Streptomyces rimosus Aminoglycoside 3"-Phosphotransferase VIII: Comparisons with Aminoglycoside 3"-Phosphotransferases of Aminoglycoside-Producing Strains and 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.     

Dependence of aminoglycoside 3'-phosphotransferase VIII activity on protein serine/threonine kinases in Streptomyces rimosus

In Streptomyces rimosus, selection with aminoglycoside kanamycin triggers "silent" aminoglycoside 3'-phosphotransferase (aph) VIII gene. Expression of aphVIII was accompanied by amplification of a chromosomal DNA fragment, which contained aphVIII. Earlier, S. rimosus aphVIII gene was isolated, sequenced, and deduced APHVIII protein sequence was reported. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibody, we demonstrate that one of the abundant proteins phosphorylated by endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 is APHVIII. Phosphoamino acid assay has shown phosphorylation of two seryl residues in APH molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca2+ was 1.84-fold as much as that detected without Ca2+. As shown by in the gel self-phosphorylation and in the substrate-containing gel phosphorylation analyses, two serine PKs with molecular masses of 74 kDa and 55 kDa were active against APHVIII. The 55-kDa PK showed a clear Ca2+ and calmodulin dependency in activity. The specific kanamycin phosphotransferase activity of exhaustedly phosphorylated APHVIII was 3.72-fold as much as that detected in the preparation of nonphosphorylated enzyme. These results suggest involvement of PKs under study in the modulation of APHVIII aminoglycoside phosphorylating activity and in the generation of kanamycin resistance in S. rimosus.     

Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus

We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca²⁺-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.     

Ca2+-dependent modulation of antibiotic resistance in Streptomyces lividans 66 and Streptomyces coelicolor A3(2)

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca²⁺ ions. The inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca²⁺-dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent STPK reduced the Ca²⁺-induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.     

Dependence of aminoglycoside 3′-phosphotransferase VIII activity on serine/threonine protein kinases in <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

In Streptomyces rimosus, selection for resistance to the aminoglycoside antibiotic kanamycin triggers the normally silent aminoglycoside 3-phosphotransferase VIII gene (aphVIII). The expression of APHVIII is accompanied by amplification of the chromosomal DNA fragment containing the aphVIII gene. Earlier, S. rimosus aphVIII gene was isolated and sequenced. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibodies, we have demonstrated that endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 actively phosphorylate two serine residues in the APHVIII molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca²⁺ is 1.84-fold greater than that without Ca²⁺. Analysis of ingel autophosphorylation and phosphorylation of the substrate incorporated into the gel matrix has shown that modification of APHVIII is catalyzed by two serine/threonine PKs (74 kDa and 55 kDa). The activity of 55-kDa PK is dependent on Ca²⁺ and calmodulin. The specific kanamycin phosphotransferase activity of exhaustively phosphorylated APHVIII is 3.72 times higher than that of the unmodified enzyme. It is proposed that the above PKs may be involved in the regulation of kanamycin resistance in S. rimosus.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 255–263.Original Russian Text Copyright © 2005 by Elizarov, Sergienko, Sizova, Danilenko.     

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.     

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.     

Molecular cloning and characterization of the human NIMA-related protein kinase 3 gene (NEK3)

NEKs (NIMA-related kinases) are a group of protein kinases sharing high amino acid sequence identities with NIMA (never in mitosis gene a) which control mitosis in Aspergillus nidulans. We have cloned a cDNA for human NEK3, a novel human gene structurally related to NIMA, by RT-PCR. Its open reading frame encodes a protein of 489 amino acid residues with the calculated molecular mass of 56.0 kDa and a predicted pI of 6.58. Phylogenetic analysis suggests that mouse and human NEK3s constitute a subfamily within the NIMA family of protein kinases. The expression pattern of NEK3 was studied by RT-PCR and a high level of expression was detected in testis, ovary, and brain, with low-level expression being detected in most of the tissues studied. NEK3 mRNA was detected in all the proliferating cell lines studied, and the amount did not change during the cell cycle. The human NEK3 gene was assigned to human chromosome 13 by somatic cell hybrids and 13q14.2 by radiation hybrid mapping.     

Isolation and characterization of the structural gene encoding elongation factor 3

The unique yeast translational factor EF-3 participates in the elongation cycle by stimulating the function of EF-1 alpha in binding aminoacyl-tRNA to the ribosome. We have isolated the structural gene encoding EF-3 from the yeast Saccharomyces cerevisiae. The YEF3 gene is found in one copy per haploid genome and is essential for vegetative growth. DNA sequence analysis reveals that the YEF3 gene contains an open reading frame of 1044 codons. The deduced amino acid sequence has two repeats of a nucleotide-binding motif. Each of these repeats shows similarity to the nucleotide-binding motif of hydrophilic, membrane-associated ATPases including human multidrug resistant protein MDR. Factor 3 manifests ribosome-dependent ATP hydrolysis. Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity and EF-3 protein levels by 3-5-fold. Yeast strains containing elevated EF-3 protein levels also exhibit increased sensitivity to the aminoglycoside antibiotics hygromycin and paromomycin. These drugs are known to increase translational errors. These observations suggest that EF-3 may affect translational accuracy.     

Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families

The amino acid sequence of bovine lung cGMP-dependent protein kinase has been determined by degradation and alignment of two primary overlapping sets of peptides generated by cleavage at methionyl or arginyl residues. The protein contains 670 residues in a single N alpha-acetylated chain corresponding to a molecular weight of 76 331. The function of the molecule is considered in six segments of sequence which may correspond to four folding domains. From the amino terminus, the first segment is related to the dimerizing property of the protein. The second and third segments appear to have evolved from an ancestral tandem internal gene duplication, generating twin cGMP-binding domains which are homologous to twin domains in the regulatory subunits of cAMP-dependent protein kinase and to the cAMP-binding domain of the catabolite gene activator of Escherichia coli. The fourth and fifth segments may comprise one domain which is homologous to the catalytic subunits of cAMP-dependent protein kinase, of calcium-dependent phosphorylase b kinase, and of certain oncogenic viral protein tyrosine kinases. The regulatory, amino-terminal half of cGMP-dependent protein kinase appears to be related to a family of smaller proteins that bind cAMP for diverse purposes, whereas the catalytic, carboxyl-terminal half is related to a family of protein kinases of varying specificity and varying sensitivity to regulators. These data suggest that ancestral gene splicing events may have been involved in the fusion of two families of proteins to generate the allosteric character of this chimeric enzyme.     

The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 A crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.     

Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides

Methylation of either of two residues (G-1405 or A-1408) within bacterial 16 S ribosomal RNA results in high level resistance to specific combinations of aminoglycoside antibiotics. The product of a gene that originated in Micromonospora purpurea (an actinomycete that produces gentamicin) gives resistance to kanamycin plus gentamicin by converting residue G-1405 to 7-methylguanosine. Resistance to kanamycin plus apramycin results from conversion of residue A-1408 to 1-methyladenosine catalysed by the product of a gene from Streptomyces tenjimariensis.     

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.     

Genetic variation of herpesvirus saimiri subgroup A transforming protein and its association with cellular src.

Herpesvirus saimiri strain 11 of subgroup A contains a gene called the saimiri transformation-associated protein, STP, which is not required for viral replication but is required for in vitro immortalization and for the lymphoma-inducing capacity of the virus. To assess the effects of sequence variation on STP function, STP genes from six subgroup A isolates were cloned and sequenced. Sequence comparisons revealed extensive amino acid substitutions within the central region, but the acidic amino terminus and the hydrophobic carboxyl terminus were well conserved. Amino acid identities varied from 73 to 99% among all two-way comparisons. The highly conserved YAEV/I motif at amino acid residues 115 to 118 was preceded by negatively charged glutamic acid residues and thus matched very well the consensus sequence for binding to SH2 domains of src family kinases. The STPs of these subgroup A strains were shown to associate with cellular src and to be an in vitro substrate for src kinase. Mutational analysis of STP-A11 showed that binding to src kinase required the tyrosine residue at 115, showing that YAEV/I is a likely binding motif for src. Also, tyrosine phosphorylation of STP-A11 by src led to subsequent binding to lck and fyn in vitro. Thus, the association of STP with src is likely to be important for T-cell transformation by subgroup A strains of herpesvirus saimiri.     

Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.     

Characterization of the autophosphorylating kinase,PkaF, in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2) M130

Streptomyces coelicolor, the model species for morphologically complex actinomycete bacteria, has unique characteristics such as morphological and physiological differentiation, which are controlled by various factors and several protein kinases. From the whole genomic sequence of S. coelicolor A3(2), 44 putative serine/threonine (Ser/Thr) protein kinases were identified, and the pkaF gene was chosen as the best-conserved protein for typical Ser/Thr protein kinases. pkaF encodes a 667-amino acid protein with a predicted N-terminal Ser/Thr kinase domain and four repeated C-terminal penicillin-binding domains and Ser/Thr kinase-associated (PASTA) domains. Based on PCR, a pkaF gene was cloned and heterologously expressed. PkaF expressed in Escherichia coli had the bigger molecular size than the expected value (75 kDa) and was further purified by Ni²⁺-NTA agarose affinity column chromatography to homogeneity. The purified PkaF was autophosphorylated through the transfer of the γ-phosphate group of ATP. The extent of phosphorylation was proportional to the amount of PkaF, and the phospho-PkaF was dephosphorylated by the addition of the cell lysate of S. coelicolor A3(2). Although no change was observed in the pkaF disruptant, overexpression of pkaF induced severe repression of morphogenesis and actinorhodin production, but not undecylprodigiosin production, implying that PkaF specifically regulates morphogenesis and actinorhodin production in S. coelicolor.     

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.