Acid Recognition Sites Research Articles

Synthesis and Characterization of Boronate Affinity Three-Dimensionally Ordered Macroporous Materials.

Sample pretreatment is a key step for qualitative and quantitative analysis of trace substances in complex samples. Cis-dihydroxyl (cis-diol) group-containing substances exist widely in biological samples and can be selectively bound by boronate affinity adsorbents. Based on this, in this article, we proposed a simple method for the preparation of novel spherical three-dimensionally ordered macropore (3DOM) materials based on a combination of the boronate affinity technique and colloidal crystal template method. The prepared 3DOM materials were characterized using Fourier transform-infrared spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and thermo-gravimetric analysis, and results showed that they possessed the characteristics of a high specific surface area, high porosity, and more boronic acid recognition sites. The adsorption performance evaluation results showed that the maximum adsorption capacity of the boron affinity 3DOMs on ovalbumin (OVA) could reach to 438.79 mg/g. Kinetic and isothermal adsorption experiments indicated that the boronate affinity 3DOM material exhibited a high affinity and selectivity towards OVA and adenosine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the proteins in egg whites was conducted and proved that the glycoprotein in the egg whites could be separated and enriched with a good performance. Therefore, a novel boronate affinity 3DOM material a with highly ordered and interconnected pore structure was prepared and could be applied in the separation and enrichment of molecules with cis-diol groups from complex samples with a good selectivity, efficiency, and high throughput.

A biocompatible chitosan-based fluorescent polymer for efficient H2O2 detection in living cells and water samples

As a biomarker of oxidative stress, hydrogen peroxide (H2O2) plays a complex role in organisms, including regulating cell signaling, respiration, the immune system, and other life processes. Therefore, it is important to develop a tool that can simply and effectively monitor H2O2 levels in organisms and the environment. In this work, naphthalene fluorophores with a borate structure were introduced into chitosan (CTS) azide, and a CTS-based fluorescence sensor (CTS-HP) was designed for sensitive H2O2 detection. The biocompatibility and degradability of CTS endowed CTS-HP with reduced biotoxicity compared with organic fluorescent dyes, and the substitution degree of fluorophores on the CTS chains was 0.703. The randomly coiled chain structure of the CTS-HP probe enabled the boronic acid recognition sites on the fluorophores to achieve the enrichment of analyte H2O2 through a synergistic effect. Therefore, the probe CTS-HP (10 μg mL−1) exhibited a 21-fold fluorescence enhancement and good detection limit (LOD = 8.98 nM) in H2O2 solution, reaching the maximum fluorescence response faster (within 16 min). The probe also successfully achieved the fluorescence imaging of endogenous and exogenous H2O2 in zebrafish and living cells and labeled the recovery experiment of H2O2 in real water samples (recoveries rates of 90.93–102.9 % and RSD < 3.09 %).

Fast responding of mitochondrial HClO using a coumarin-based fluorescent probe in living cells and zebrafish

As an important reactive oxygen species (ROS), hypochlorous acid (HClO) plays pivotal role in the human immune system and ensures the normal function of the organisms. HClO is derived from mitochondria and can regulate intracellular redox balance. Nevertheless, abnormal HClO levels is related to immune system disorders, neurodegenerative injury and cancer. Therefore, fast and sensitive monitoring HClO in mitochondria is extremely significant for some diseases indication. Herein, we designed a mitochondrial-targeted probe HDCl to detect HClO in living cells, which was a hybrid of coumarin, quaternized pyridine (mitochondrial target site) and N, N-dimethylthiocarbamate (DMTC, hypochlorous acid recognition site). The probe HDCl present high sensitivity, high selectivity and ultrafast (<5 s) responding properties for HClO in phosphate buffer solution (PBS) with the very low detection limit of 2.8 nM and trigger a significant enhanced fluorescent signal at 641 nm. Additionally, with a massive Stokes shift (176 nm), HDCl has been successfully applied to imaging HClO in mitochondria of living cells and in zebrafish.

Boronic Acid-Based Dendrimers with Various Surface Properties for Bacterial Recognition with Adjustable Selectivity.

