Optimizing pH Response of Affinity between Protein G and IgG Fc: HOW ELECTROSTATIC MODULATIONS AFFECT PROTEIN-PROTEIN INTERACTIONS*S?

Protein-protein interaction in response to environmental conditions enables sophisticated biological and biotechnological processes. Aiming toward the rational design of a pH-sensitive protein-protein interaction, we engineered pH-sensitive mutants of streptococcal protein G B1, a binder to the IgG constant region. We systematically introduced histidine residues into the binding interface to cause electrostatic repulsion on the basis of a rigid body model. Exquisite pH sensitivity of this interaction was confirmed by surface plasmon resonance and affinity chromatography employing a clinically used human IgG. The pH-sensitive mechanism of the interaction was analyzed and evaluated from kinetic, thermodynamic, and structural viewpoints. Histidine-mediated electrostatic repulsion resulted in significant loss of exothermic heat of the binding that decreased the affinity only at acidic conditions, thereby improving the pH sensitivity. The reduced binding energy was partly recovered by “enthalpy-entropy compensation.” Crystal structures of the designed mutants confirmed the validity of the rigid body model on which the effective electrostatic repulsion was based. Moreover, our data suggested that the entropy gain involved exclusion of water molecules solvated in a space formed by the introduced histidine and adjacent tryptophan residue. Our findings concerning the mechanism of histidine-introduced interactions will provide a guideline for the rational design of pH-sensitive protein-protein recognition.Molecular interactions govern a number of biological processes, including metabolism, signal transduction, and immunoreaction. A better understanding of the molecular basis for these interactions is crucial for a complete elucidation of biological phenomena and redesign of interactions for drug discovery and industrial biotechnology applications. Interactions between biomolecules are generally characterized by their affinity, specificity, and environmental responsiveness, such as sensitivity to pH. Such pH-dependent ligand binding enables biological processes to function in an “on and off” manner in response to environmental conditions, resulting in sophisticated systems of regulation (e.g. pheromone production (1, 2), immune systems (3-5), and mechanisms of virus survival (6)).From an industrial perspective, pH sensitivity is advantageous to various fields, such as drug delivery systems for medications (7), biosensing techniques (8, 9), and affinity chromatography (10, 11). Although structure-based protein design is a promising technique for improving molecular function (12-15), it is yet difficult to specifically modulate pH sensitivity of a protein-protein interaction without an associated loss of inherent function and/or structural stability. Some naturally occurring proteins undergo substantial conformational change by pH shift, thereby achieving pH-dependent binding for small molecules (2, 4, 16, 17). However, artificial design of an equivalent mechanism involving conformational change is highly problematic. Indeed, proteins have multiple degrees of freedom and consist of a large number of atoms. Therefore, given that the resulting protein must maintain both its innate binding ability and structural stability, the system appears too complicated for rational design. By contrast to the method based on conformational change, a rigid body-based model (i.e. introduction of electrostatic repulsion or attraction into a binding interface between rigid protein domains) could be a more promising approach for pH switching. Naturally occurring proteins with pH sensitivity generally conserve histidine residues (18-21), which function as a pH switch at slightly acidic conditions (pH ∼6.5) near the pK_a of the histidine side chain. In the presence of a histidine residue at a binding interface, dissociation under acidic conditions would be driven by electrostatic repulsion between rigid domains without conformational change (Fig. 1). This mechanism is rather simple and applicable to protein engineering (22, 23). However, to our knowledge, it still remains unclear how systematic design should be carried out and, in particular, how histidine-mediated electrostatic repulsion influences protein-protein interactions. Indeed, very little experimental data are available for the molecular basis of histidine-introduced protein binders.Open in a separate windowFIGURE 1.A schematic model for introduction of histidine-mediated electrostatic repulsion into the binding interface between protein G (GB) and Fc. Protein G residues positioned closely to basic side chains (depicted as B) on Fc were systematically identified by distance calculations and mutated into histidine to cause electrostatic repulsion under acidic conditions. The inset shows an example of candidate positions for the mutation.To better understand the design methodology for a pH-sensitive protein-protein interaction, we generated a number of pH-sensitive streptococcal protein G B1 (24) mutants by rationally introducing histidine residues onto the binding surface. Protein G, a bacterial Fc (fragment of crystallizable region) receptor to the constant region of IgG, has been used as an affinity chromatography binder for antibody immobilization and purification. Protein G has an acidic pH optimum for binding relative to another bacterial Fc receptor, Staphylococcus aureus protein A. The harsh elution conditions are likely to induce acidic conformational changes in antibodies (25, 26) during the purification procedure, causing aggregation that is problematic for pharmaceutical applications. The usefulness of the histidine-mediated electrostatic repulsion for antibody purification was examined by constructing affinity chromatography columns. Using the designed mutants, we analyzed the molecular basis of the histidine-mediated interaction from a kinetic, thermodynamic, and structural perspective. The observed data revealed functional and structural consequences for the introduction of histidine residues. Analysis of our results provides a guideline for the design of pH-dependent protein-protein interactions.     

Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of α-Synuclein: IMPLICATIONS FOR THE PATHOGENESIS OF PARKINSON DISEASE*S?

