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The macrolides are a class of antibiotic, characterized by a large macrocyclic lactone ring that can be inactivated by macrolide phosphotransferase enzymes. We present structures for MPH(2')-I and MPH(2')-II in the apo state, and in complex with GTP analogs and six different macrolides. These represent the first structures from the two main classes of macrolide phosphotransferases. The structures show that the enzymes are related to the aminoglycoside phosphotransferases, but are distinguished from them by the presence of a large interdomain linker that contributes to an expanded antibiotic binding pocket. This pocket is largely hydrophobic, with a negatively charged patch located at a conserved aspartate residue, rationalizing the broad-spectrum resistance conferred by the enzymes. Complementary mutation studies provide insights into factors governing substrate specificity. A comparison with macrolides bound to their natural target, the 50S ribosome, suggests avenues for next-generation antibiotic development.

Trehalose has been shown to protect bacterial cells from environmental stress. Its uptake and osmoprotective effect inClostridium perfringenswere investigated by comparing wild typeC. perfringensATCC 13124 with a fluoroquinolone- (gatifloxacin-) resistant mutant. In a chemically defined medium, trehalose and sucrose supported the growth of the wild type but not that of the mutant. Microarray data and qRT-PCR showed that putative genes for the phosphorylation and transport of sucrose and trehalose (via phosphoenolpyruvate-dependent phosphotransferase systems, PTS) and some regulatory genes were downregulated in the mutant. The wild type had greater tolerance than the mutant to salts and low pH; trehalose and sucrose further enhanced the osmotolerance of the wild type to NaCl. Expression of the trehalose-specific PTS was lower in the fluoroquinolone-resistant mutant. Protection ofC. perfringensfrom environmental stress could therefore be correlated with the ability to take up trehalose.

Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring. DOI: http://dx.doi.org/10.7554/eLife.21422.001

The UDP-GlcNAc:lysosomal enzyme, N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-PT), is an α2 β2 γ2 hexamer that mediates the initial step in the formation of the mannose 6-phosphate targeting signal on newly synthesized lysosomal acid hydrolases. The GNPTAB gene encodes the 1256 amino acid long α/β precursor which is normally cleaved at K928 in the early Golgi by Site-1 protease (S1P). Here, we show that removal of the so-called 'spacer-1' domain (residues 86-322) results in cleavage almost exclusively at a second S1P consensus sequence located upstream of K928. In addition, GlcNAc-1-PT lacking spacer-1 exhibits enhanced phosphorylation of several non-lysosomal glycoproteins, while the phosphorylation of lysosomal acid hydrolases is not altered. In view of these effects on the maturation and function of GlcNAc-1-PT, we suggest renaming `spacer-1' the `regulatory-1' domain.

Membrane-anchored receptors are essential cellular signaling elements for stimulus sensing, propagation, and transmission inside cells. However, the contributions of lipid interactions to the function and dynamics of embedded receptor kinases have not been described in detail. In this study, we used amide hydrogen/deuterium exchange mass spectrometry, a sensitive biophysical approach, to probe the dynamics of a membrane-embedded receptor kinase, EnvZ, together with functional assays to describe the role of lipids in receptor kinase function. Our results reveal that lipids play an important role in regulating receptor function through interactions with transmembrane segments, as well as through peripheral interactions with nonembedded domains. Specifically, the lipid membrane allosterically modulates the activity of the embedded kinase by altering the dynamics of a glycine-rich motif that is critical for phosphotransfer from ATP. This allostery in EnvZ is independent of membrane composition and involves direct interactions with transmembrane and periplasmic segments, as well as peripheral interactions with nonembedded domains of the protein. In the absence of the membrane-spanning regions, lipid allostery is propagated entirely through peripheral interactions. Whereas lipid allostery impacts the phosphotransferase function of the kinase, extracellular stimulus recognition is mediated via a four-helix bundle subdomain located in the cytoplasm, which functions as the osmosensing core through osmolality-dependent helical stabilization. Our findings emphasize the functional modularity in a membrane-embedded kinase, separated into membrane association, phosphotransferase function, and stimulus recognition. These components are integrated through long-range communication relays, with lipids playing an essential role in regulation.

The lysosomal storage disorder mucolipidosis III γ is caused by defects in the γ subunit of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the enzyme that tags lysosomal enzymes with the mannose 6-phosphate lysosomal targeting signal. In patients with this disorder, most of the newly synthesized lysosomal enzymes are secreted rather than being sorted to lysosomes, resulting in increased levels of these enzymes in the plasma. Several missense mutations in GNPTG, the gene encoding the γ subunit, have been reported in mucolipidosis III γ patients. However, in most cases the impact of these mutations on γ subunit function has remained unclear. Here we report that the variants c.316G>A (p.G106S), c.376G>A (p.G126S) and c.425G>A (p.C142Y) cause misfolding of the γ subunit, while another variant, c.857C>T (p.T286M), does not appear to alter γ subunit function. The misfolded γ subunits were retained in the ER and failed to rescue the lysosomal targeting of lysosomal acid glycosidases.