The need for a selective bacterial recognition method is evident to overcome the global problem of antibiotic resistance. Even though researchers have focused on boronic acid-based nanoprobes that immediately form boronate esters with saccharides at room temperature, the mechanism has not been well studied. We have developed boronic acid-modified poly(amidoamine) (PAMAM) dendrimers with various surface properties to investigate the mechanism of bacterial recognition. The boronic acid-based nanoprobes showed selectivity toward strains, species, or a certain group of bacteria by controlling their surface properties. Our nanoprobes showed selectivity toward Gram-positive bacteria or Escherichia coli K12W3110 without having to modify the boronic acid recognition sites. The results were obtained in 20 min and visible to the naked eye. Selectivity toward Gram-positive bacteria was realized through electrostatic interaction between the bacterial surface and the positively charged nanoprobes. In this case, the recognition target was lipoteichoic acid on the bacterial surface. On the other hand, pseudo-zwitterionic nanoprobes showed selectivity for E. coli K12W3110, indicating that phenylboronic acid did not recognize the outermost O-antigen on the lipopolysaccharide layer. Boronic acid-based nanoprobes with optimized surface properties are expected to be a powerful clinical tool to recognize multidrug-resistant strains or highly pathogenic bacteria.

Amino Acid Specificity of Ancestral Aminoacyl-tRNA Synthetase Prior to the Last Universal Common Ancestor Commonote commonote

Extant organisms commonly use 20 amino acids in protein synthesis. In the translation system, aminoacyl-tRNA synthetase (ARS) selectively binds an amino acid and transfers it to the cognate tRNA. It is postulated that the amino acid repertoire of ARS expanded during the development of the translation system. In this study we generated composite phylogenetic trees for seven ARSs (SerRS, ProRS, ThrRS, GlyRS-1, HisRS, AspRS, and LysRS) which are thought to have diverged by gene duplication followed by mutation, before the evolution of the last universal common ancestor. The composite phylogenetic tree shows that the AspRS/LysRS branch diverged from the other five ARSs at the deepest node, with the GlyRS/HisRS branch and the other three ARSs (ThrRS, ProRS and SerRS) diverging at the second deepest node. ThrRS diverged next, and finally ProRS and SerRS diverged from each other. Based on the phylogenetic tree, sequences of the ancestral ARSs prior to the evolution of the last universal common ancestor were predicted. The amino acid specificity of each ancestral ARS was then postulated by comparison with amino acid recognition sites of ARSs of extant organisms. Our predictions demonstrate that ancestral ARSs had substantial specificity and that the number of amino acid types amino-acylated by proteinaceous ARSs was limited before the appearance of a fuller range of proteinaceous ARS species. From an assumption that 10 amino acid species are required for folding and function, proteinaceous ARS possibly evolved in a translation system composed of preexisting ribozyme ARSs, before the evolution of the last universal common ancestor.

The cell-penetrating YopM protein-functionalized quantum dot-plasmid DNA conjugate as a novel gene delivery vector

Non-viral gene delivery systems have great potential for safe and efficient gene therapy, while inefficient cellular and nuclear uptake remain as the major hurdles. Novel approaches are needed to enhance the transfection efficiency of non-viral vectors. In accordance with this need, the objective of this study was to construct a non-viral vector that could achieve gene delivery without using additional lipid-based transfection agent. We aimed to impart self-delivery property to a non-viral vector by using the cell and nucleus penetrating properties of YopM proteins from the three Yersinia spp. (Y. pestis, Y. enterocolotica and Y. pseudotuberculosis). Plasmid DNA (pDNA) encoding green fluorescent protein (GFP) was labeled with quantum dots (QDs) via peptide-nucleic acid (PNA) recognition site. Recombinant YopM protein was then attached to the conjugate via a second PNA recognition site. The YopM ̶ QDs ̶ pDNA conjugate was transfected into HeLa cells without using additional transfection reagent. All three conjugates produced GFP fluorescence, indicating that the plasmid was successfully delivered to the nucleus. As control, naked pDNA was transfected into the cells by using a commercial transfection reagent. The Y. pseudotuberculosis YopM-functionalized conjugate achieved the highest GFP expression, compared to other two YopM proteins and the transfection reagent. To the best of our knowledge, YopM protein was used for the first time in a non-viral gene delivery vector.

