Tyr51: Key Determinant of the Low Thermostability of the Colwellia psychrerythraea Cold-Shock Protein.

Cold-shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of psychrophile Colwellia psychrerythraea 34H, ( Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs, including a five ß-stranded barrel, with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10-7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of the Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed for the formation of a K3-E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 and 642 s-1), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3-17), helix II (residues 39-53), helix III (residues 60-64), and helix IV (residues 68-78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe(45) in helix II and Phe(18) in the α1α2 loop and a hydrogen bonding between Ser(15) in helix I and Ile(20) in the α1α2 loop, resulting in its high thermal stability. Phe(45)-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser(58) in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains.

Functional Roles of Aromatic Residues and Helices of Papiliocin in its Antimicrobial and Anti-inflammatory Activities.

A cecropin-like peptide, papiliocin, isolated from the swallowtail butterfly Papilio xuthus, possesses high selectivity against gram-negative bacteria. Since Trp(2) and Phe(5) are highly conserved residues in cecropin-like peptides, we investigated the role of Trp(2) and Phe(5) in antibacterial activity. Substitution of Trp(2) and Phe(5) in papiliocin with Ala (papiliocin-2A and papiliocin-5A) revealed that Trp(2) is a key residue in its antibacterial activities. In order to understand the structural requirements for papiliocin function and to design shorter, but more potent, peptide antibiotics, we designed papiliocin constructs, PapN (residues Arg(1)-Ala(22) from the N-terminal amphipathic helix). PapN exhibited significant broad-spectrum antibacterial activities without cytotoxicity. Bactericidal kinetics of peptides against E.coli showed that papiliocin completely and rapidly killed E.coli in less than 10 minutes at 2× MIC concentration, while papiliocin-2A and papiliocin-5A killed four times more slowly than papiliocin. The PapN series peptides permeabilized bacterial membranes less effectively than papiliocin, showing no antibacterial activities in an hour. The results imply that the Trp(2) and Phe(5) in the amphipathic N-terminal helix are important in the rapid permeabilization of the gram-negative bacterial membrane. The hydrophobic C-terminal residues permeabilize the hydrophobic bacterial cell membrane synergistically with these aromatic residues, providing selectivity against gram-negative bacteria.

Role of phenylalanine and valine10 residues in the antimicrobial activity and cytotoxicity of piscidin-1.

Piscidin-1 (Pis-1) is a linear antibacterial peptide derived from mast cells of aquacultured hybrid striped bass that comprises 22 amino acids with a phenylalanine-rich amino-terminus. Pis-1 exhibits potent antibacterial activity against pathogens but is not selective for distinguishing between bacterial and mammalian cells. To determine the key residues for its antibacterial activity and those for its cytotoxicity, we investigated the role of each Phe residue near the N-terminus as well as the Val10 residue located near the boundary of the hydrophobic and hydrophilic sectors of the helical wheel diagram. Fluorescence dye leakage and tryptophan fluorescence experiments were used to study peptide-lipid interactions, showing comparable depths of insertion of substituted peptides in different membranes. Phe2 was found to be the most deeply inserted phenylalanine in both bacterial- and mammalian-mimic membranes. Each Phe was substituted with Ala or Lys to investigate its functional role. Phe2 plays key roles in the cytotoxicity as well as the antibacterial activities of Pis-1, and Phe6 is essential for the antibacterial activities of Pis-1. We also designed and synthesized a piscidin analog, Pis-V10K, in which Lys was substituted for Val10, resulting in an elevated amphipathic α-helical structure. Pis-V10K showed similar antibacterial activity (average minimum inhibitory concentration (MIC)  = 1.6 µM) to Pis-1 (average MIC  = 1.5 µM). However, it exhibited much lower cytotoxicity than Pis-1. Lys10-substituted analogs, Pis-F1K/V10K, Pis-F2K/V10K, and Pis-F6K/V10K in which Lys was substituted for Phe retained antibacterial activity toward standard and drug-resistant bacterial strains with novel bacterial cell selectivity. They exert anti-inflammatory activities via inhibition of nitric oxide production, TNF-α secretion, and MIP-1 and MIP-2 production. They may disrupt the binding of LPS to toll-like receptors, eventually suppressing MAPKs-mediated signaling pathways. These peptides may be good candidates for the development of peptide antibiotics with potent antibacterial activity but without cytotoxicity.