Dihydroxyacetone (Dha) kinases are a family of sequence-conserved enzymes which utilize either ATP (in animals, plants and eubacteria) or phosphoenolpyruvate (PEP, in eubacteria) as their source of high-energy phosphate. The kinases consist of two domains/subunits: DhaK, which binds Dha covalently in hemiaminal linkage to the Nε2 of a histidine, and DhaL, an eight-helix barrel that contains the nucleotide-binding site. The PEP-dependent kinases comprise a third subunit, DhaM, which rephosphorylates in situ the firmly bound ADP cofactor. DhaM serves as the shuttle for the transfer of phosphate from the bacterial PEP: carbohydrate phosphotransferase system (PTS) to the Dha kinase. The DhaL and DhaK subunits of the PEP-dependent Escherichia coli kinase act as coactivator and corepressor of DhaR, a transcription factor from the AAA+ family of enhancerbinding proteins. In Gram-positive bacteria genes for homologs of DhaK and DhaL occur in operons for putative transcription factors of the TetR and DeoR families. Proteins with the Dha kinase fold can be classified into three families according to phylogeny and function: Dha kinases, DhaK and DhaL homologs (paralogs) associated with putative transcription regulators of the TetR and DeoR families, and proteins with a circularly permuted domain order that belong to the DegV family

Summary The aim of this work was to investigate the potential role of pyrophosphate: fructose 6-phosphate phosphotransferase during cold-induced sweetening in potatoes. The enzyme purified to near homogeneity from mature tubers of Solanum tuberosum cv. Record was reversibly inhibited by low temperature. Under optimal assay conditions the temperature coefficient of the enzyme increased from 1.3 at 25 °C to 2.6 at 5 °C. Low temperature had no significant effect on the affinity of the fully-activated enzyme for its substrates fructose 6-phosphate, pyrophosphate, fructose 1,6-bisphosphate and phosphate. However, the concentration of fructose 2,6-bisphosphate required for half-maximal activation increased about 8-fold as the temperature was lowered from 25 °C to 2 °C. In addition, the extent of activation by this metabolite increased from about 5-fold to 50-fold over the same temperature range. These effects may restrict the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate at low temperature, and thus contribute to the accumulation of hexose phosphates and subsequent sucrose synthesis in the cold.

Tannerella forsythia is a Gram-negative periodontal pathogen lacking the ability to undergo de novo synthesis of amino sugars N -acetylmuramic acid (MurNAc) and N -acetylglucosamine (GlcNAc) that form the disaccharide repeating unit of the peptidoglycan backbone. T. forsythia relies on the uptake of these sugars from the environment, which is so far unexplored. Here, we identified a novel transporter system of T. forsythia involved in the uptake of MurNAc across the inner membrane and characterized a homolog of the Escherichia coli MurQ etherase involved in the conversion of MurNAc-6-phosphate (MurNAc-6-P) to GlcNAc-6-P. The genes encoding these components were identified on a three-gene cluster spanning Tanf_08375 to Tanf_08385 located downstream from a putative peptidoglycan recycling locus. We show that the three genes, Tanf_08375, Tanf_08380, and Tanf_08385, encoding a MurNAc transporter, a putative sugar kinase, and a MurQ etherase, respectively, are transcriptionally linked. Complementation of the Tanf_08375 and Tanf_08380 genes together in trans , but not individually, rescued the inability of an E. coli mutant deficient in the phosphotransferase (PTS) system-dependent MurNAc transporter MurP as well as that of a double mutant deficient in MurP and components of the PTS system to grow on MurNAc. In addition, complementation with this two-gene construct in E. coli caused depletion of MurNAc in the medium, further confirming this observation. Our results show that the products of Tanf_08375 and Tanf_08380 constitute a novel non-PTS MurNAc transporter system that seems to be widespread among bacteria of the Bacteroidetes phylum. To the best of our knowledge, this is the first identification of a PTS-independent MurNAc transporter in bacteria. IMPORTANCE In this study, we report the identification of a novel transporter for peptidoglycan amino sugar N -acetylmuramic acid (MurNAc) in the periodontal pathogen T. forsythia . It has been known since the late 1980s that T. forsythia is a MurNAc auxotroph relying on environmental sources for this essential sugar. Most sugar transporters, and the MurNAc transporter MurP in particular, require a PTS phosphorelay to drive the uptake and concurrent phosphorylation of the sugar through the inner membrane in Gram-negative bacteria. Our study uncovered a novel type of PTS-independent MurNAc transporter, and although so far, it seems to be unique to T. forsythia , it may be present in a range of bacteria both of the oral cavity and gut, especially of the phylum Bacteroidetes .

Targeting soluble acid hydrolases to lysosomes requires the addition of mannose 6-phosphate residues on their N-glycans. This process is initiated by GlcNAc-1-phosphotransferase, a multi-subunit enzyme encoded by the GNPTAB and GNPTG genes. The GNPTAB gene products (the α and s subunits) are responsible for recognition and catalysis of hydrolases whereas the GNPTG gene product (the γ subunit) enhances mannose phosphorylation of a subset of hydrolases. Here we identify and characterize a zebrafish gnptg insertional mutant and show that loss of the gamma subunit reduces mannose phosphorylation on a subset glycosidases but does not affect modification of several cathepsin proteases. We further show that glycosidases, but not cathepsins, are hypersecreted from gnptg(-/-) embryonic cells, as evidenced by reduced intracellular activity and increased circulating serum activity. The gnptg(-/-) embryos lack the gross morphological or craniofacial phenotypes shown in gnptab-deficient morphant embryos to result from altered cathepsin activity. Despite the lack of overt phenotypes, decreased fertilization and embryo survival were noted in mutants, suggesting that gnptg associated deposition of mannose 6-phosphate modified hydrolases into oocytes is important for early embryonic development. Collectively, these findings demonstrate that loss of the zebrafish GlcNAc-1-phosphotransferase γ subunit causes enzyme-specific effects on mannose phosphorylation. The finding that cathepsins are normally modified in gnptg(-/-) embryos is consistent with data from gnptab-deficient zebrafish suggesting these proteases are the key mediators of acute pathogenesis. This work also establishes a valuable new model that can be used to probe the functional relevance of GNPTG mutations in the context of a whole animal.