CD4 binding loop responsible for the neutralization of human monoclonal neutralizing antibody Y498

Broad and potent human monoclonal neutralizing antibodies have considerable potential in the prevention and treatment of acquired immunodeficiency syndrome (AIDS). To identify the key amino acid recognition site contacted with neutralizing antibody Y498, peptides were panned from the PhD-12 peptide library and predicted using online software. Then, four key amino acid sites, G367, D368, E370, and V372 located on the CD4 binding loop on gp120 of envelope of human immunodeficiency virus-1 (HIV-1), were found to determine the neutralization of antibody Y498. Residue E370 is in the deep part of the CD4 binding loop, which affects Y498-mediated neutralization. This form of recognition leads to a somewhat limiting neutralization spectrum of neutralizing antibody Y498, although it has some neutralization ability. Further study of the interactions between the neutralizing antibody Y498 and its epitope on the surface of the virus may facilitate vaccine development and so prevent new AIDS cases.

Two Synthetic Replicators Compete To Process a Dynamic Reagent Pool.

Complementary building blocks, comprising a set of four aromatic aldehydes and a set of four nucleophiles-three anilines and one hydroxylamine-combine through condensation reactions to afford a dynamic covalent library (DCL) consisting of the eight starting materials and 16 condensation products. One of the aldehydes and, consequently, all of the DCL members derived from this compound bear an amidopyridine recognition site. Exposure of this DCL to two maleimides, Mp and Mm, each equipped with a carboxylic acid recognition site, results in the formation of a series of products through irreversible 1,3-dipolar cycloaddition reactions with the four nitrones present in the DCL. However, only the two cycloadducts in the product pool that incorporate both recognition sites, Tp and Tm, are self-replicators that can harness the DCL as feedstock for their own formation, facilitating their own synthesis via autocatalytic and cross-catalytic pathways. The ability of these replicators to direct their own formation from the components present in the dynamic reagent pool in response to the input of instructions in the form of preformed replicators is demonstrated through a series of quantitative 19F{1H} NMR spectroscopy experiments. Simulations establish the critical relationships between the kinetic and thermodynamic parameters of the replicators, the initial reagent concentrations, and the presence or absence of the DCL and their influence on the competition between Tp and Tm. Thus, we establish the rules that govern the behavior of the competing replicators under conditions where their formation is coupled tightly to the processing of a DCL.

A Critical Cross-Catalytic Relationship Determines the Outcome of Competition in a Replicator Network.

A network of two synthetic replicators exhibits a critical unidirectional cross-catalytic relationship that directs competing replication processes. In this network, nitrone N bearing a 6-methylamidopyridine recognition site can participate in 1,3-dipolar cycloaddition reactions with two maleimides that differ in the relative position of their carboxylic acid recognition site: either para (Mp) or meta (Mm) relative to the maleimide ring. These cycloaddition reactions create replicators trans-Tp and trans-Tm. In isolation, trans-Tp templates its own formation with an efficiency that is markedly greater than that of trans-Tm. Kinetic fitting and simulations reveal that this efficiency arises from a higher template-mediated rate constant for the cycloaddition and lower stability of the trans-Tp template duplex, compared to trans-Tm. By contrast, in a situation where Mp and Mm compete for a limited quantity of N, the normally less efficient trans-Tm outcompetes trans-Tp. Through a series of comprehensive kinetic 19F{1H} NMR spectroscopy experiments, this system-level outcome is traced to a critical cross-catalytic pathway, whereby the presence of trans-Tp templates the formation of trans-Tm, but not vice versa. Replicator trans-Tm also reduces the efficiency of its competitor trans-Tp by sequestering trans-Tp in a heteroduplex that is more stable than homoduplex [Tp·Tp]. The addition of different templates as instructions reveals that, while the outcome of competition between replicators can be altered selectively, it is limited by the reaction environment employed. These results represent a conceptual and practical framework for the examination of selectivity in replication networks operating outside well-stirred batch reactor conditions.