Cytotoxic activity of 3,6-dihydroxyflavone in human cervical cancer cells and its therapeutic effect on c-Jun N-terminal kinase inhibition.

Previously we have shown that 3,6-dihydroxyflavone (3,6-DHF) is a potent agonist of the human peroxisome proliferator-activated receptor (hPPAR) with cytotoxic effects on human cervical cancer cells. To date, the mechanisms by which 3,6-DHF exerts its antitumor effects on cervical cells have not been clearly defined. Here, we demonstrated that 3,6-DHF exhibits a novel antitumor activity against HeLa cells with IC50 values of 25 µM and 9.8 µM after 24 h and 48 h, respectively. We also showed that the anticancer effects of 3,6-DHF are mediated via the toll-like receptor (TLR) 4/CD14, p38 mitogen-activated protein kinase (MAPK), Jun-N terminal kinase (JNK), extracellular-signaling regulated kinase (ERK), and cyclooxygenase (COX)-2 pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. We found that 3,6-DHF showed a similar IC50 (113 nM) value to that of the JNK inhibitor, SP600125 (IC50 = 118 nM) in a JNK1 kinase assay. Binding studies revealed that 3,6-DHF had a strong binding affinity to JNK1 (1.996 × 105 M-1) and that the 6-OH and the carbonyl oxygen of the C ring of 3,6-DHF participated in hydrogen bonding interactions with the carbonyl oxygen and the amide proton of Met111, respectively. Therefore, 3,6-DHF may be a candidate inhibitor of JNKs, with potent anticancer effects.

Structure and flexibility of the thermophilic cold-shock protein of Thermus aquaticus.

The thermophilic bacterium Thermus aquaticus is a well-known source of Taq polymerase. Here, we studied the structure and dynamics of the T. aquaticus cold-shock protein (Ta-Csp) to better understand its thermostability using NMR spectroscopy. We found that Ta-Csp has a five-stranded ß-barrel structure with five salt bridges which are important for more rigid structure and a higher melting temperature (76 °C) of Ta-Csp compared to mesophilic and psychrophilic Csps. Microsecond to millisecond time scale exchange processes occur only at the ß1-ß2 surface region of the nucleic acid binding site with an average conformational exchange rate constant of 674 s(-1). The results imply that thermophilic Ta-Csp has a more rigid structure and may not need high structural flexibility to accommodate nucleic acids upon cold shock compared to its mesophile and psychrophile counterparts.

Structure and backbone dynamics of vanadate-bound PRL-3: comparison of 15N nuclear magnetic resonance relaxation profiles of free and vanadate-bound PRL-3.

Phosphatases of regenerating liver (PRLs) constitute a novel class of small, prenylated phosphatases with oncogenic activity. PRL-3 is particularly important in cancer metastasis and represents a potential therapeutic target. The flexibility of the WPD loop as well as the P-loop of protein tyrosine phosphatases is closely related to their catalytic activity. Using nuclear magnetic resonance spectroscopy, we studied the structure of vanadate-bound PRL-3, which was generated by addition of sodium orthovanadate to PRL-3. The WPD loop of free PRL-3 extended outside of the active site, forming an open conformation, whereas that of vanadate-bound PRL-3 was directed into the active site by a large movement, resulting in a closed conformation. We suggest that vanadate binding induced structural changes in the WPD loop, P-loop, helices α4-α6, and the polybasic region. Compared to free PRL-3, vanadate-bound PRL-3 has a longer α4 helix, where the catalytic R110 residue coordinates with vanadate in the active site. In addition, the hydrophobic cavity formed by helices α4-α6 with a depth of 14-15 Å can accommodate a farnesyl chain at the truncated prenylation motif of PRL-3, i.e., from R169 to M173. Conformational exchange data suggested that the WPD loop moves between open and closed conformations with a closing rate constant k(close) of 7 s(-1). This intrinsic loop flexibility of PRL-3 may be related to their catalytic rate and may play a role in substrate recognition.

Tyrosine-mediated two-dimensional peptide assembly and its role as a bio-inspired catalytic scaffold.