The bacterial phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) consists of cascading phosphotransferases that couple the simultaneous import and phosphorylation of a variety of sugars to the glycolytic conversion of phosphoenolpyruvate (PEP) to pyruvate. As the primary route of glucose uptake in E. coli, the PTS plays a key role in regulating central carbon metabolism and carbon catabolite repression, and is a frequent target of metabolic engineering interventions. Here we show that Enzyme I, the terminal phosphotransferase responsible for the conversion of PEP to pyruvate, is responsible for a significant in vivo flux in the reverse direction (pyruvate to PEP) during both gluconeogenic and glycolytic growth. We use 13C alanine tracers to quantify this back-flux in single and double knockouts of genes relating to PEP synthetase and PTS components. Our findings are relevant to metabolic engineering design and add to our understanding of gene-reaction connectivity in E. coli., Enzyme I, a component of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), converts phosphoenolpyruvate to pyruvate. Here, the authors show that Enzyme I facilitates also the reverse reaction during both gluconeogenic and glycolytic growth in E. coli.

The Golgi enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2 hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous non-lysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the α subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif.

Background Oenococcus oeni is a lactic acid bacterium that is specialised for growth in the ecological niche of wine, where it is noted for its ability to perform the secondary, malolactic fermentation that is often required for many types of wine. Expanding the understanding of strain-dependent genetic variations in its small and streamlined genome is important for realising its full potential in industrial fermentation processes. Results Whole genome comparison was performed on 191 strains of O. oeni; from this rich source of genomic information consensus pan-genome assemblies of the invariant (core) and variable (flexible) regions of this organism were established. Genetic variation in amino acid biosynthesis and sugar transport and utilisation was found to be common between strains. Furthermore, we characterised previously-unreported intra-specific genetic variations in the natural competence of this microbe. Conclusion By assembling a consensus pan-genome from a large number of strains, this study provides a tool for researchers to readily compare protein-coding genes across strains and infer functional relationships between genes in conserved syntenic regions. This establishes a foundation for further genetic, and thus phenotypic, research of this industrially-important species. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2604-7) contains supplementary material, which is available to authorized users.

Rifampin (RIF) is a first-line drug used for the treatment of tuberculosis and other bacterial infections. Various RIF resistance mechanisms have been reported, and recently an RIF-inactivation enzyme, RIF phosphotransferase (RPH), was reported to phosphorylate RIF at its C21 hydroxyl at the cost of ATP. However, the underlying molecular mechanism remained unknown. Here, we solve the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations. LmRPH comprises three domains: an ATP-binding domain (AD), an RIF-binding domain (RD), and a catalytic His-containing domain (HD). Structural analyses reveal that the C-terminal HD can swing between the AD and RD, like a toggle switch, to transfer phosphate. In addition to its catalytic role, the HD can bind to the AD and induce conformational changes that stabilize ATP binding, and the binding of the HD to the RD is required for the formation of the RIF-binding pocket. A line of hydrophobic residues forms the RIF-binding pocket and interacts with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moieties of RIF. The R group of RIF points toward the outside of the pocket, explaining the low substrate selectivity of RPH. Four residues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and Gln337, were found to play essential roles in the phosphorylation of RIF; among these the His825 residue may function as the phosphate acceptor and donor. Our study reveals the molecular mechanism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins.

In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

Transposon insertion into Listeria monocytogenes Imo2665, which encodes an EIIC of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), was found to prevent D-arabitol utilization. We confirm this result with a deletion mutant and show that Lmo2665 is also required for D-xylitol utilization. We therefore called this protein EIICAxl. Both pentitols are probably catabolized via the pentose phosphate pathway (PPP) because Imo2665 belongs to an operon, which encodes the three PTSAxl components, two sugar-P dehydrogenases, and most PPP enzymes. The two dehydrogenases oxidize the pentitol-phosphates produced during PTS-catalyzed transport to the PPP intermediate xylulose-5-P. L. monocytogenes contains another PTS, which exhibits significant sequence identity to PTSAxl. Its genes are also part of an operon encoding PPP enzymes. Deletion of the EIIC-encoding gene (Imo0508) affected neither D-arabitol nor D-xylitol utilization, although D-arabitol induces the expression of this operon. Both operons are controlled by MtIR/LicR-type transcription activators (Lmo2668 and Lnno0501, respectively). Phosphorylation of Lmo0501 by the soluble PTSAxl components probably explains why D-arabitol also induces the second pentitol operon. Listerial virulence genes are submitted to strong repression by PTS sugars, such as glucose. However, D-arabitol inhibited virulence gene expression only at high concentrations, probably owing to its less efficient utilization compared to glucose.

The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates, and is involved in signal transduction. It comprises two general phosphotransferase proteins (EI and HPr) and a—species dependent—variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC). EI and HPr transfer phosphoryl groups from PEP to the IIA units. IIA and IIB sequentially transfer phosphates to the sugar, which is translocated by the IIC unit. The ratio of phosphorylated to non-phosphorylated IIA and IIB varies with transport activity, and the phosphorylation state of some of the IIA and IIB serves as signal input for regulation of catabolite repression, intermediate metabolism, gene expression and chemotaxis in response to the availability of carbohydrates and PEP (glycolytic activity). PTS occur in about one-third of all eubacteria and in a few archaebacteria but not in animals and plants. Uniqueness and pleiotropic function make the PTS a potential target for anti-infectives. The PTS transporter for mannose is utilized as a gate for the penetration of bacteriophage lambda DNA across, and insertion of certain bacteriocins (small antimicrobial peptides) into the inner membrane. The PTS of Escherichia coli is in the focus of this review, but occasionally comparisons with other species are made. The topics are: History; Modular design of the E. coli PTS; Structure function and catalytic mechanism of the protein modules; Regulation of and by the PTS; The PTS in pathogenicity and virulence; Computational models; Metabolic engineering.