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln⁷⁹ and Gln¹⁰⁹ serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln⁷⁹ and Gln¹⁰⁹. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn.Parkinson disease (PD)² is a progressive movement disorder that is caused by the loss of dopaminergic neurons in the substantia nigra, the part of the brain responsible for controlling movement. Clinically, PD is manifested in symptoms that include tremors, rigidity, and difficulty in initiating movement (bradykinesia). Pathologically, PD is characterized by the presence of intraneuronal, cytoplasmic inclusions known as Lewy bodies (LB), which are composed primarily of the protein “α-synuclein” (α-syn) (1) and are seen in the post-mortem brains of PD patients with the sporadic or familial forms of the disease (2). α-Syn is a presynaptic protein of 140 residues with a “natively” unfolded structure (3). Three missense point mutations in α-syn (A30P, E46K, and A53T) are associated with the early-onset, dominant, inherited form of PD (4, 5). Moreover, duplication or triplication of the α-syn gene has been linked to the familial form of PD, suggesting that an increase in α-syn expression is sufficient to cause PD. Together, these findings suggest that α-syn plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn oligomerization and fibrillogenesis in vivo remain poorly understood. In vitro aggregation studies have shown that the mutations associated with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but only E46K and A53T α-syn show higher propensity to fibrillize than wild-type (WT) α-syn (6-8). This suggests that oligomerization, rather than fibrillization, is linked to early-onset familial PD (9). Our understanding of the molecular composition and biochemical state of α-syn in LBs has provided important clues about protein-protein interactions and post-translational modifications that may play a role in modulating oligomerization, fibrillogenesis, and LB formation of the protein. In addition to ubiquitination (10), phosphorylation (11, 12), nitration (13, 14), and C-terminal truncation (15, 16), analysis of post-mortem brain tissues from PD and Lewy bodies in dementia patients has confirmed the colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked α-syn monomers and higher molecular aggregates in LBs within dopaminergic neurons (17, 18). Tissue transglutaminase catalyzes a calcium-dependent transamidating reaction involving glutamine and lysine residues, which results in the formation of a covalent cross-link via ε-(γ-glutamyl) lysine bonds (Fig. 2F). To date, seven different isoforms of tTGs have been reported, of which only tTG2 seems to be expressed in the human brain (19), whereas tTG1 and tTG3 are more abundantly found in stratified squamous epithelia (20). Subsequent immuno-histochemical, colocalization, and immunoprecipitation studies have shown that the levels of tTG and cross-linked α-syn species are increased in the substantia nigra of PD brains (17). These findings, combined with the known role of tTG in cross-linking and stabilizing bimolecular assemblies, led to the hypothesis that tTG plays an important role in the initiation and propagation of α-syn fibril formation and that it contributes to fibril stability in LBs. This hypothesis was initially supported by in vitro studies demonstrating that tTG catalyzes the polymerization of the α-syn-derived non-amyloid component (NAC) peptide via intermolecular covalent cross-linking of residues Gln⁷⁹ and Lys⁸⁰ (21) and by other studies suggesting that tTG promotes the fibrillization of amyloidogenic proteins implicated in the pathogenesis of other neurodegenerative diseases such as Alzheimer disease, supranuclear palsy, Huntington disease, and other polyglutamine diseases (22-24). However, recent in vitro studies with full-length α-syn have shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn and inhibits, rather than promotes, its fibrillization in vitro (25, 26). The structural basis of this inhibitory effect and the exact residues involved in tTG-mediated cross-linking of α-syn, as well as structural and functional consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native (—) and cross-linked (- - -) α-syn, showing that most tTG-catalyzed cross-linking products of WT or disease-associated mutant forms of α-syn are intramolecularly linked (predominant peak with two cross-links), and up to three intramolecular cross-links can occur (left shoulder). The abbreviations M and m/cl are used to designate native and cross-linked α-synuclein, respectively. D and E, kinetic analysis of α-syn (A30P) cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic depiction of the tTG-catalyzed chemical reaction (isodipeptide formation) between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn and investigated how cross-linking these residues affects the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Using single-site mutagenesis and mass spectrometry applied to exhaustive proteolytic digests of native and cross-linked monomeric α-syn, we identified Gln¹⁰⁹ and Gln⁷⁹ as the major tTG substrates. We demonstrate that the altered electrophoretic mobility of the intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of tTG-catalyzed cross-linking of Gln¹⁰⁹ to lysine residues in the N terminus of α-syn, which leads to the formation of more compact monomers. Consistent with previous studies, we show that intramolecularly cross-linked α-syn forms off-pathway oligomers that are distinct from those formed by the wild-type protein and that do not convert to fibrils within the time scale of our experiments (3-5 days). We also show that membrane-bound α-syn is a substrate of tTG and that intramolecular cross-linking does not interfere with the ability of monomeric α-syn to adopt an α-helical conformation upon binding to synthetic membranes. These studies provide novel mechanistic insight into the sequence and structural basis of events that allow tTG to inhibit α-syn fibrillogenesis, and they shed light on the potential role of tTG-catalyzed cross-linking in modulating the physiological and pathogenic properties of α-syn.     

Promiscuous Usage of Nucleotides by the DNA Helicase of Bacteriophage T7: DETERMINANTS OF NUCLEOTIDE SPECIFICITY*S?