Substituent-Modulated Affinities of Halobenzene Derivatives to the HIV-1 Integrase Recognition Site. Analyses of the Interaction Energies by Parallel Quantum Chemical and Polarizable Molecular Mechanics

The C-X bond of halobenzenes (X = Cl, Br) has a dual character, its electron density being depleted in its prolongation and built-up on its sides. We have recently considered three protein or nucleic acid recognition sites of halobenzenes and quantified the energy gains that either electron-attracting substituents or electron-donating ones contribute due to such a character (El Hage et al., paper in revision). Nonadditivity was found to impact the total interaction energies. We focus here on one recognition site, that of the HIV-1 integrase, in which the halobenzene ring of the drug elvitegravir is sandwiched between a guanine and a cytosine base. We perform energy-decomposition analyses of the ab initio quantum-chemistry (QC) binding energies of the parent halobenzene ring and its derivatives with this G-C base pair. In these complexes, the nonadditivity of ΔE could be traced back mostly to the polarization contribution Epol. In view of large-scale applications to the entirety of the complex formed between the integrase, the viral DNA, and the whole drug, the analyses were performed in parallel with a polarizable molecular mechanics method, SIBFA. This method could faithfully reproduce most features of the QC energies. This is due to its use of QC-derived distributed multipoles and polarizabilities, which enable us to account for both nonisotropy and nonadditivity.

Recombinant expression and purification of the antimicrobial peptide magainin‐2

Magainin-2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram-positive and gram-negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N-terminal of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2-LK-CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK-CBM3. Its functionality was confirmed by testing its activity against gram-negative bacteria.

Hypomorphic Mutation in the Site-1 Protease Mbtps1 Endows Resistance to Persistent Viral Infection in a Cell-Specific Manner

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), which naturally persists in rodents, represents a model for HIV, HBV, and HCV. Cleavage of the viral glycoprotein precursor by membrane-bound transcription factor peptidase, site 1 (Mbtps1 or site-1 protease), is crucial for the life cycle of arenaviruses and therefore represents a potential target for therapy. Recently, we reported a viable hypomorphic allele of Mbtps1 (woodrat) encoding a protease with diminished enzymatic activity. Using the woodrat allele, we examine the role of Mbtps1 during persistent LCMV infection. Surprisingly, Mbtps1 inhibition limits persistent but not acute viral infection and is associated with an organ/cell type-specific decrease in viral titers. Analysis of bone marrow-derived dendritic cells from woodrat mice supports their specific role in resolving persistent viral infection. These results support in vivo targeting of Mbtps1 in the treatment of arenavirus infections and demonstrate a critical role for dendritic cells in persistent viral infections.

Antibodies Causing Thrombocytopenia in Patients Given RGD-Mimetic Platelet Inhibitors Recognize Drug-Induced (and drug-specific) Conformational Epitopes Adjacent to the RGD Recognition Site of Gpiib/Iiia.