Triclosan is a widely used biocide effective against different microorganisms. At bactericidal concentrations, triclosan appears to affect multiple targets, while at bacteriostatic concentrations, triclosan targets FabI. The site-specific antibiotic-like mode-of-action and a widespread use of triclosan in household products claimed to possibly induce cross-resistance to other antibiotics. Thus, we set out to define more systematically the genes conferring resistance to triclosan; A genomic library of Escherichia coli strain W3110 was constructed and enriched in a selective medium containing a lethal concentration of triclosan. The genes enabling growth in the presence of triclosan were identified by using a DNA microarray and confirmed consequently by ASKA clones overexpressing the selected 62 candidate genes. Among these, forty-seven genes were further confirmed to enhance the resistance to triclosan; these genes, including the FabI target, were involved in inner or outer membrane synthesis, cell-surface material synthesis, transcriptional activation, sugar phosphotransferase (PTS) systems, various transporter systems, cell division, and ATPase and reductase/dehydrogenase reactions. In particular, overexpression of pgsA, rcsA, or gapC conferred to E. coli cells a similar level of triclosan resistance induced by fabI overexpression. These results indicate that triclosan may have multiple targets other than well-known FabI and that there are several undefined novel mechanisms for the resistance development to triclosan, thus probably inducing cross antibiotic resistance.

Bacteriophage T7 encodes a serine/threonine-specific protein kinase that phosphorylates multiple cellular proteins during infection of Escherichia coli. Recombinant T7 protein kinase (T7PK), normally purified in phosphorylated form, exhibits a modest level of phosphotransferase activity. A procedure is described that provides dephosphorylated T7PK with an enhanced ability to phosphorylate protein substrates, including translation initiation factor IF1 and the nuclease domain of ribonuclease III. Mass spectrometric analysis identified Thr12 as the site of IF1 phosphorylation in vitro. T7PK undergoes Mg(2+)-dependent autophosphorylation on Ser216 in vitro, which also is modified in vivo. The inability to isolate the presumptive autophosphorylation-resistant T7PK Ser216Ala mutant indicates a toxicity of the phosphotransferase activity and suggests a role for Ser216 modification in limiting T7PK activity during infection.

5′-Nucleotides including 5′-inosinic acid have characteristic taste and important application in various foods as flavour potentiators. The selective nucleoside acid phosphatase/phosphotransferase (AP/PTase) can catalyse the synthesis of 5′-nucleotides by transfer of phosphate groups. In this study, a 747-bp gene encoding AP/PTase from Escherichia blattae was synthesised. After expression, the recombinant AP/PTase was purified using nickel–NTA. The optimal temperature and pH of this enzyme were 30 °C and 5.0, respectively. The activity was partially inhibited by metal ions such as Hg 2+ , Ag + and Cu 2+ , but not by chelating reagents such as EDTA. The values of K m and V max for inosine were 40 mM and 3.5 U/mg, respectively. Using this purified enzyme, 16.83 mM of 5′-IMP was synthesised from 37 mM of inosine and the molar yield reached 45.5%. Homology modelling and docking simulation were discussed.

The present review devoted to the analysis of recent literature on genetic determination and the domain organization of the newly discovered two-component signaling systems in pro- and eukaryotes. These structures are involved in the regulation of numerous morphological and physiological processes in plants. RR-proteins, it the key elements of signaling systems, they launch a cascade of phosphotransferase reactions and directly or indirectly regulate the transcription and activity other proteins, including enzymes, in response to hormones or environmental factors. Modern views on the molecular and genetic mechanisms of photoperiodic response, circadian rhythms and anti-stress responses in plants are set out in these positions. The relationship between gene expression and photoreceptor sensitivity of plants to photoperiod traced. We present our own data obtained on the isogenic lines of wheat, where been showed dependence expression of structural genes of enzymes on the allelic composition of individual PRR-loci and the duration action of low temperature.

Many Proteobacteria possess the paralogous PTS(Ntr), in addition to the sugar transport phosphotransferase system (PTS). In the PTS(Ntr) phosphoryl-groups are transferred from phosphoenolpyruvate to protein EIIA(Ntr) via the phosphotransferases EI(Ntr) and NPr. The PTS(Ntr) has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTS(Ntr) plays a role in potassium homeostasis. In particular, EIIA(Ntr) binds to and stimulates activity of a two-component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high-affinity K(+) transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTS(Ntr). The pho regulon, which comprises more than 30 genes, is activated by the two-component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA(Ntr) are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild-type level in mutants deficient for EIIA(Ntr) phosphorylation suggesting that non-phosphorylated EIIA(Ntr) modulates pho. Protein interaction analyses reveal binding of EIIA(Ntr) to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA(Ntr) is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli.

The control of several catabolic operons in bacteria by transcription antitermination is mediated by RNA-binding proteins that consist of an RNA-binding domain and two reiterated phosphotransferase system regulation domains (PRDs). The Bacillus subtilis GlcT antitermination protein regulates the expression of the ptsG gene, encoding the glucose-specific enzyme II of the phosphotransferase system. In the absence of glucose, GlcT becomes inactivated by enzyme II-dependent phosphorylation at its PRD1, whereas the phosphotransferase HPr phosphorylates PRD2. However, here we demonstrate by NMR analysis and mass spectrometry that HPr also phosphorylates PRD1 in vitro but with low efficiency. Size exclusion chromatography revealed that non-phosphorylated PRD1 forms dimers that dissociate upon phosphorylation. The effect of HPr on PRD1 was also investigated in vivo. For this purpose, we used GlcT variants with altered domain arrangements or domain deletions. Our results demonstrate that HPr can target PRD1 when this domain is placed at the C terminus of the protein. In agreement with the in vitro data, HPr exerts a negative control on PRD1. This work provides the first insights into how specificity is achieved in a regulator that contains duplicated regulatory domains with distinct dimerization properties that are controlled by phosphorylation by different phosphate donors. Moreover, the results suggest that the domain arrangement of the PRD-containing antitermination proteins is under selective pressure to ensure the proper regulatory output, i.e. transcription antitermination of the target genes specifically in the presence of the corresponding sugar.