In two-dimensional interfacial assemblies, there is an interplay between molecular ordering and interface geometry, which determines the final morphology and order of entire systems. Here we present the interfacial phenomenon of spontaneous facet formation in a water droplet driven by designed peptide assembly. The identified peptides can flatten the rounded top of a hemispherical droplet into a plane by forming a macroscopic two-dimensional crystal structure. Such ordering is driven by the folding geometry of the peptide, interactions of tyrosine and crosslinked stabilization by cysteine. We discover the key sequence motifs and folding structures and study their sequence-specific assembly. The well-ordered, densely packed, redox-active tyrosine units in the YYACAYY (H-Tyr-Tyr-Ala-Cys-Ala-Tyr-Tyr-OH) film can trigger or enhance chemical/electrochemical reactions, and can potentially serve as a platform to fabricate a molecularly tunable, self-repairable, flat peptide or hybrid film.

Anti-inflammatory activity of rhamnetin and a model of its binding to c-Jun NH2-terminal kinase 1 and p38 MAPK.

Rhamnetin (1), a commonly occurring plant O-methylated flavonoid, possesses antioxidant properties. To address the potential therapeutic efficacy of 1, its anti-inflammatory activity and mode of action in mouse macrophage-derived RAW264.7 cells stimulated with lipopolysaccharide (LPS) or interferon (IFN)-γ were investigated. Rhamnetin (1) suppressed mouse tumor necrosis factor (mTNF)-α, mouse macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine production in LPS-stimulated macrophages. A nontoxic dose of 1 suppressed nitric oxide production. It was found that the anti-inflammatory effects of 1 are mediated by actions on the p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and cyclooxygenase (COX)-2 pathways in LPS- or IFN-γ-stimulated RAW264.7 cells. It was determined that 1 binds to human JNK1 (9.7 × 10(8) M(-1)) and p38 MAPK (2.31 × 10(7) M(-1)) with good affinity. The binding model showed interactions with the 3'- and 4'-hydroxy groups of the B-ring and the 5-hydroxy group of the A-ring of 1. Further, 1 exerted an anti-inflammatory effect, reducing the levels of pro-inflammatory cytokines and mediators.

De novo design and synthesis of ultra-short peptidomimetic antibiotics having dual antimicrobial and anti-inflammatory activities.

BACKGROUND: Much attention has been focused on the design and synthesis of potent, cationic antimicrobial peptides (AMPs) that possess both antimicrobial and anti-inflammatory activities. However, their development into therapeutic agents has been limited mainly due to their large size (12 to 50 residues in length) and poor protease stability. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to overcome the issues described above, a set of ultra-short, His-derived antimicrobial peptides (HDAMPs) has been developed for the first time. Through systematic tuning of pendant hydrophobic alkyl tails at the N(π)- and N(τ)-positions on His, and the positive charge of Arg, much higher prokaryotic selectivity was achieved, compared to human AMP LL-37. Additionally, the most potent HDAMPs showed promising dual antimicrobial and anti-inflammatory activities, as well as anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and proteolytic resistance. Our results from transmission electron microscopy, membrane depolarization, confocal laser-scanning microscopy, and calcein-dye leakage experiments propose that HDAMP-1 kills microbial cells via dissipation of the membrane potential by forming pore/ion channels on bacterial cell membranes. CONCLUSION/SIGNIFICANCE: The combination of the ultra-short size, high-prokaryotic selectivity, potent anti-MRSA activity, anti-inflammatory activity, and proteolytic resistance of the designed HDAMP-1, -3, -5, and -6 makes these molecules promising candidates for future antimicrobial therapeutics.

Binding model for eriodictyol to Jun-N terminal kinase and its anti-inflammatory signaling pathway.

The anti-inflammatory activity of eriodictyol and its mode of action were investigated. Eriodictyol suppressed tumor necrosis factor (mTNF)-α, inducible nitric oxide synthase (miNOS), interleukin (mIL)-6, macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine release in LPS-stimulated macrophages. We found that the anti-inflammatory cascade of eriodictyol is mediated through the Toll-like Receptor (TLR)4/CD14, p38 mitogen-activated protein kinases (MAPK), extracellular-signal-regulated kinase (ERK), Jun-N terminal kinase (JNK), and cyclooxygenase (COX)-2 pathway. Fluorescence quenching and saturation-transfer difference (STD) NMR experiments showed that eriodictyol exhibits good binding affinity to JNK, 8.79 × 10(5) M(-1). Based on a docking study, we propose a model of eriodictyol and JNK binding, in which eriodictyol forms 3 hydrogen bonds with the side chains of Lys55, Met111, and Asp169 in JNK, and in which the hydroxyl groups of the B ring play key roles in binding interactions with JNK. Therefore, eriodictyol may be a potent anti-inflammatory inhibitor of JNK.