Abstract 889▪▪This icon denotes an abstract that is clinically relevant.RGD-mimetic GPIIb/IIIa inhibitors (fibans) react with the arginine-glycine-aspartic acid (RGD) recognition site of alphaIIb/beta3 integrin (GPIIb/IIIa), thereby preventing the activated integrin from reacting with fibrinogen and participating in platelet thrombus formation. This class of drugs has been shown to inhibit significantly the incidence of adverse complications following angioplasty. However, between 0.2 and 2.0% of treated patients develop acute, sometimes severe thrombocytopenia (TP) within a few hours of treatment. Platelet destruction in these cases appears to be caused by naturally occurring antibodies that recognize GPIIb/IIIa in a complex with the drug being administered (Blood 100: 2071, 2002). It has been speculated that epitopes recognized by these antibodies are the result of structural changes induced in GPIIb/IIIa when a fiban drug binds.To test this hypothesis, we studied drug-dependent binding to GPIIb/IIIa of antibodies from patients who developed acute thromboytopenia after being treated with the RGD-mimetics eptifibatide (n = 3), tirofiban (n = 3), xemilofiban (n = 1) or orbofiban (n = 1), each of which binds to a well-defined site in the “head” region of GPIIb/IIIa at the junction of the GPIIb beta propeller and GPIIIa beta A domains. Reactions of each of the human antibodies with GPIIb/IIIa were drug-specific except for the xemilofiban- and orbofiban antibodies, which exhibited some degree of cross-reactivity. None of the human antibodies recognized GPIIb/IIIa in a complex with RGDW peptide. In preliminary studies, binding sites for a family of monoclonal antibodies (mAbs) were mapped to various domains of GPIIb/IIIa using chimeric GPIIb/IIIa constructs in which one or more structural domains were switched between human and rat. We then examined the ability of these mAbs to interfere with drug-dependent binding to GPIIb/IIIa of antibodies from patients with thrombocytopenia induced by the RGD-mimetic drugs. Two eptifibatide-dependent antibodies were inhibited by mAbs specific for the beta propeller domain of GPIIb, whereas the tirofiban- and one of the eptifibatide-specific antibodies were inhibited by mAbs that recognize the beta A domain of GPIIIa. The xemilofiban- and orbofiban-dependent antibodies were inhibited to varying degrees by mAbs to both the GPIIb beta propeller and the GPIIIa beta A domains. Monoclonal antibodies specific for more distal regions of GPIIb/IIIa had no effect on drug-dependent antibody binding.The findings indicate 1) Antibodies causing thrombocytopenia in patients treated with fibans recognize various “neoepitopes” created in domains of GPIIb/IIIa near to, but not coincident with the drug binding site; 2) Different RGD-mimetic drugs, despite their structural similarity, induce slightly different structural changes in the GPIIb/IIIa “head” region that can be differentiated by the human antibodies and 3) Epitopes recognized by the human antibodies are distinct from classical ligand-induced binding sites (LIBS) produced by RGD peptide. Why these antibodies occur naturally in some individuals is an interesting question that deserves further study. Disclosures:No relevant conflicts of interest to declare.

Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum

The cathelicidin derived human peptide LL37 has a broad spectrum of antimicrobial and immunomodulatory activities. The large variety of biological activities makes LL37 a very promising candidate for clinical applications. The production of biologically active LL37 in large amounts with reduced costs can only be achieved using recombinant techniques. In this work, LL37 has been cloned to the N- and C-termini of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; both constructions (LL37-LK-CBM3 and LK-CBM3-LL37) were cloned into the pET-21a vector. A formic acid recognition site was introduced between the two modules, allowing the isolation of LL37 after chemical cleavage.The recombinant proteins were expressed in Escherichia coli BL21 (DE3) and solubilized with Triton X-100. The purification was achieved using cellulose CF11 fibers, taking advantage of the CBM3 specific affinity for cellulose; after hydrolysis with formic acid, LL37 was further purified by reverse-phase HPLC, as confirmed by MALDI-TOF mass spectrometry. The production and purification methodology developed in this work compares advantageously to other protocols previously described, having fewer purification steps. Only the recombinant LL37 obtained from the C-terminally fused protein (LK-CBM3-LL37) showed antibacterial activity against E. coli K12, with a MIC of 180μg/ml.

Protein–RNA interactions: Structural analysis and functional classes

A data set of 89 protein-RNA complexes has been extracted from the Protein Data Bank, and the nucleic acid recognition sites characterized through direct contacts, accessible surface area, and secondary structure motifs. The differences between RNA recognition sites that bind to RNAs in functional classes has also been analyzed. Analysis of the complete data set revealed that van der Waals interactions are more numerous than hydrogen bonds and the contacts made to the nucleic acid backbone occur more frequently than specific contacts to nucleotide bases. Of the base-specific contacts that were observed, contacts to guanine and adenine occurred most frequently. The most favored amino acid-nucleotide pairings observed were lysine-phosphate, tyrosine-uracil, arginine-phosphate, phenylalanine-adenine and tryptophan-guanine. The amino acid propensities showed that positively charged and polar residues were favored as expected, but also so were tryptophan and glycine. The propensities calculated for the functional classes showed trends similar to those observed for the complete data set. However, the analysis of hydrogen bond and van der Waal contacts showed that in general proteins complexed with messenger RNA, transfer RNA and viral RNA have more base specific contacts and less backbone contacts than expected, while proteins complexed with ribosomal RNA have less base-specific contacts than the expected. Hence, whilst the types of amino acids involved in the interfaces are similar, the distribution of specific contacts is dependent upon the functional class of the RNA bound.