The gene that encodes the catabolite repressor/activator, Cra (FruR), of Pseudomonas putida is divergent from the fruBKA operon for the uptake of fructose via the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS(Fru)). The expression of the fru cluster has been studied in cells growing on substrates that change the intracellular concentrations of fructose-1-P (F1P), the principal metabolic intermediate that counteracts the DNA-binding ability of Cra on an upstream operator. While the levels of the regulator were not affected by any of the growth conditions tested, the transcription of fruB was stimulated by fructose but not by the gluconeogenic substrate, succinate. The analysis of the P(fruB) promoter activity in a strain lacking the Cra protein and the determination of key metabolites revealed that this regulator represses the expression of PTS(Fru) in a fashion that is dependent on the endogenous concentrations of F1P. Because FruB (i.e. the EI-HPr-EIIA(Fru) polyprotein) can deliver a high-energy phosphate to the EIIA(Ntr) (PtsN) enzyme of the PTS(Ntr) branch, the cross-talk between the two phosphotransferase systems was examined under metabolic regimes that allowed for the high or low transcription of the fruBKA operon. While fructose caused cross-talk, succinate prevented it almost completely. Furthermore, PtsN phosphorylation by FruB occurred in a Δcra mutant regardless of growth conditions. These results traced the occurrence of the cross-talk to intracellular pools of Cra effectors, in particular F1P. The Cra/F1P duo seems to not only control the expression of the PTS(Fru) but also checks the activity of the PTS(Ntr) in vivo.

The organisation of genes conferring utilisation of raffinose family oligosaccharides (RFOs) has been analysed in several probiotic bacteria from the Bifidobacterium and Lactobacillus genera. Glycoside hydrolase family 36 (GH36) α-galatosidase encoding genes occur together with sugar transport systems of the glycoside–pentoside–hexuronide cation symporter family (GPH), sugar phosphotransferase systems (PTSs) or ATP-binding cassette systems (ABCs) highlighting the diversity of RFO uptake. The GH36 genes are often clustered together with sucrose hydrolases or phosphorylases ensuring the degradation of RFO to monosaccharides. Differential proteomics and transcriptomics data from our laboratories implicated ABC transporters in the uptake of RFO in both Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Interestingly, only one of three GH36 encoding genes in B. animalis subsp. lactis Bl-04 was upregulated upon growth on RFO, suggesting that the other two gene products may have dif...

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (

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.

Phosphatidylinositol-specific phospholipase C (PI-PLC) is a virulence factor produced and secreted by many Gram positive bacteria (1). When secreted by extracellular pathogens (e.g., Bacillus and Staphylococcus strains (2–5) this enzyme catalyzes the cleavage of glycan-phosphatidylinositol (GPI) anchored proteins. PI-PLC removal of the protective GPI-anchored proteins on the exterior surface of mammalian cells allows cellular damage to occur, and the diacylglycerol, translocating across the bilayer (6), can interfere with cellular signaling processes (7). Phosphatidylinositol molecules are also good substrates for this enzyme and provide an easier system to explore mechanistic details of these enzymes (1). PI-PLC cleaves its substrate via an intramolecular phosphotransferase reaction on the PI moiety to form a cyclic inositol phosphate molecule and diacylglycerol (8). Subsequent hydrolysis of the cyclic phosphodiester produces inositol 1-phosphate if PI is the substrate (9, 10). The PI-PLC from S. aureus is unusual in exhibiting significant activity towards PI at acidic pH (4) – a property that may contribute to the virulence of the organism, which is often in an acidic milieu (11). Crystal structures of the PI-PLC enzymes from Bacillus species (12, 13) and Listeria monocytogenes (14), an intracellular pathogen, show very similar distorted TIM barrels with an active site close to the barrel rim. Each has a short helix at the rim that contains at least one lysine, important for binding to negatively charged interfaces, and a tryptophan that, in the case of the Bacillus PI-PLC, has been proposed to insert into target membranes (15–17). Another key structural feature is a rim loop near the active site that contains one large hydrophobic residue (either a tryptophan or phenylalanine) amid many small flexible residues. This surface-exposed hydrophobic residue is also thought to aid in anchoring the protein to membranes (15, 16). Several of the bacterial PI-PLC enzymes exhibit kinetic activation by non-substrate phospholipids. Specifically, inclusion of PC in vesicles containing PI lead to large increases in the specific activity of these secreted PI-PLC enzymes (9, 18, 19). The detailed mechanism for PC activation is not the same for the different enzymes. B. thuringiensis PI-PLC has a discrete site for PC binding (20), while the L. monocytogenes PI-PLC requires PC to dilute cationic enzyme/anionic lipid aggregates that trap the enzyme in a nonproductive state (19). The PI-PLC from S. aureus can also exhibit a significant increase in specific activity towards PI in vesicles containing PC (vide infra). We have solved the structure of S. aureus PI-PLC strain FPR3757 at two different pH values: (i) pH 4.6, where the enzyme shows low activity (but significantly higher than other bacterial PI-PLC enzymes), and (ii) pH 7.5, where it is very active towards PI/PC (but not pure PI) vesicles. A large conformational change in the rim mobile loop between the acidic and basic pH structures depends on a titratable π-histidine cation interaction. π-Cation interactions are well known in proteins, however these interactions commonly exist in protein-protein interfaces, enzyme-drug interactions, helix stabilization and protein-DNA interactions (21–23). π-Cation complexes with protonated histidine as the cation are also known (24). Most of these interactions result in subtle structural changes; typically they mediate ligand binding or helix stabilization (25, 26). However, a recent structure of the transferrin receptor showed a large change in structure in the dimer interface region upon transferrin binding whereby a protonated surface histidine flipped into the protein to engage in π-stacking with a phenylalanine and tyrosine (27). Observation of the titratable intramolecular π-cation interaction in S. aureus PI-PLC suggests higher activity at acidic pH is connected with easier release of water-soluble product inositol 1,2-(cyclic)phosphate (cIP). This structural feature also provides an explanation for how PC may activate this bacterial PI-PLC. The presence of that zwitterionic phospholipid in a substrate-containing interface provides cationic choline moieties that can compete with cationic His258 for interactions with the π system of Phe249 allowing the latter to partition into membranes. The higher activity once PC is added indicates that this membrane conformation must differ from the one where only PI is present.