The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.Helicases are molecular machines that translocate unidirectionally along single-stranded nucleic acids using the energy derived from nucleotide hydrolysis (1–3). The gene 4 protein encoded by bacteriophage T7 consists of a helicase domain and a primase domain, located in the C-terminal and N-terminal halves of the protein, respectively (4). The T7 helicase functions as a hexamer and has been used as a model to study ring-shaped replicative helicases. In the presence of dTTP, T7 helicase binds to single-stranded DNA (ssDNA)³ as a hexamer and translocates 5′ to 3′ along the DNA strand using the energy of hydrolysis of dTTP (5–7). T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; however, dTTP hydrolysis is optimally coupled to DNA unwinding (5).Most hexameric helicases use rATP to fuel translocation and unwind DNA (3). T7 helicase does hydrolyze rATP but with a 20-fold higher K_m as compared with dTTP (5, 8). It has been suggested that T7 helicase actually uses rATP in vivo where the concentration of rATP is 20-fold that of dTTP in the Escherichia coli cell (8). However, hydrolysis of rATP, even at optimal concentrations, is poorly coupled to translocation and unwinding of DNA (9). Other ribonucleotides (rCTP, rGTP, and rUTP) are either not hydrolyzed or the poor hydrolysis observed is not coupled to DNA unwinding (8). Furthermore, Patel et al. (10) found that the form of T7 helicase found in vivo, an equimolar mixture of the full-length gp4 and a truncated form lacking the zinc binding domain of the primase, prefers dTTP and dATP. Therefore, in the present study we have restricted our examination of nucleotides to the deoxyribonucleotides.The nucleotide binding site of the replicative DNA helicases, such as T7 gene 4 protein, bind nucleotides at the subunit interface (Fig. 1) located between two RecA-like subdomains that bind ATP (11, 12). The location of the nucleotide binding site at the subunit interface provides multiple interactions of residues with the bound NTP. A number of cis- and trans-acting amino acids stabilize the bound nucleotide in the nucleotide binding site and also provide for communication between subunits (13–15). Earlier reports revealed that the arginine finger (Arg-522) in T7 helicase is positioned to interact with the γ-phosphate of the bound nucleotide in the adjacent subunit (12, 16). However, His-465 (phosphate sensor), Glu-343 (catalytic base), and Asp-424 (Walker motif B) interacts with the γ-phosphate of the bound nucleotide in the same subunit (12, 17, 18). The arginine finger and the phosphate sensor have been proposed to couple NTP hydrolysis to DNA unwinding. Substitution of Glu-343, the catalytic base, eliminates dTTP hydrolysis (19), and substitution of Asp-424 with Asn leads to a severe reduction in dTTP hydrolysis (20). The conserved Lys-318 in Walker motif A interacts with the β-phosphate of the bound nucleotide and plays an important role in dTTP hydrolysis (21).Open in a separate windowFIGURE 1.Crystal structure of T7 helicase. A, crystal structure of the hexameric helicase C-terminal domain of gp4 (17). The structure reveals a ring-shaped molecule with a central core through which ssDNA passes. The inset shows the interface between two subunits of the helicase with adenosine 5′-{β,γ-imidol}-triphosphate in the nucleotide binding site. B, the nucleotide binding site of a monomer of the gp4 with the crucial amino acid residues reported earlier and in the present study is shown in sticks. The crystal structures of the T7 gene 4 helicase domain (12) with bound dTTP (C) and dATP (D). The structures shown are the nucleotide binding site of T7 helicase as viewed in Pymol by analyzing the PDB files 1cr1 and 1cr2 (12). Arg-504 and Tyr-535 sandwiches the base of the bound dNTP. Additionally, Arg-504 forms a hydrogen bridge with dTTP. Arg-363 interacts specifically with the 3-OH group of bound dATP. AMPPNP, adenosine 5′-(β,γ-imino)triphosphate.Considering the wealth of information on the above residues that are involved in the hydrolysis of dTTP and the coupling of hydrolysis to unwinding, it is intriguing that little information is available on nucleotide specificity. Several crystal structures of T7 helicase in complex with a nucleotide triphosphate are available. However, most of structures were crystallized with a non-hydrolyzable analogue of dTTP or the nucleotide was diffused into the crystal. The crystal structure of the T7 helicase domain bound with dTTP or dATP was reported by Sawaya et al. (12). These structures assisted us in identifying two basic residues (Arg-363 and Arg-504) in close proximity to the sugar and base of the bound nucleotide whose orientation suggested that these residues could be involved in nucleotide selection. Arg-504 together with Tyr-535 sandwich the base of the bound nucleotide at the subunit interface of the hexameric helicase (Fig. 1). Arg-504 and Tyr-535 are structurally well conserved in various helicases (12). However, Arg-504 could make a hydrogen bridge with the OH group of thymidine, thus suggesting a role in dTTP specificity. On the other hand, Arg-363 is in close proximity (∼3.4 Å) to the sugar 3′-OH of bound dATP, whereas in the dTTP-bound structure this residue is displaced by 7.12 Å (Fig. 1) from the equivalent position. Consequently Arg-363 could play a role in dATP binding. The crystal structures do not provide any information on different interaction of residues with the phosphates of dATP and dTTP. However, alignment of the residues in the P-loops of different hexameric helicases reveals that the serine adjacent to the invariant lysine at position 319 (Ser-319) is conserved in bacteriophages, whereas bacterial helicases have a conserved threonine in the equivalent position (supplemental Fig. 1). Bacterial helicases use rATP in the DNA unwinding reactions. whereas T7 helicase preferentially uses dTTP, and bacteriophage T4 gene 41 uses rGTP or rATP (22).Although considerable information is available on the role of residues in nucleotide binding and dTTP hydrolysis, very little is known on the determinants of nucleotide specificity. In the present study we made an attempt to address the role of a few selected residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity, especially dTTP and dATP, both of which are hydrolyzed and mediate DNA unwinding. We show that under physiological conditions T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.     

Enhancing the Activity of a Protein by Stereospecific Unfolding: CONFORMATIONAL LIFE CYCLE OF INSULIN AND ITS EVOLUTIONARY ORIGINS*S??