Relationship between structural flexibility and function in the C-terminal region of the heparin-binding domain of VEGF165.

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Previously, we reported that triamterene (Trm) inhibits VEGF-amyloid ß (Aß) interactions without affecting other biological activities of VEGF or Aß [Jeong, K.-W., et al. (2011) Biochemistry 50, 4843-4854]. We further showed that molecular motions in the N-terminal disordered loop region of the heparin-binding domain (HBD) are important for interaction with Trm. To investigate the importance of motion at the C-terminal domain of HBD, we constructed a binding model of HBD with heparin octasaccharide (HOS) based on measurements of chemical shift changes and docking studies. Furthermore, the dynamic properties of the HBD-HOS and HBD-Trm-HOS complexes were assessed by measuring spin relaxation rates. The results showed that the HOS-binding site is composed of two basic clusters consisting of side chains of residues R13, R14, and K15 and residues K30, R35, and R49. When HOS binds, values for the heteronuclear nuclear Overhauser effect near HOS-binding sites increased dramatically. CPMG (Carr-Purcell-Meiboom-Gill sequence) experiments as well as an R2 relaxation experiment were undertaken to understand millisecond time-scale motions in HBD. There is large relaxation dispersion of residues at Trm- and HOS-binding sites in free HBD. C-Terminal residues such as S34, C48, and D51 near the HOS-binding sites continued to exhibit slow conformational motions in the HBD-Trm complex, while those slow motions disappeared in the bound conformation of HBD with HOS. Collectively, our results demonstrate that the inherent structural flexibilities of the C-terminal region of the HBD are important in the heparin binding process and that Trm does not inhibit VEGF-heparin interactions necessary for the biological activities of VEGF.

Structure-activity relationships of cecropin-like peptides and their interactions with phospholipid membrane.

Cecropin A and papiliocin are novel 37-residue cecropin-like antimicrobial peptides isolated from insect. We have confirmed that papiliocin possess high bacterial cell selectivity and has an α-helical structure from Lys(3) to Lys(21) and from Ala(25) to Val(35), linked by a hinge region. In this study, we demonstrated that both peptides showed high antimicrobial activities against multi-drug resistant Gram negative bacteria as well as fungi. Interactions between these cecropin-like peptides and phospholipid membrane were studied using CD, dye leakage experiments, and NMR experiments, showing that both peptides have strong permeabilizing activities against bacterial cell membranes and fungal membranes as well as Trp(2) and Phe(5) at the N-terminal helix play an important role in attracting cecropin-like peptides to the negatively charged bacterial cell membrane. Cecropin-like peptides can be potent peptide antibiotics against multi-drug resistant Gram negative bacteria and fungi.

Structural and dynamic features of cold-shock proteins of Listeria monocytogenes, a psychrophilic bacterium.

Cold-shock proteins (Csps), proteins expressed when the ambient temperature drops below the growth-supporting temperature, bind to single-stranded nucleic acids and act as RNA chaperones to regulate translation. Listeria monocytogenes is a psychrophilic food-borne pathogen that is problematic for the food industry. Structures of Csps from psychrophilic bacteria have not yet been studied. Despite dramatic differences in the thermostability of Csps of various thermophilic microorganisms, these proteins share a high degree of primary sequence homology and a high degree of three-dimensional structural similarity. Here, we investigated the structural and dynamic features as well as the thermostability of L. monocytogenes CspA (Lm-CspA). Lm-CspA has a five-stranded ß-barrel structure with hydrophobic core packing and two salt bridges. When heptathymidine (dT(7)) binds, values for the heteronuclear nuclear Overhauser effect and order parameters of residues in surface loop regions near nucleic acid binding sites increase dramatically. Moreover, Carr-Purcell-Meiboom-Gill experiments showed that slow motions observed for the nucleic acid binding residues K7, W8, F15, F27, and R56 disappeared in Lm-CspA-dT(7). Lm-CspA is less thermostable than mesophilic and thermophilic Csps, with a lower melting temperature (40 °C). The structural flexibility that accompanies longer surface loops and less hydrophobic core packing and a number of salt bridges and unfavorable electrostatic repulsion are likely key factors in the low thermostability of Lm-CspA. This implies that the large conformational flexibility of psychrophilic Lm-CspA, which more easily accommodates nucleic acids at low temperature, is required for RNA chaperone function under cold-shock conditions and for the cold adaptation of L. monocytogenes.