Arginine kinase (ATP: l -arginine ω- N -phosphotransferase, EC2.7.3.3.; AK) is an enzyme with crucial functions in the energy metabolism of insects and other invertebrates as well as some protozoa and has been proposed as a parasiticidal and pesticidal drug target. In this study we report the identification, cDNA cloning, genomic gene structure and functional expression of an AK gene from the parasitic sheep blowfly Lucilia ( L. ) cuprina . The blowfly AK-encoding gene is devoid of introns and present in a single copy in the L. cuprina genome. AK is present in L. cuprina flies as a highly expressed soluble enzyme and is more abundant in the thorax, where it represents up to 2% of the total soluble protein, compared to head or abdomen. Guanidino substrate specificity studies show that L. cuprina AK is stereospecific for the l -form over the d -form of its specific substrate arginine. Furthermore, the presence of a free or esterified carboxylic group as well as an unsubstituted α-amine group are essential for acceptance of the l -arginine substrate while modifications in the aliphatic side chain are better tolerated. The apparent Michaelis–Menten constants K M and the molecular size of the recombinant enzyme are virtually identical to that of the native enzyme suggesting that the gene cloned in this study encodes the highly expressed AK enzyme of the sheep blowfly.

Rice sheath blight disease caused by Rhizoctonia solani Kuhn results in significant yield and quality losses in rice growing areas worldwide. The glycolytic pathway is important in the resistance response to R. solani infection in rice. This study examined one of the regulatory steps in this pathway catalyzed by pyrophosphate-fructose-6-phosphate-phosphotransferase (PFP) and 6-phosphofructokinase (PFK). PFP and PFK activity in R. solani -infected rice plants increased. The mRNA expression of PFP/PFK isozymes showed that PFK 1, 2, 4 and 5 in the resistant line at 1 dpi were high as compared to the gradual increase observed in the expression of all PFP isozymes. Also PFK 1, PFK 3, PFK 4, PFK 5, PFP 2 and PFP 5 were adaptive to sheath blight disease infection and linked to defence response while, the expressions of PFK 2, PFP 1, PFP 3 and PFP 4 although adaptive, were not specific to R. solani infection. These observations provide evidence that (a) both PFP and PFK have isozymes that play an adaptive role after R. solani infection but while those of PFK are expressed at higher levels within a short time after infection those of PFP are expressed gradually, (b) the adaptive activation of PFP in R. solani -infected rice plants is correlated with the paired expression of its α- and β- subunits as shown by PFP 2 and PFP 5, and (c) the expression of some α- subunits is not specific to R. solani infection as shown by PFP 1, PFP 3 and PFP 4.

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in C4 photosynthesis and is also found in C3 plants. It is post-translationally modified by the PPDK regulatory protein (RP) that possesses both kinase and phosphotransferase activities. Phosphorylation and dephosphorylation of PPDK lead to inactivation and activation respectively. Arabidopsis thaliana contains two genes that encode chloroplastic (RP1) and cytosolic (RP2) isoforms of RP, and although RP1 has both kinase and phosphotransferase activities, to date RP2 has only been shown to act as a kinase. Here we demonstrate that RP2 is able to catalyse the dephosphorylation of PPDK, although at a slower rate than RP1 under the conditions of our assay. From yeast two-hybrid analysis we propose that RP1 binds to the central catalytic domain of PPDK, and that additional regions towards the carboxy and amino termini are required for a stable interaction between RP2 and PPDK. For 21 highly conserved amino acids in RP1, mutation of 15 of these reduced kinase and phosphotransferase activity, while mutation of six residues had no impact on either activity. We found no mutant in which only one activity was abolished. However, in some chimaeric fusions that comprised the amino and carboxy termini of RP1 and RP2 respectively, the kinase reaction was severely compromised but phosphotransferase activity remained unaffected. These findings are consistent with the findings that both RP1 and RP2 modulate reversibly the activity of PPDK, and possess one bifunctional active site or two separate sites in close proximity.

The phosphoenolpyruvate-(PEP)-dependent-carbohydrate:phosphotransferase systems (PTSs) of enteric bacteria constitute a complex transport and sensory system. Such a PTS usually consists of two cytoplasmic energy-coupling proteins, Enzyme I (EI) and HPr, and one of more than 20 different carbohydrate-specific membrane proteins named Enzyme II (EII), which catalyze the uptake and concomitant phosphorylation of numerous carbohydrates. The most prominent representative is the glucose-PTS, which uses a PTS-typical phosphorylation cascade to transport and phosphorylate glucose. All components of the glucose-PTS interact with a large number of non-PTS proteins to regulate the carbohydrate flux in the bacterial cell. Several aspects of the glucose-PTS have been intensively investigated in various research projects of many groups. In this article we will review our recent findings on a Glc-PTS-dependent metalloprotease, on the interaction of EIICB(Glc) with the regulatory peptide SgrT, on the structure of the membrane spanning C-domain of the glucose transporter and on the modeling approaches of ptsG regulation, respectively, and discuss them in context of general PTS research.

