lost in 1500 bacterial generations in the absence of antibiotics. The implication is that, in the absence of selection, bacteria harboring an enzyme that catalyzes the consumption of key metabolites could experience the loss of the plasmid that encodes for the given enzyme.     

Acyl Carrier Protein-specific 4��-Phosphopantetheinyl Transferase Activates 10-Formyltetrahydrofolate Dehydrogenase

4′-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4′-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO₂. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G₁ arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.     

Substrate promiscuity of an aminoglycoside antibiotic resistance enzyme via target mimicry

The misuse of antibiotics has selected for bacteria that have evolved mechanisms for evading the effects of these drugs. For aminoglycosides, a group of clinically important bactericidal antibiotics that target the A-site of the 16S ribosomal RNA, the most common mode of resistance is enzyme-catalyzed chemical modification of the drug. While aminoglycosides are structurally diverse, a single enzyme can confer resistance to many of these antibiotics. For example, the aminoglycoside kinase APH(3')-IIIa, produced by pathogenic Gram-positive bacteria such as enterococci and staphylococci, is capable of detoxifying at least 10 distinct aminoglycosides. Here we describe the crystal structures of APH(3')-IIIa in complex with ADP and kanamycin A or neomycin B. These structures reveal that the basis for this enzyme's substrate promiscuity is the presence of two alternative subsites in the antibiotic binding pocket. Furthermore, comparison between the A-site of the bacterial ribosome and APH(3')-IIIa shows that mimicry is the second major factor in dictating the substrate spectrum of APH(3')-IIIa. These results suggest a potential strategy for drug design aimed at circumventing antibiotic resistance.     

Fluorinated aminoglycosides and their mechanistic implication for aminoglycoside 3'-phosphotransferases from Gram-negative bacteria

Aminoglycoside 3'-phosphotransferases [APH(3')s] are important bacterial resistance enzymes for aminoglycoside antibiotics. These enzymes phosphorylate the 3'-hydroxyl of these antibiotics, a reaction that inactivates the drug. A series of experiments were carried out to shed light on the details of the turnover chemistry by these enzymes. Quench-flow pre-steady-state kinetic analyses of the reactions of Gram-negative APH(3') types Ia and IIa with kanamycin A, neamine, and their respective difluorinated analogues 4'-deoxy-4',4'-difluorokanamycin A and 4'-deoxy-4',4'-difluoroneamine were carried out, in conjunction with measurements of thio effect and viscosity studies. The fluorinated analogues were shown to be severely impaired as substrates for these enzymes. The magnitude of the effect of the impairment of the fluorinated substrates was in the same range as when the D198A mutant APH(3')-Ia was studied with nonfluorinated substrates. Residue 198 is the proposed active site base that promotes the aminoglycoside hydroxyl for phosphorylation. These findings collectively argue that the Gram-negative APH(3')s show significant nucleophilic participation in the transition state for the phosphate transfer reaction.     

The COOH terminus of aminoglycoside phosphotransferase (3')-IIIa is critical for antibiotic recognition and resistance.

The aminoglycoside phosphotransferases (APHs) are widely distributed among pathogenic bacteria and are employed to covalently modify, and thereby detoxify, the clinically relevant aminoglycoside antibiotics. The crystal structure for one of these aminoglycoside kinases, APH(3')-IIIa, has been determined in complex with ADP and analysis of the electrostatic surface potential indicates that there is a large anionic depression present adjacent to the terminal phosphate group of the nucleotide. This region also includes a conserved COOH-terminal alpha-helix that contains the COOH-terminal residue Phe(264). We report here mutagenesis and computer modeling studies aimed at examining the mode of aminoglycoside binding to APH(3')-IIIa. Specifically, seven site mutants were studied, five from the COOH-terminal helix (Asp(261), Glu(262), and Phe(264)), and two additional residues that line the wall of the anionic depression (Tyr(55) and Arg(211)). Using a molecular modeling approach, six ternary complexes of APH(3')-IIIa.ATP with the antibiotics, kanamycin, amikacin, butirosin, and ribostamycin were independently constructed and these agree well with the mutagenesis data. The results obtained show that the COOH-terminal carboxylate of Phe(264) is critical for proper function of the enzyme. Furthermore, these studies demonstrate that there exists multiple binding modes for the aminoglycosides, which provides a molecular basis for the broad substrate- and regiospecificity observed for this enzyme.     

Cloning, overexpression, and purification of aminoglycoside antibiotic nucleotidyltransferase (2'')-Ia: conformational studies with bound substrates

Aminoglycoside nucleotidyltransferase (2')-Ia [ANT (2')-Ia] was cloned from Pseudomonas aeruginosa and purified from overexpressing Escherichia coli BL21(DE3) cells. The first enzyme-bound conformation of an aminoglycoside antibiotic in the active site of an aminoglycoside nucleotidyltransferase was determined using the purified aminoglycoside nucleotidyltransferase (2' ')-Ia. The conformation of the aminoglycoside antibiotic isepamicin, a psuedo-trisaccharide, bound to aminoglycoside nucleotidyltransferase (2' ')-Ia has been determined using NMR spectroscopy. Molecular modeling, employing experimentally determined interproton distances, resulted in two different enzyme-bound conformations (conformer 1 and conformer 2) of isepamicin. Conformer 1 was by far the major conformer defined by the following average glycosidic dihedral angles: PhiBC = -65.26 +/- 1.63 degrees and PsiBC = -54.76 +/- 4.64 degrees. Conformer 1 was further subdivided into one major (conformer 1a) and two minor components (conformers 1b and 1c) based on the comparison of glycosidic dihedral angles PhiAB and PsiAB. The arrangement of substrates in the enzyme.metal-ATP.isepamicin complex was determined on the basis of the measured effect of the paramagnetic substrate analogue Cr(H2O)4ATP on the relaxation rates of substrate protons which were used to determine relative distances of isepamicin protons to the Cr3+. Both conformers of isepamicin yielded arrangements that satisfied the NOE restraints and the observed paramagnetic effects of Cr(H2O)4ATP. It has been suggested that aminoglycosides use both electrostatic interactions and hydrogen bonds in binding to RNA and that the contacts made by the A and B rings to RNA are the most important for binding [Fourmy, D., Recht, M. I., Blanchard, S. C., and Puglisi, J. D. (1996) Science 274, 1367-1371]. Comparisons based on the determined conformations of enzyme-bound aminoglycoside antibiotics also suggested that interactions of rings A and B with enzymes may be the major determinant in aminoglycoside binding to enzymes [Serpersu, E. H., Cox, J. R., DiGiammarino, E. L., Mohler, M. L., Ekman, D. R., Akal-Strader, A., and Owston, M. (2000) Cell Biochem. Biophys. (in press)]. The conformation of isepamicin bound to the aminoglycoside nucleotidyltransferase (2' ')-Ia, determined in this work, lent further support to this theory. Furthermore, comparison of enzyme-bound conformations of isepamicin to the RNA-bound conformation of gentamycin C1a also showed remarkable similarities between the enzyme-bound and RNA-bound aminoglycoside antibiotic conformations. These studies should aid in the design of effective inhibitors possessing a broad range of aminoglycoside-modifying enzymes as targets.