Backbone dynamics of an atypical orphan response regulator protein, Helicobacter pylori 1043.

An atypical orphan response regulator protein, HP1043 (HP-RR) in Helicobacter pylori, is proven to be essential for cell growth and does not require the well known phosphorelay scheme. HP-RR was identified as a symmetric dimer with two functional domains, an N-terminal regulatory domain (HP-RR(r)) and a C-terminal effector domain (HP-RR(e)). HP-RR is a new class of response regulator, as a phosphorylation-independent regulator. Previously, we have presented a detailed three-dimensional structure of HP-RR using NMR spectroscopy and X-ray crystallography. In this study, in order to understand the functional importance of flexibilities in HP-RR(r) and HP-RR(e), T1, T2, heteronuclear NOE experiments have been performed and backbone dynamics of HP-RR(r) and HP-RR(e) were investigated. HP-RR(r) is a symmetric dimer and the interface region, α4-ß5-α5 of dimer, showed high rigidity (high S (2) values). Site of rearrangements associated with phosphorylation of HP-RR(r) (Ser(75): R ex = 3.382, Ile(95): R ex = 5.228) showed slow chemical exchanges. HP-RR(e) is composed of three α-helices flanked on two sides by anti-parallel ß-sheets. Low order parameters as well as conformational exchanges in the centers of loop regions known as the DNA binding site and transcription site of HP-RR(e) suggested that flexibility of HP-RR(e) is essential for interaction with DNA. In conclusion, backbone dynamics information for HP-RR implies that structural flexibilities in HP-RR(r) are necessary for the phosphorylation site and the dynamic nature of HP-RR(e) is essential for the regulation of interaction between protein and DNA.

Insight into the antimicrobial activities of coprisin isolated from the dung beetle, Copris tripartitus, revealed by structure-activity relationships.

The novel 43-residue, insect defensin-like peptide coprisin, isolated from the dung beetle, Copris tripartitus, is a potent antibiotic with bacterial cell selectivity, exhibiting antimicrobial activities against Gram-positive and Gram-negative bacteria without exerting hemolytic activity against human erythrocytes. Tests against Staphylococcus aureus using fluorescent dye leakage and depolarization measurements showed that coprisin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of coprisin in aqueous solution by nuclear magnetic resonance spectroscopy, which showed that coprisin has an amphipathic α-helical structure from Ala(19) to Arg(28), and ß-sheets from Gly(31) to Gln(35) and Val(38) to Arg(42). Coprisin has electropositive regions formed by Arg(28), Lys(29), Lys(30), and Arg(42) and ITC results proved that coprisin and LPS have electrostatically driven interactions. Using measurements of nitric oxide release and inflammatory cytokine production, we provide the first verification of the anti-inflammatory activity and associated mechanism of an insect defensin, demonstrating that the anti-inflammatory actions of the defensin-like peptide, coprisin, are initiated by suppressing the binding of LPS to toll-like receptor 4, and subsequently inhibiting the phosphorylation of p38 mitogen-activated protein kinase and nuclear translocation of NF-kB. In conclusion, we have demonstrated that an amphipathic helix and an electropositive surface in coprisin may play important roles in its effective interaction with bacterial cell membranes and, ultimately, in its high antibacterial activity and potent anti-inflammatory activity. In addition to elucidating the antimicrobial action of coprisin, this work may provide insight into the mechanism of action of insect defense systems.

Biochemical characterization and FAD-binding analysis of oleate hydratase from Macrococcus caseolyticus.