A central tenet of molecular biology holds that the function of a protein is mediated by its structure. An inactive ground-state conformation may nonetheless be enjoined by the interplay of competing biological constraints. A model is provided by insulin, well characterized at atomic resolution by x-ray crystallography. Here, we demonstrate that the activity of the hormone is enhanced by stereospecific unfolding of a conserved structural element. A bifunctional β-strand mediates both self-assembly (within β-cell storage vesicles) and receptor binding (in the bloodstream). This strand is anchored by an invariant side chain (Phe^B24); its substitution by Ala leads to an unstable but native-like analog of low activity. Substitution by d-Ala is equally destabilizing, and yet the protein diastereomer exhibits enhanced activity with segmental unfolding of the β-strand. Corresponding photoactivable derivatives (containing l- or d-para-azido-Phe) cross-link to the insulin receptor with higher d-specific efficiency. Aberrant exposure of hydrophobic surfaces in the analogs is associated with accelerated fibrillation, a form of aggregation-coupled misfolding associated with cellular toxicity. Conservation of Phe^B24, enforced by its dual role in native self-assembly and induced fit, thus highlights the implicit role of misfolding as an evolutionary constraint. Whereas classical crystal structures of insulin depict its storage form, signaling requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, we envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity. The cryptic threat of misfolding poses a universal constraint in the evolution of polypeptide sequences.How insulin binds to the insulin receptor (IR)² is not well understood despite decades of investigation. The hormone is a globular protein containing two chains, A (21 residues) and B (30 residues) (Fig. 1A). In pancreatic β-cells, insulin is stored as Zn²⁺-stabilized hexamers (Fig. 1B), which form microcrystal-line arrays within specialized secretory granules (1). The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. The monomer is proposed to undergo a change in conformation upon receptor binding (2). In this study, we investigated a site of conformational change in the B-chain (Phe^B24) (arrow in Fig. 1A). In classical crystal structures, this invariant aromatic side chain (tawny in Fig. 1B) anchors an antiparallel β-sheet at the dimer interface (blue in Fig. 1C). Total chemical synthesis is exploited to enable comparison of corresponding d- and l-amino acid substitutions at this site, an approach designated “chiral mutagenesis” (3-5). In the accompanying article, the consequences of this conformational change are investigated by photomapping of the receptor-binding surface (6). Together, these studies redefine the interrelation of structure and activity in a protein central to the hormonal control of metabolism.Open in a separate windowFIGURE 1.Sequence and structure of insulin. A, sequences of the B-chain (upper) and A-chain (lower) with disulfide bridges as indicated. The arrow indicates invariant Phe^B24. The B24-B28 β-strand is highlighted in blue. B, crystal structure of the T₆ zinc insulin hexamer (Protein Data Bank code 4INS): ribbon model (left) and space-filling model (right). The B24-B28 β-strand is shown in blue, and the side chain of Phe^B24 is highlighted in tawny. The B-chain is otherwise dark gray; the A-chain, light gray; and zinc ions, magenta. Also shown at the left are the side chains of His^B10 at the axial zinc-binding sites. C, cylinder model of the insulin dimer showing the B24-B26 antiparallel β-sheet (blue) anchored by the B24 side chain (tawny circle). The A- and B-chains are shown in light and dark gray, respectively. The protomer at the left is shown in the R-state, in which the central α-helix of the B-chain is elongated (B3-B19 in the frayed R^f protomer of T₃R^f₃ hexamers and B1-B19 in the R protomer of R₆ hexamers). The three types of zinc insulin hexamers share similar B24-B26 antiparallel β-sheets as conserved dimerization elements.The structure of an insulin monomer in solution resembles a crystallographic protomer (Fig. 2A) (7-9). The A-chain contains an N-terminal α-helix, non-canonical turn, and second helix; the B-chain contains an N-terminal segment, central α-helix, and C-terminal β-strand. The β-strand is maintained in an isolated monomer wherein the side chain of Phe^B24 (tawny in Fig. 2A), packing against the central α-helix of the B-chain, provides a “plug” to seal a crevice in the hydrophobic core (Fig. 2B). Anomalies encountered in previous studies of insulin analogs suggest that Phe^B24 functions as a conformational switch (4, 7, 10-14). Whereas l-amino acid substitutions at B24 generally impair activity (even by such similar residues as l-Tyr) (15), a seeming paradox is posed by the enhanced activities of nonstandard analogs containing d-amino acids (10-12).

TABLE 1

Previous studies of insulin analogs

Aromatic Prenylation in Phenazine Biosynthesis: DIHYDROPHENAZINE-1-CARBOXYLATE DIMETHYLALLYLTRANSFERASE FROM STREPTOMYCES ANULATUS*S?