The molecular mechanisms of human cytomegalovirus (CMV) resistance to both ganciclovir and maribavir reported so far rely predominantly on the presence of mutations within UL97 phosphotransferase. The accurate interpretation of genotypic antiviral resistance assay results requires the clear distinction between resistance mutations and natural interstrain sequence variations. The objective of this work was to extend the catalog of CMV UL97 phosphotransferase natural polymorphisms. The full-length UL97 gene sequence analysis from 4 laboratory strains and 35 clinical samples from patients who had not received any previous anti-CMV treatment was performed. At the nucleotide level, the interstrain identity was >98%. At the amino acid level, ten natural polymorphisms never previously described were identified. Together with all previous results reported in the literature, a new map of UL97 phosphotransferase natural polymorphism could be settled in the era of ganciclovir and maribavir.

Bacterial phosphoenolpyruvate-dependent phosphotransferase systems (PTS) play multiple roles in addition to sugar transport. Recent studies revealed that enzyme IIA(Ntr) of the nitrogen PTS regulates the intracellular concentration of K(+) by direct interaction with TrkA and KdpD. In this study, we show that dephosphorylated NPr of the nitrogen PTS interacts with Escherichia coli LpxD which catalyzes biosynthesis of lipid A of the lipopolysaccharide (LPS) layer. Mutations in lipid A biosynthetic genes such as lpxD are known to confer hypersensitivity to hydrophobic antibiotics such as rifampin; a ptsO (encoding NPr) deletion mutant showed increased resistance to rifampin and increased LPS biosynthesis. Taken together, our data suggest that unphosphorylated NPr decreases lipid A biosynthesis by inhibiting LpxD activity.

The central carbon metabolic pathway is the most important among metabolic pathways in all microorganisms since it produces energy and precursors for biosynthesis. In this study, a dynamic model for central carbon metabolism in Escherichia coli (E. coli) consisting of the phosphotransferase (PTS) system, glycolysis, pentose-phosphate pathway (PPP), and storage materials was obtained by ameliorating the model proposed by Chassagnole et al. (2002). In order to improve the performance of the model, principal parameters were estimated through the experimental measurements of intracellular concentrations of metabolites under transient conditions. Through dynamic metabolic control analysis (MCA), the tendencies of the metabolic fluxes at branch points were investigated, and the key parameters and enzyme kinetics that most dominantly affected the productivity of the desired metabolites were determined.

An analysis of the apical domain of the Group-I and Group-II chaperonins shows that they have structural similarities to two different protein folds: a "swivel-domain'' phosphotransferase and a thioredoxin-like peroxiredoxin. There is no significant sequence similarity that supports either similarity and the degree of similarity based on structure is comparable but weak for both relationships. Based on possible evolutionary transitions, we deduced that a phosphotransferase origin would require both a large insertion and deletion of structure whereas a peroxiredoxin origin requires only a peripheral rearrangement, similar to an internal domain-swap. We postulate that this change could have been triggered by the insertion of a peroxiredoxin into the ATPase domain that led to the modern chaperonin domain arrangement. The peroxidoxin fold is the most highly embellished member of the thioredoxin super-family and the insertion event may have "overloaded'' the core, leading to a rearrangement. A peroxiredoxin origin for the domain also provides a functional explanation, as the peroxiredoxins can act as chaperones when they adopt a multimeric ring complex, similar to the chaperonin subunit configuration. In addition, several of the GroEL apical domain hydrophobic residues which interact with the unfolded protein are located in a position that corresponds to the protein substrate binding region of the peroxiredoxin fold. We suggest that the origin of the ur-chaperonin from a thioredoxin/peroxiredoxin fold might also account for the number of thioredoxin-fold containing proteins that interact with chaperonins, such as tubulin and phosducin-like proteins.

GlcNAc-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate, a recognition marker essential for efficient transport of lysosomal hydrolases to lysosomes. The enzyme complex is composed of six subunits (α(2)β(2)γ(2)). The α- and β-subunits are catalytically active, whereas the function of the γ-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse γ-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed γ-subunits were localized in the cis-Golgi apparatus. Secreted forms of γ-subunits were detectable in media of cultured cells as well as in human serum. The γ-subunit contains two in vivo used N-glycosylation sites at positions 88 and 115, equipped with high mannose-type oligosaccharides. (35)S pulse-chase experiments and size exclusion chromatography revealed that the majority of non-glycosylated γ-subunit mutants were integrated in high molecular mass complexes, failed to exit the endoplasmic reticulum (ER), and were rapidly degraded. The substitution of cysteine 245 involved in dimerization of γ-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric γ-subunits failed, however, to associate with other GlcNAc-1-phosphotransferase subunits. The data provide evidence that assembly of the GlcNAc-1-phosphotransferase complex takes place in the ER and requires dimerization of the γ-subunits.

Corynebacterium glutamicum R has two β-glucoside phosphoenolpyruvate, carbohydrate phosphotransferase systems (PTS) encoded by bglF and bglF2 located in the respective clusters, bglF-bglA-bglG and bglF2-bglA2-bglG2 . Previously, we reported that whereas β-glucoside-dependent induction of bglF is strongly repressed by glucose, glucose repression of bglF2 is very weak. Here, we reveal the mechanism behind the different effects of glucose on the two bgl genes. Deletion of the ribonucleic antiterminator sequence and transcriptional terminator located upstream of the translation initiation codon of bglF markedly relieved the glucose repression of a bglF-lacZ fusion, indicating that glucose affects the antitermination mechanism that is responsible for the β-glucoside-dependent induction of the bglF cluster. The glucose repression of bglF mRNA was also relieved by introducing a multicopy plasmid carrying the bglG gene encoding an antiterminator of the bglF cluster. Moreover, replacement of the GUG translation initiation codon of bglG with AUG was effective in relieving the glucose repression of bglF and bglG . Inversely, expression of bglF2 and bglG2 was subject to strict glucose repression in a mutant strain in which the AUG translation initiation codon of bglG2 encoding antiterminator of the bglF2 cluster was replaced with GUG. These results suggest that the translation initiation efficiency of the antiterminator proteins, at least in part, determines whether the target genes are subject to glucose repression. We also found that bglF expression was induced by glucose in the BglG-overexpressing strains, which may be explained by the ability of BglF to transport glucose.