A putative fatty acid hydratase gene from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was a 68 kDa dimer with a molecular mass of 136 kDa. The enzymatic products formed from fatty acid substrates by the putative enzyme were isolated with high purity (>99%) by solvent fractional crystallization at low temperature. After the identification by GC-MS, the purified hydroxy fatty acids were used as standards to quantitatively determine specific activities and kinetic parameters for fatty acids as substrates. Among the fatty acids evaluated, specific activity and catalytic efficiency (k(cat)/K(m)) were highest for oleic acid, indicating that the putative fatty acid hydratase was an oleate hydratase. Hydration occurred only for cis-9-double and cis-12-double bonds of unsaturated fatty acids without any trans-configurations. The maximum activity for oleate hydration was observed at pH 6.5 and 25 °C with 2% (v/v) ethanol and 0.2 mM FAD. Without FAD, all catalytic activity was abolished. Thus, the oleate hydratase is an FAD-dependent enzyme. The residues G29, G31, S34, E50, and E56, which are conserved in the FAD-binding motif of fatty acid hydratases (GXGXXG((A/S))X((15-21))E((D))), were selected by alignment, and the spectral properties and kinetic parameters of their alanine-substituted variants were analyzed. Among the five variants, G29A, G31A, and E56A showed no interaction with FAD and exhibited no activity. These results indicate that G29, G31, and E56 are essential for FAD-binding.

Discovery of novel selective inhibitors of Staphylococcus aureus ß-ketoacyl acyl carrier protein synthase III.

ß-Ketoacyl-acyl carrier protein synthase III (KAS III) is a condensing enzyme in bacterial fatty acid synthesis and a potential target while designing novel antibiotics. In our previous report, we discovered the lead compound YKAs3003, which serves as an inhibitor of Escherichia coli KAS III (ecKAS III), and determined a reliable pharmacophore map from in silico screening. In this study, we determined two pharmacophore maps from receptor-oriented pharmacophore-based in silico screening of the x-ray structure of Staphylococcus aureus KAS III (saKAS III) to identify potent saKAS III inhibitors. We discovered a new potential inhibitor (6) with broad-spectrum antimicrobial activity and 0.8 nM binding affinity for saKAS III, proving the reliability of our pharmacophore map. Using optimization procedures, we identified three new antimicrobial saKAS III inhibitors: 6c (2,4-dichloro-benzoic acid (2,3,4-trihydroxy-benzylidene)-hydrazide), 6e (4-[(3-chloro-pyrazin-2-yl)-hydrazonomethyl]-benzene-1,3-diol), and 6 (4-[(5-trifluoromethyl-pyridin-2-yl)-hydrazonomethyl]-benzene-1,3-diol). All three inhibitors have a novel 4-hydrazonomethyl-benzene-1,3-diol core structure. These inhibitors exhibited high binding affinity to saKAS III and highly selective antimicrobial activities against S. aureus and methicillin-resistant S. aureus, with minimal inhibitory concentration values of 1-2 µg/mL.

Structure and function of papiliocin with antimicrobial and anti-inflammatory activities isolated from the swallowtail butterfly, Papilio xuthus.

Papiliocin is a novel 37-residue cecropin-like peptide isolated recently from the swallowtail butterfly, Papilio xuthus. With the aim of identifying a potent antimicrobial peptide, we tested papiliocin in a variety of biological and biophysical assays, demonstrating that the peptide possesses very low cytotoxicity against mammalian cells and high bacterial cell selectivity, particularly against Gram-negative bacteria as well as high anti-inflammatory activity. Using LPS-stimulated macrophage RAW264.7 cells, we found that papiliocin exerted its anti-inflammatory activities by inhibiting nitric oxide (NO) production and secretion of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2, producing effects comparable with those of the antimicrobial peptide LL-37. We also showed that the innate defense response mechanisms engaged by papiliocin involve Toll-like receptor pathways that culminate in the nuclear translocation of NF-κB. Fluorescent dye leakage experiments showed that papiliocin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of papiliocin in 300 mm dodecylphosphocholine micelles by NMR spectroscopy, showing that papiliocin has an α-helical structure from Lys(3) to Lys(21) and from Ala(25) to Val(36), linked by a hinge region. Interactions between the papiliocin and LPS studied using tryptophan blue-shift data, and saturation transfer difference-NMR experiments revealed that Trp(2) and Phe(5) at the N-terminal helix play an important role in attracting papiliocin to the cell membrane of Gram-negative bacteria. In conclusion, we have demonstrated that papiliocin is a potent peptide antibiotic with both anti-inflammatory and antibacterial activities, and we have laid the groundwork for future studies of its mechanism of action.

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus.

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn(2+) that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K(m) and dramatic decrease of k(cat), and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.