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae

We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C4-C6 and C3-C8 chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.

Second sialic acid binding site in Newcastle disease virus hemagglutinin-neuraminidase: implications for fusion.

Paramyxoviruses are the leading cause of respiratory disease in children. Several paramyxoviruses possess a surface glycoprotein, the hemagglutinin-neuraminidase (HN), that is involved in attachment to sialic acid receptors, promotion of fusion, and removal of sialic acid from infected cells and progeny virions. Previously we showed that Newcastle disease virus (NDV) HN contained a pliable sialic acid recognition site that could take two states, a binding state and a catalytic state. Here we present evidence for a second sialic acid binding site at the dimer interface of HN and present a model for its involvement in cell fusion. Three different crystal forms of NDV HN now reveal identical tetrameric arrangements of HN monomers, perhaps indicative of the tetramer association found on the viral surface.

Structure of the Rho Transcription Terminator: Mechanism of mRNA Recognition and Helicase Loading

In bacteria, one of the major transcriptional termination mechanisms requires a RNA/DNA helicase known as the Rho factor. We have determined two structures of Rho complexed with nucleic acid recognition site mimics in both free and nucleotide bound states to 3.0 Å resolution. Both structures show that Rho forms a hexameric ring in which two RNA binding sites—a primary one responsible for target mRNA recognition and a secondary one required for mRNA translocation and unwinding—point toward the center of the ring. Rather than forming a closed ring, the Rho hexamer is split open, resembling a “lock washer” in its global architecture. The distance between subunits at the opening is sufficiently wide (12 Å) to accommodate single-stranded RNA. This open configuration most likely resembles a state poised to load onto mRNA and suggests how related ring-shaped enzymes may be breached to bind nucleic acids.

Probing the sialic acid binding site of the hemagglutinin-neuraminidase of Newcastle disease virus: identification of key amino acids involved in cell binding, catalysis, and fusion.

We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (HN) from Newcastle disease virus. This multifunctional protein is responsible for binding to cellular sialyl-glycoconjugate receptors, promotion of fusion through interaction with the second viral surface fusion (F) glycoprotein, and processing progeny virions by removal of sialic acid from newly synthesized viral coat proteins. Our structural studies suggest that HN possesses a single sialic acid recognition site that can be switched between being a binding site and a catalytic site. Here we examine the effect of mutation of several conserved amino acids around the binding site on the hemagglutination, neuraminidase, and fusion functions of HN. Most mutations around the binding site result in loss of neuraminidase activity, whereas the effect on receptor binding is more variable. Residues E401, R416, and Y526 appear to be key for receptor binding. The increase in fusion promotion seen in some mutants that lack receptor binding activity presents a conundrum. We propose that in these cases HN may be switched into a fusion-promoting state through a series of conformational changes that propagate from the sialic acid binding site through to the HN dimer interface. These results further support the single-site model and suggest certain residues to be important for the triggering of fusion.

D-Serine inhibits serine palmitoyltransferase, the enzyme catalyzing the initial step of sphingolipid biosynthesis

Serine palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L-serine to produce 3-ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [ 3H]KDS from L-[ 3H]serine was performed using purified SPT. D-Serine inhibited [ 3H]KDS production as effectively as non-radioactive L-serine, whereas neither D-alanine nor D-threonine showed any significant effect. Incubation of purified SPT with [palmitoyl 1- 14C]palmitoyl coenzyme A and D-serine did not produce [ 14C]KDS, while the control incubation with L-serine did. These results suggest that D-serine competes with L-serine for the amino acid recognition site of SPT, but that D-serine is not utilized by this enzyme to produce KDS.