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C–C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The K_m value for dimethylallyl diphosphate was determined as 116 μm. For dihydro-PCA, half-maximal velocity was observed at 35 μm. K_cat was calculated as 0.435 s^-1. PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives.The transfer of isoprenyl moieties to aromatic acceptor molecules gives rise to an astounding diversity of secondary metabolites in bacteria, fungi, and plants, including many compounds that are important in pharmacotherapy. However, surprisingly little biochemical and genetic data are available on the enzymes catalyzing the C-prenylation of aromatic substrates. Recently, a new family of aromatic prenyltransferases was discovered in streptomycetes (1), Gram-positive soil bacteria that are prolific producers of antibiotics and other biologically active compounds (2). The members of this enzyme family show a new type of protein fold with a unique α-β-β-α architecture (3) and were therefore termed ABBA prenyltransferases (1). Only 13 members of this family can be identified by sequence similarity searches in the data base at present, and only four of them have been investigated biochemically (3–6). Up to now, only phenolic compounds have been identified as aromatic substrates of ABBA prenyltransferases. We now report the discovery of a new member of the ABBA prenyltransferase family, catalyzing the transfer of a dimethylallyl moiety to C-9 of 5,10-dihydrophenazine 1-carboxylate (dihydro-PCA).² Streptomyces strains produce many of prenylated phenazines as natural products. For the first time, the present paper reports the identification of a prenyltransferase involved in their biosynthesis.Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces several prenylated phenazines, among them endophenazine A and B (Fig. 1A) (7). We wanted to investigate which type of prenyltransferase might catalyze the prenylation reaction in endophenazine biosynthesis. In streptomycetes and other microorganisms, genes involved in the biosynthesis of a secondary metabolite are nearly always clustered in a contiguous DNA region. Therefore, the prenyltransferase of endophenazine biosynthesis was expected to be localized in the vicinity of the genes for the biosynthesis of the phenazine core (i.e. of PCA).Open in a separate windowFIGURE 1.A, prenylated phenazines from S. anulatus 9663. B, biosynthetic gene cluster of endophenazine A.In Pseudomonas, an operon of seven genes named phzABCDEFG is responsible for the biosynthesis of PCA (8). The enzyme PhzC catalyzes the condensation of phosphoenolpyruvate and erythrose-4-phosphate (i.e. the first step of the shikimate pathway), and further enzymes of this pathway lead to the intermediate chorismate. PhzD and PhzE catalyze the conversion of chorismate to 2-amino-2-deoxyisochorismate and the subsequent conversion to 2,3-dihydro-3-hydroxyanthranilic acid, respectively. These reactions are well established biochemically. Fewer data are available about the following steps (i.e. dimerization of 2,3-dihydro-3-hydroxyanthranilic acid, several oxidation reactions, and a decarboxylation, ultimately leading to PCA via several instable intermediates). From Pseudomonas, experimental data on the role of PhzF and PhzA/B have been published (8, 9), whereas the role of PhzG is yet unclear. Surprisingly, the only gene cluster for phenazine biosynthesis described so far from streptomycetes (10) was found not to contain a phzF orthologue, raising the question of whether there may be differences in the biosynthesis of phenazines between Pseudomonas and Streptomyces.Screening of a genomic library of the endophenazine producer strain S. anulatus now allowed the identification of the first complete gene cluster of a prenylated phenazine, including the structural gene of dihydro-PCA dimethylallyltransferase.     

Identification of the Lipopolysaccharide Core of Yersinia pestis and Yersinia pseudotuberculosis as the Receptor for Bacteriophage ��A1122

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.     

Selective Inhibition of Carotenoid Cleavage Dioxygenases: PHENOTYPIC EFFECTS ON SHOOT BRANCHING*S?