A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395, and its activity was studied in Escherichia coli. The 3.9-kb operon comprising three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EII(Mtl)) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD), and a mannitol operon repressor (MtlR). The transport of [(3)H]mannitol by the cloned mannitol operon in E. coli was 13.8 ± 1.4 nmol/min/mg protein. The insertional inactivation of EII(Mtl) abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram-positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon.

Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme is generally composed of two catalytic (α and/or α') and two regulatory (β) subunits, forming a differently assembled tetramer. The free and catalytically active α/α' subunits can be present in cells under some circumstances. We present here the isolation of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2β subunits were constructed and cloned into expression vectors, and the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2β subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz), and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2β subunits have similar features to their mammalian orthologs.

In a scientific career spanning from 1955 to 2000, my research focused on phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. Grounded in basic enzymology, and initially pursuing the steady-state rate behavior of isolated preparations of these critically important gluconeogenic enzymes, our key findings were confirmed and extended by in situ enzyme rate experiments exploiting isolated liver perfusions. These efforts culminated in the discovery of the liver cytosolic isozyme of carboxykinase, known today as (GTP)PEPCK-C (EC4.1.1.32) and also revealed a biosynthetic function and multicomponent nature of glucose-6-phosphatase (EC3.1.3.9). Discovery that glucose-6-phosphatase possessed an intrinsically biosynthetic activity, now known as carbamyl-P:glucose phosphotransferase - along with a deeper consideration of the enzyme's hydrolytic activity as well as the action of liver glucokinase resulted in the evolution of Tuning/Retuning Hypothesis for blood glucose homeostasis in health and disease. This THEN & NOW review shares with the reader the joy and exhilaration of major scientific discovery and also contrasts the methodologies and approaches on which I relied with those currently in use.

Twenty-four mutacin-producing Streptococcus mutans strains were screened for their propensity to produce class II one-peptide bacteriocin using a deferred antagonism assay. Streptococcus salivarius and 3 mutants defective in their mannose phosphotransferase systems (mannose-PTS) were used as sensitive strains to identify which mannose-PTS could act as the docking site for class II one-peptide bacteriocin activity. We observed that only 2 strains of S. mutans, T9 and 3B, potentially produce class II one-peptide bacteriocin, namely mutacins I-T9 and R-3B, but with no preference for any mannose-PTS complex as a target.

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.

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 (kcat/Km) 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.

Directed evolution by random PCR mutagenesis of the gene for the aminoglycoside 2-IIa phosphotransferase generated R92H/D268N and N196D/D268N mutant enzymes, resulting in elevated levels of resistance to amikacin and isepamicin but not to other aminoglycoside antibiotics. Increases in the activities of the mutant phosphotransferases for isepamicin are the result of decreases in Km values, while improved catalytic efficiency for amikacin is the result of both a decrease in Km values and an increase in turnover of the antibiotic. Enzymes with R92H, D268N, and D268N single amino acid substitutions did not result in elevated MICs for aminoglycosides. Since the introduction of streptomycin into clinical use in 1944, aminoglycoside antibiotics have been used for treatment of serious bacterial infections for more than 70 years (8, 14). In response to their extensive use, bacterial isolates acquired resistance to many aminoglycosides, rendering some of them obsolete (15, 19). The major mechanism of resistance to aminoglycoside antibiotics in clinical bacterial isolates is the production of three types of aminoglycoside-modifying enzymes, aminoglycoside phosphotransferases (APHs), aminoglycoside acetyltransferases (AACs), and aminoglycoside nucleotidyl

Capuramycin-related compounds, including A-500359s and A-503083s, are nucleoside antibiotics that inhibit the enzyme bacterial translocase I involved in peptidoglycan cell wall biosynthesis. Within the biosynthetic gene cluster for the A-500359s exists a gene encoding a putative aminoglycoside 3-phosphotransferase that was previously demonstrated to be highly expressed during the production of A-500359s and confers selective resistance to capuramycins when expressed in heterologous hosts. A similar gene (capP) was identified within the biosynthetic gene cluster for the A-503083s, and CapP is now shown to similarly confer selective resistance to capuramycins. Recombinant CapP was produced and purified from Escherichia coli, and the function of CapP is established as an ATP-dependent capuramycin phosphotransferase that regio-specifically transfers the gamma-phosphate to the 3''-hydroxyl of the unsaturated hexuronic acid moiety of A-503083 B. Kinetic analysis with the three major A-503083 congeners suggests that CapP preferentially phosphorylates A-503083s containing an aminocaprolactam moiety attached to the hexuronic acid, and bi-substrate kinetic analysis was consistent with CapP employing a sequential kinetic mechanism similar to most known aminoglycoside 3-phosphotransferases. The purified CapP product lost its antibiotic activity against Mycobacterium smegmatis, and this loss in bioactivity is primarily due to a 272-fold increase in the IC(50) in the bacterial translocase I-catalyzed reaction. The results establish CapP-mediated phosphorylation as a mechanism of resistance to capuramycins and now set the stage to explore this strategy of resistance as a potential mechanism inherent to pathogens and provide the impetus for preparing second generation analogues as a preemptive strike to such resistance strategies.

Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3') isozymes and an APH(2'') enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3') and APH(2'') enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.