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.     

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.     

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.     

Establishing the principles of recognition in the adenine-binding region of an aminoglycoside antibiotic kinase [APH(3')-IIIa

The protein-based molecular recognition of the adenine ring has implications throughout biological systems. In this paper, we discuss the adenine-binding region of an aminoglycoside antibiotic kinase [APH(3')-IIIa], which serves as an excellent model system for proteins that bind the adenine ring. This enzyme employs a hydrogen-bonding network involving water molecules along with enzyme backbone/side-chain atoms and a pi-pi stacking interaction to recognize the adenine ring. Our approach utilized site-directed mutagenesis, adenosine analogues and a variety of biophysical methods to probe the contacts in the adenine-binding region of APH(3')-IIIa. The results point to the polar nature of an adenine-Met90 contact in this binding pocket and the important role that Met90, the "gatekeeper" residue in structurally similar Ser/Thr protein kinases, plays in adenine binding. The results also suggest that small changes in the structure of the adenine ring can lead to significant changes in the ability of these analogues to occupy the adenine-binding region of the enzyme. Additional computational experiments indicate that both size and electronic factors are important in the binding of aromatic systems in this interaction-rich pocket. The principles governing adenine recognition established in this study may be applied to other protein-ligand complexes and used to navigate future studies directed at discovering potent and selective inhibitors of APH-type enzymes.     

Thermodynamics of aminoglycoside binding to aminoglycoside-3'-phosphotransferase IIIa studied by isothermal titration calorimetry

The aminoglycoside-3'-phosphotransferase IIIa [APH(3')-IIIa] phosphorylates aminoglycoside antibiotics and renders them ineffective against bacteria. APH(3')-IIIa is the most promiscuous aminoglycoside phosphotransferase enzyme, and it modifies more than 10 different aminoglycoside antibiotics. A wealth of information exists about the enzyme; however, thermodynamic properties of enzyme-aminoglycoside complexes are still not known. This study describes the determination of the thermodynamic parameters of the binary enzyme-aminoglycoside and the ternary enzyme-metal-ATP-aminoglycoside complexes of structurally related aminoglycosides using isothermal titration calorimetry. Formation of the binary enzyme-aminoglycoside complexes is enthalpically driven and exhibits a strongly disfavored entropic contribution. Formation of the ternary enzyme-metal-ATP-aminoglycoside complexes yields much smaller negative DeltaH values and more favorable entropic contributions. The presence of metal-ATP generally increases the affinity of aminoglycosides to the enzyme. This is consistent with the kinetic mechanism of the enzyme in which ordered binding of substrates occurs. However, the observed DeltaH values neither correlate with kinetic parameters k(cat), K(m), and k(cat)/K(m) nor correlate with the molecular size of the substrates. Comparison of the thermodynamic properties of the complexes formed by structurally similar aminoglycosides indicated that the 2'- and the 6'-amino groups of the substrates are involved in binding to the enzyme. Thermodynamic properties of the complexes formed by aminoglycosides differing only at the 3'-hydroxyl group suggested that the absence of this group does not alter the thermodynamic parameters of the ternary APH(3')-IIIa-metal-ATP-aminoglycoside complex. Our results also indicate that protonation of ligand and protein ionizable groups is coupled to the complex formation between aminoglycosides and APH(3')-IIIa. Comparison of DeltaH values for different aminoglycoside-enzyme complexes indicates that enzyme and substrates undergo significant conformational changes in complex formation.     

A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases

Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.     

ATP binding enables broad antibiotic selectivity of aminoglycoside phosphotransferase(3')-IIIa: an elastic network analysis

The bacterial enzyme aminoglycoside phosphotransferase(3′)-IIIa (APH) confers resistance against a wide range of aminoglycoside antibiotics. In this study, we use the Gaussian network model to investigate how the binding of nucleotides and antibiotics influences the dynamics and thereby the ligand binding properties of APH. Interestingly, in NMR experiments, the dynamics differ significantly in various APH complexes, although crystallographic studies indicate that no larger conformational changes occur upon ligand binding. Isothermal titration calorimetry also shows different thermodynamic contributions to ligand binding. Formation of aminoglycoside-APH complexes is enthalpically driven, while the enthalpic change upon aminoglycoside binding to the nucleotide-APH complex is much smaller. The differential effects of nucleotide binding and antibiotic binding to APH can be explained theoretically by single-residue fluctuations and correlated motions of the enzyme. The surprising destabilization of β-sheet residues upon nucleotide binding, as seen in hydrogen/deuterium exchange experiments, shows that the number of closest neighbors does not fully explain residue flexibility. Additionally, we must consider correlated motions of dynamic protein domains, which show that not only connectivity but also the overall protein architecture is important for protein dynamics.     

Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins

The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3')-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.     

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.     

Mapping of the ATP-binding domain of human fructosamine 3-kinase-related protein by affinity labelling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine

The modification of proteins by reducing sugars through the process of non-enzymatic glycation is one of the principal mechanisms by which hyperglycaemia may precipitate the development of diabetic complications. Fn3K (fructosamine 3-kinase) and Fn3KRP (Fn3K-related protein) are two recently discovered enzymes that may play roles in metabolizing early glycation products. However, although the activity of these enzymes towards various glycated substrates has been established, very little is known about their structure-function relationships or their respective mechanisms of action. Furthermore, their only structural similarities noted to date with members of other kinase families has been with the bacterial aminoglycoside kinases. In the present study, we employed affinity labelling with the ATP analogue FSBA {5'-p-[(fluorosulfonyl)benzoyl]adenosine} to probe the active-site topology of Fn3KRP as an example of this enigmatic family of kinases. FSBA was found to modify Fn3KRP at five distinct sites; four of these were predicted to be localized in close proximity to its ATP-binding site, based on alignments with the aminoglycoside kinase APH(3')-IIIa, and examination of its published tertiary structure. The results of the present studies provide evidence that Fn3KRP possesses an ATP-binding domain that is structurally related to that of both the aminoglycoside kinases and eukaryotic protein kinases.     

Allosteric inhibition of aminoglycoside phosphotransferase by a designed ankyrin repeat protein

Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.     

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.     

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.     

Branched aminoglycosides: biochemical studies and antibacterial activity of neomycin B derivatives

The C5'-OH group in neomycin B was glycosylated with a variety of mono- and di-saccharides to probe the effect of introduction of additional binding elements on antibacterial activity and interaction with the aminoglycosides modifying enzyme APH(3')-IIIa. The designed structures show antibacterial activity superior to that of neomycin B against pathogenic and resistant strains, while in parallel they demonstrate poor substrate activity with APH(3')-IIIa.     

The crystal structure of aminoglycoside-3'-phosphotransferase-IIa,an enzyme responsible for antibiotic resistance

A major factor in the emergence of antibiotic resistance is the existence of enzymes that chemically modify common antibiotics. The genes for these enzymes are commonly carried on mobile genetic elements, facilitating their spread. One such class of enzymes is the aminoglycoside phosphotransferase (APH) family, which uses ATP-mediated phosphate transfer to chemically modify and inactivate aminoglycoside antibiotics such as streptomycin and kanamycin. As part of a program to define the molecular basis for aminoglycoside recognition and inactivation by such enzymes, we have determined the high resolution (2.1A) crystal structure of aminoglycoside-3'-phosphotransferase-IIa (APH(3')-IIa) in complex with kanamycin. The structure was solved by molecular replacement using multiple models derived from the related aminoglycoside-3'-phosphotransferase-III enzyme (APH(3')-III), and refined to an R factor of 0.206 (R(free) 0.238). The bound kanamycin molecule is very well defined and occupies a highly negatively charged cleft formed by the C-terminal domain of the enzyme. Adjacent to this is the binding site for ATP, which can be modeled on the basis of nucleotide complexes of APH(3')-III; only one change is apparent with a loop, residues 28-34, in a position where it could fold over an incoming nucleotide. The three rings of the kanamycin occupy distinct sub-pockets in which a highly acidic loop, residues 151-166, and the C-terminal residues 260-264 play important parts in recognition. The A ring, the site of phosphoryl transfer, is adjacent to the catalytic base Asp190. These results give new information on the basis of aminoglycoside recognition, and on the relationship between this phosphotransferase family and the protein kinases.     

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.     

Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.     

Streptomyces rimosus aminoglycoside-3'-phosphotransferase VIII: comparisons with aminoglycoside-3'-phosphotransferases of aminoglycoside-producing strains with eukaryotic protein kinases

The nucleotide sequence was established for the aphVIII aminoglycoside phosphotransferase gene of an oxytetracycline-producing Streptomyces rimosus strain. The gene is 804 bp in size and possibly codes for APHVIII of 267 residues. Heterologous expression of aphVIII was studied in Escherichia coli and Chlamydomonas reinhardtii. The deduced APHVIII sequence was compared with known sequences of aminoglycoside phosphotransferases of aminoglycoside-producing actinomycete strains and of eukaryotic protein kinases. A local homology of 38 residues was found between APHVIII and actinomycete serine-threonine protein kinases in the conserved region possibly involved in ATP binding. APHVIII differed from aminoglycoside 3'-phosphotransferases of aminoglycoside-producing actinomycete strains and of clinical isolates, and can be classed to a separate group.