Members of the carotenoid cleavage dioxygenase family catalyze the oxidative cleavage of carotenoids at various chain positions, leading to the formation of a wide range of apocarotenoid signaling molecules. To explore the functions of this diverse enzyme family, we have used a chemical genetic approach to design selective inhibitors for different classes of carotenoid cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized in which the distance between an iron-chelating hydroxamic acid and an aromatic ring was varied; these compounds were screened as inhibitors of four different enzyme classes, either in vitro or in vivo. Potent inhibitors were found that selectively inhibited enzymes that cleave carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar concentrations. Application of certain inhibitors at 100 μm to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage.Carotenoids are synthesized in plants and micro-organisms as photoprotective molecules and are key components in animal diets, an example being β-carotene (pro-vitamin A). The oxidative cleavage of carotenoids occurs in plants, animals, and micro-organisms and leads to the release of a range of apocarotenoids that function as signaling molecules with a diverse range of functions (1). The first gene identified as encoding a carotenoid cleavage dioxygenase (CCD)² was the maize Vp14 gene that is required for the formation of abscisic acid (ABA), an important hormone that mediates responses to drought stress and aspects of plant development such as seed and bud dormancy (2). The VP14 enzyme cleaves at the 11,12 position (Fig. 1) of the epoxycarotenoids 9′-cis-neoxanthin and/or 9-cis-violaxanthin and is now classified as a 9-cis-epoxycarotenoid dioxygenase (NCED) (3), a subclass of the larger CCD family.Open in a separate windowFIGURE 1.Reactions catalyzed by the carotenoid cleavage dioxygenases. a, 11,12-oxidative cleavage of 9′-cis-neoxanthin by NCED; b, oxidative cleavage reactions on β-carotene and zeaxanthin.Since the discovery of Vp14, many other CCDs have been shown to be involved in the production of a variety of apocarotenoids (Fig. 1). In insects, the visual pigment retinal is formed by oxidative cleavage of β-carotene by β-carotene-15,15′-dioxygenase (4). Retinal is produced by an orthologous enzyme in vertebrates, where it is also converted to retinoic acid, a regulator of differentiation during embryogenesis (5). A distinct mammalian CCD is believed to cleave carotenoids asymmetrically at the 9,10 position (6) and, although its function is unclear, recent evidence suggests a role in the metabolism of dietary lycopene (7). The plant volatiles β-ionone and geranylacetone are produced from an enzyme that cleaves at the 9,10 position (8) and the pigment α-crocin found in the spice saffron results from an 7,8-cleavage enzyme (9).Other CCDs have been identified where biological function is unknown, for example, in cyanobacteria where a variety of cleavage specificities have been described (10-12). In other cases, there are apocarotenoids with known functions, but the identity or involvement of CCDs have not yet been described: grasshopper ketone is a defensive secretion of the flightless grasshopper Romalea microptera (13), mycorradicin is produced by plant roots during symbiosis with arbuscular mycorrhyza (14), and strigolactones (15) are plant metabolites that act as germination signals to parasitic weeds such as Striga and Orobanche (16).Recently it was discovered that strigolactones also function as a branching hormone in plants (17, 18). The existence of such a branching hormone has been known for some time, but its identity proved elusive. However, it was known that the hormone was derived from the action of at least two CCDs, max3 and max4 (more axillary growth) (19), because deletion of either of these genes in Arabidopsis thaliana, leads to a bushy phenotype (20, 21). In Escherichia coli assays, AtCCD7 (max3) cleaves β-carotene at the 9,10 position and the apocarotenoid product (10-apo-β-carotene) is reported to be further cleaved at 13,14 by AtCCD8 (max4) to produce 13-apo-β-carotene (22). Also recent evidence suggests that AtCCD8 is highly specific, cleaving only 10-apo-β-carotene (23). How the production of 13-apo-β-carotene leads to the synthesis of the complex strigolactone is unknown. The possibility remains that the enzymes may have different specificities and cleavage activities in planta. In addition, a cytochrome P450 enzyme (24) is believed to be involved in strigolactone synthesis and acts in the pathway downstream of the CCD genes. Strigolactone is thought to effect branching by regulating auxin transport (25). Because of the involvement of CCDs in strigolactone synthesis, the possibility arises that plant architecture and interaction with parasitic weeds and mycorrhyza could be controlled by the manipulation of CCD activity.Although considerable success has been obtained using genetic approaches to probe function and substrate specificity of CCDs in their native biological contexts, particularly in plant species with simple genetic systems or that are amenable to transgenesis, there are many systems where genetic approaches are difficult or impossible. Also, when recombinant CCDs are studied either in vitro or in heterologous in vivo assays, such as in E. coli strains engineered to accumulate carotenoids (26), they are often active against a broad range of substrates (5, 21, 27), and in many cases the true in vivo substrate of a particular CCD remains unknown. Therefore additional experimental tools are needed to investigate both apocarotenoid and CCD functions in their native cellular environments.In the reverse chemical genetics approach, small molecules are identified that are active against known target proteins; they are then applied to a biological system to investigate protein function in vivo (28, 29). This approach is complementary to conventional genetics since the small molecules can be applied easily to a broad range of species, their application can be controlled in dose, time, and space to provide detailed studies of biological functions, and individual proteins or whole protein classes may be targeted by varying the specificity of the small molecules. Notably, functions of the plant hormones gibberellin, brassinosteroid, and abscisic acid have been successfully probed using this approach by adapting triazoles to inhibit specific cytochrome P450 monooxygenases involved in the metabolism of these hormones (30).In the case of the CCD family, the tertiary amines abamine (31) and the more active abamineSG (32) were reported as specific inhibitors of NCED, and abamine was used to show new functions of abscisic acid in legume nodulation (33). However, no selective inhibitors for other types of CCD are known. Here we have designed a novel class of CCD inhibitor based on hydroxamic acids, where variable chain length was used to direct inhibition of CCD enzymes that cleave carotenoids at specific positions. We demonstrate the use of such novel inhibitors to control shoot branching in a model plant.     

Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)- and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY*S?

Linoleate (10R)-dioxygenase (10R-DOX) of Aspergillus fumigatus was cloned and expressed in insect cells. Recombinant 10R-DOX oxidized 18:2n-6 to (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE; ∼90%), (8R)-hydroperoxylinoleic acid (8R-HPODE; ∼10%), and small amounts of 12S(13R)-epoxy-(10R)-hydroxy-(8E)-octadecenoic acid. We investigated the oxygenation of 18:2n-6 at C-10 and C-8 by site-directed mutagenesis of 10R-DOX and 7,8-linoleate diol synthase (7,8-LDS), which forms ∼98% 8R-HPODE and ∼2% 10R-HPODE. The 10R-DOX and 7,8-LDS sequences differ in homologous positions of the presumed dioxygenation sites (Leu-384/Val-330 and Val-388/Leu-334, respectively) and at the distal site of the heme (Leu-306/Val-256). Leu-384/Val-330 influenced oxygenation, as L384V and L384A of 10R-DOX elevated the biosynthesis of 8-HPODE to 22 and 54%, respectively, as measured by liquid chromatography-tandem mass spectrometry analysis. The stereospecificity was also decreased, as L384A formed the R and S isomers of 10-HPODE and 8-HPODE in a 3:2 ratio. Residues in this position also influenced oxygenation by 7,8-LDS, as its V330L mutant augmented the formation of 10R-HPODE 3-fold. Replacement of Val-388 in 10R-DOX with leucine and phenylalanine increased the formation of 8R-HPODE to 16 and 36%, respectively, whereas L334V of 7,8-LDS was inactive. Mutation of Leu-306 with valine or alanine had little influence on the epoxyalcohol synthase activity. Our results suggest that Leu-384 and Val-388 of 10R-DOX control oxygenation of 18:2n-6 at C-10 and C-8, respectively. The two homologous positions of prostaglandin H synthase-1, Val-349 and Ser-353, are also critical for the position and stereospecificity of the cyclooxygenase reaction.Linoleate diol synthases (LDS)² and linoleate 10R-DOX are fungal fatty acid dioxygenases of the myeloperoxidase gene family (1-3). LDS have dual enzyme activities and transform 18:2n-6 sequentially to 8R-HPODE in an 8R-dioxygenase reaction and to 5,8-, 7,8-, or 8,11-DiHODE in hydroperoxide isomerase reactions. These oxylipins affect sporulation, development, and pathogenicity of Aspergilli (4-6). Fatty acid dioxygenases of the myeloperoxidase gene family also occur in vertebrates, plants, and algae (7-9). The most thoroughly investigated vertebrate enzymes are ovine PGHS-1 and mouse PGHS-2 with known crystal structures (10-12). PGHS transforms 20:4n-6 to PGG₂ in a cyclooxygenase and PGG₂ to PGH₂ in a peroxidase reaction. Aspirin and other nonsteroidal anti-inflammatory drugs inhibit the cyclooxygenase reaction. This is of paramount medical importance (13, 14), and PGHS-1 and -2 are commonly known as COX-1 and -2 (15). α-DOX occur in plants and algae, and biosynthesis of α-DOX in plants is elicited by pathogens (7). α-DOX oxidizes fatty acids to unstable (2R)-hydroperoxides, which readily break down nonenzymatically to fatty acid aldehydes and CO₂ (7).LDS, 10R-DOX, PGHS, and α-DOX oxygenate fatty acids to different products, but their oxygenation mechanisms have mechanistic similarities. Sequence alignment shows that many critical amino acid residues for the cyclooxygenase reaction are conserved in LDS, 10R-DOX, and α-DOX. These include the proximal histidine heme ligand, the distal histidine, and the catalytic important tyrosine (Tyr-385) of PGHS-1. The latter is oxidized to a tyrosyl radical, which initiates the cyclooxygenase reaction by abstraction of the pro-S hydrogen at C-13 of 20:4n-6 (16). In analogy, LDS and 10R-DOX catalyze stereospecific abstraction of the pro-S hydrogen at C-8 of 18:2n-6 (3), whereas α-DOX abstracts the pro-R hydrogen at C-2 of fatty acids (17). Site-directed mutagenesis of the conserved tyrosine homologues of Tyr-385 and proximal heme ligands abolishes the dioxygenase activities of 7,8-LDS and α-DOX (17, 18). The orientation of the substrate at the dioxygenation site differs. The carboxyl groups of fatty acids are positioned in a hydrophobic grove close to the tyrosine residue of α-DOX (19). In contrast, the ω ends of eicosanoic fatty acids are buried deep inside the cyclooxygenase channel so that C-13 lies in the vicinity of Tyr-385 (20). Several observations suggest that 18:2n-6 may also be positioned with its ω end embedded in the interior of 7,8-LDS of Gaeumannomyces graminis (18).7,8-LDS of G. graminis and Magnaporthe grisea and 5,8-LDS of Aspergillus nidulans have been sequenced (5, 8, 21). Gene targeting revealed the catalytic properties of 5,8-LDS, 8,11-LDS, and 10R-DOX in Aspergillus fumigatus and A. nidulans (3). Homologous genes can be found in other Aspergilli spp. Alignment of the two 7,8-LDS amino acid sequences with 5,8-LDS, 8,11-LDS, and 10R-DOX sequences of five Aspergilli revealed several conserved regions with single amino acid differences between the enzymes with 8R-DOX and 10R-DOX activities, as illustrated by the selected sequences in Fig. 1. Leu-306, Leu-384, and Val-388 of 10R-DOX are replaced in 5,8- and 7,8-LDS by valine, valine, and leucine residues, respectively. Whether these amino acids are important for the oxygenation mechanism is unknown, and this is one topic of the present investigation. The predicted secondary structure of 10R-DOX suggests that Leu-384 of 10R-DOX can be present in an α-helix with Val-388 close to its border. This α-helix is homologous to helix 6 of PGHS-1, which contains Val-349 and Ser-353 at the homologous positions of Leu-384 and Val-388 (Fig. 1).Open in a separate windowFIGURE 1.Alignments of partial amino acid sequences of five heme containing fatty acid dioxgenases and a comparison of the predicted secondary structure of 10R-DOX with ovine PGHS-1. A, top, amino acids residues at the presumed peroxidase and hydroperoxide isomerase sites. The last two residues, His and Asn, are conserved in all myeloperoxidases (1). Middle and bottom, amino acid residues of the presumed dioxygenation sites are shown. Conserved residues in all sequences are in boldface, and mutated residues of 10R-DOX and/or 7,8-LDS are marked by an asterisk. B, alignment of partial amino acid sequences of 10R-DOX with ovine PGHS-1, and a secondary structure prediction of the 10R-DOX sequence. The secondary structure of 10R-DOX was predicted by PSIPRED (43) and the secondary structure of ovine PGHS-1 from its crystal structure (Protein Data Bank code 1diy; cf. Ref 19). In short, our first strategy for site-directed mutagenesis was to switch hydrophobic residues between the enzymes with 10R- and 8R-DOX activities and to assess the effects on the DOX and hydroperoxide isomerase activities (10R-DOX/7,8-LDS: Leu-306/Val-256, Leu-384/Val-330, Val-388/Leu-334, and Ala-426/Ile-375) and to switch one hydrophobic/charged residue (Ala-435/Glu-384). Only catalytically active pairs would provide clear information on their importance for the position of dioxygenation (e.g. L384V of 10R-DOX and V330L of 7,8-LDS, both of which were active). Unfortunately, replacements of 7,8-LDS often led to inactivation or very low activity (e.g. V330A, V330M, I375A, E384A). Our second strategy was to study replacements in two homologous positions of ovine PGHS-1 (Val-349 and Ser-353) with smaller and larger hydrophobic residues, i.e. at Leu-384 and Val-388 of 10R-DOX. Abbreviations used are as follows: oCOX-1, ovine cyclooxygenase-1; Af, A. fumigatus; Gg, G. graminis. The GenBank™ protein sequences were derived from {"type":"entrez-protein","attrs":{"text":"P05979","term_id":"754286265","term_text":"P05979"}}P05979, {"type":"entrez-protein","attrs":{"text":"EAL89712","term_id":"66849384","term_text":"EAL89712"}}EAL89712, {"type":"entrez-protein","attrs":{"text":"AAD49559","term_id":"258406875","term_text":"AAD49559"}}AAD49559, {"type":"entrez-protein","attrs":{"text":"EAL84400","term_id":"129555261","term_text":"EAL84400"}}EAL84400, and {"type":"entrez-protein","attrs":{"text":"ACL14177","term_id":"219382647","term_text":"ACL14177"}}ACL14177. The amino acid sequences were aligned with the ClustalW algorithm (DNAStar).The overall three-dimensional structures of myeloperoxidases are conserved. It is therefore conceivable that important residues for substrate binding in the cyclooxygenase channel of PGHS could be conserved in LDS and 10R-DOX. The three-dimensional structure of ovine PGHS-1 shows that Val-349 and Ser-353 are close to C-3 and C-4 of 20:4n-6, and residues in these positions can alter both position and stereospecificity of oxygenation (22-24). Replacement of Val-349 of PGHS-1 with alanine increased the biosynthesis of 11R-HETE, whereas V349L decreased the generation of 11R-H(P)ETE and increased formation of 15(R/S)-H(P)ETE (23, 25). V349I formed PGG₂ with 15R configuration (22, 24). Replacement of Ser-353 with threonine reduced cyclooxygenase and peroxidase activities by over 50% and increased the biosynthesis of 11R-HPETE and 15S-HPETE 4-5 times (23).There is little information on the hydroperoxide isomerase and peroxidase sites of LDS (18, 26), but the latter could be structurally related to the peroxidase site of PGHS. PGG₂ and presumably 8R-HPODE bind to the distal side of the heme group, which can be delineated by hydrophobic amino acid residues (27). Val-291 is one of these residues, which form a dome over the distal heme side of COX-1. The V291A mutant retained cyclooxygenase and peroxidase activities (27). 5,8- and 7,8-LDS also have valine residues in the homologous position, whereas 8,11-LDS and 10R-DOX have leucine residues (Fig. 1). Whether these hydrophobic residues are important for the peroxidase activities is unknown.In this study we decided to compare the two catalytic sites of 10R-DOX of A. fumigatus and 7,8-LDS (EC 1.13.11.44) of G. graminis (18). Our first aim was to find a robust expression system for 10R-DOX of A. fumigatus. The second objective was to determine whether C₁₆ and C₂₀ fatty acid substrates enter the oxygenation site of 10R-DOX “head” or “tail” first. Unexpectedly, we found that 10R-DOX oxygenated 20:4n-6 by hydrogen abstraction at both C-13 and C-10 with formation of two nonconjugated and four cis-trans-conjugated HPETEs. Our third objective was to investigate the structural differences between 10R-DOX and 7,8-LDS of G. graminis, which could explain that oxygenation of 18:2n-6 mainly occurred at C-10 and at C-8, respectively. The strategy for site-directed mutagenesis of 10R-DOX and 7,8-LDS is outlined in the legend to Fig. 1; an alignment of the amino acid sequences of 10R-DOX and 7,8-LDS is found in supplemental material.     

Thiamin (Vitamin B1) Biosynthesis and Regulation: A Rich Source of Antimicrobial Drug Targets?

Drug resistance of pathogens has necessitated the identification of novel targets for antibiotics. Thiamin (vitamin B1) is an essential cofactor for all organisms in its active form thiamin diphosphate (ThDP). Therefore, its metabolic pathways might be one largely untapped source of antibiotics targets. This review describes bacterial thiamin biosynthetic, salvage, and transport pathways. Essential thiamin synthetic enzymes such as Dxs and ThiE are proposed as promising drug targets. The regulation mechanism of thiamin biosynthesis by ThDP riboswitch is also discussed. As drug targets of existing antimicrobial compound pyrithiamin, the ThDP riboswitch might serves as alternative targets for more antibiotics.     

A comparison of larval,ovitrap and MosquiTRAP?surveillance for Aedes (Stegomyia) aegypti

In Brazil, the entomological surveillance of Aedes (Stegomyia) aegypti is performed by government-mandated larval surveys. In this study, the sensitivities of an adult sticky trap and traditional surveillance methodologies were compared. The study was performed over a 12-week period in a residential neighbourhood of the municipality of Pedro Leopoldo, state of Minas Gerais, Brazil. An ovitrap and a MosquiTRAP were placed at opposite ends of each neighbourhood block (60 traps in total) and inspections were performed weekly. The study revealed significant correlations of moderate strength between the larval survey, ovitrap and MosquiTRAP measurements. A positive relationship was observed between temperature, adult capture measurements and egg collections, whereas precipitation and frequency of rainy days exhibited a negative relationship.     

A revised key to the Neotropical cleptoparasitic anthidiine genera (Hymenoptera,Megachilinae) with notes and description of the male of? Rhynostelis?Moure?& Urban

Rhynostelis Moure & Urban is a monotypic cleptoparasitic neotropical anthidiine genus currently known from two females. Herein, we describe and illustrate for the first time the male and its genitalia and it is confirmed that Rhynostelis parasitizes nests of Eufriesea. An identification key to the genera of cleptoparasitic anthidiine from the Neotropical region is also presented.