Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.

The broad-leaf herbicide 2,4-dichlorophenoxyacetic acid turns rice into a living trap for a major insect pest and a parasitic wasp

Synthetic chemical elicitors of plant defense have been touted as a powerful means for sustainable crop protection. Yet, they have never been successfully applied to control insect pests in the field. We developed a high-throughput chemical genetics screening system based on a herbivore-induced linalool synthase promoter fused to a β-glucuronidase (GUS) reporter construct to test synthetic compounds for their potential to induce rice defenses. We identified 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin homolog and widely used herbicide in monocotyledonous crops, as a potent elicitor of rice defenses. Low doses of 2,4-D induced a strong defensive reaction upstream of the jasmonic acid and ethylene pathways, resulting in a marked increase in trypsin proteinase inhibitor activity and volatile production. Induced plants were more resistant to the striped stem borer Chilo suppressalis, but became highly attractive to the brown planthopper Nilaparvata lugens and its main egg parasitoid Anagrus nilaparvatae. In a field experiment, 2,4-D application turned rice plants into living traps for N. lugens by attracting parasitoids. • Our findings demonstrate the potential of auxin homologs as defensive signals and show the potential of the herbicide to turn rice into a selective catch crop for an economically important pest.

THE EQUILIBRIUM CONSTANT FOR THE GLYCINE SYNTHASE REACTION (ONE-CARBON, METABOLISM, FOLATE)

The equilibrium constant (K(,c)) under physiological conditions (38(DEGREES)C, 0.25 M ionic strength (I), pH 7.0) for the glycine synthase (GS) reaction (E C 2.1.2.1.0) (Equation 1) has been determined. (UNFORMATTED TABLE FOLLOWS)^ 5,10-CH(,2)-H(,4)Folate NADH NH (,4)+ CO(,2) ^ K(,c) = Eq. 1^ H(,4)Folate NAD('+) GLY ^(TABLE ENDS)^ The enzymatic instability of the GS enzyme complex itself has made it necessary to determine the overall K(,c) from the product of constants for the partial reactions of GS determined separately under the same conditions. The partial reactions are the H(,4)Folate-formaldehyde (CH(,2)(OH)(,2)) condensation reaction (Reaction 1) the K(,c) for which has been reported by this laboratory (3.0 x 10('4)), the lipoate (LipS(,2)) dehydrogenase reaction (LipDH) (Reaction 2) and the Gly-Lip^ decarboxylase reaction (Reaction 3) forming reduced lipoate (Lip(SH)(,2)), NH(,4)('+), CO(,2) and CH(,2)(OH)(,2.) (UNFORMATTED TABLE FOLLOWS)(,)^ H(,4)Fote + CH(,2)(OH)(,2) 5,10-CH(,2)-H(,4)Folate (1)^ Lip(SH)(,2) + NAD('+) LipS(,2) + NADH + H('+) (2)^ H('+) + Gly + LipS(,2) Lip(SH)(,2) + NH(,4)('+) CO(,2) + CH(,2)(OH)(,2) (3)^(TABLE ENDS)^ In this work the K(,c) for Reactions 2 and 3 are reported.^ The K(,c)' for the LipDH reaction described by other authors was reported with unexplainable conclusions regarding the pH depend- ence for the reaction. These conclusions would imply otherwise unexpected acid dissociation constants for reduced and oxidized lipoate. The pK(,a)',s for these compounds have been determined to resolve discrepancy. The conclusions are as follows: (1) The K(,c) for the LipDH reaction is 2.08 x 10('-8); (2) The pK(,a)',s for Lip(SH)(,2) are 4.77(-COOH), 9.91(-SH), 11.59(-SH); for LipS(,2) the carboxyl pK(,a)' is 4.77; (3) Contrary to previous literature, the log K(,c)' for the LipDH reaction is a linear function of the pH, a conclusion supported by the values for the dissociation constants.^ The K(,c) for Reaction 3 is the product of constants for Reactions 4-7. (UNFORMATTED TABLE FOLLOWS)^ LipSHSCH(,2)OH + H(,2)O Lip(SH)(,2) + CH(,2)(OH)(,2) (4)^ H(,2)O + LipSHSCH(,2)NH(,3)('+) LipSHSCH(,2)OH + NH(,4)('+) (5)^ LipSHSCH(,2)NH(,2) + H('+) LipSHSCH(,2)NH(,3)('+) (6)^ Gly + LipS(,2) LipSHSCH(,2)NH(,2) + CO(,2) (7)^(TABLE ENDS)^ Reactions 4-6 are non-enzymatic reactions whose constants were determined spectrophotometrically. Reaction 7 was catalyzed by the partially purified P-protein of GS with equilibrium approached from both directions. The value for K(,c) for this reaction is 8.15 x 10('-3). The combined K(,c) for Reactions 4-7 or Reaction 3 is 2.4 M.^ The overall K(,c) for the GS reaction determined by combination of values for Reactions 1-3 is 1.56 x 10('-3). ^

Amino acid in Deep Sea Drilling Project cores

Several amino acid diagenetic reactions, which take place in the deep-sea sedimentary environment, were investigated, using various Deep Sea Drilling Project (DSDP) cores. Initially it was found that essentially all the amino acids in sediments are bound in peptide linkages; but, with increasing age, the peptide bonds undergo slow hydrolysis that results in an increasingly larger fraction of amino acids in the free state. The hydrolysis half-life in calcareous sediments was estimated to be ~1-2 million years, while in non-carbonate sediment the hydrolysis rate may be considerably slower. The amino acid compositions and the extent of racemization of several amino acids were determined in various fractions isolated from the sediments. These analyses demonstrated that the mechanism, kinetics, and rate of amino acid diagenesis are highly dependent upon the physical state (i.e., free, bound, etc.) in which the amino acids exist in the sedimentary environment. In the free state, serine and threonine were found to decompose primarily by a dehydration reaction, while in the bound state (residue or HCl-insoluble fraction) a reversible aldol-cleavage reaction is the main decomposition pathway of these amino acids. The change in amino acid composition of the residue fraction with time was suggested to be due to the hydrolysis of peptide bonds, while in foraminiferal tests the compositional changes over geological time are the result of various decomposition reactions. Reversible first-order racemization kinetics are not observed for free amino acids in sediments. The explanation for these anomalous kinetics involves a complex reaction series which includes the hydrolysis of peptide bonds and the very rapid racemization of free amino acids. The racemization rates of free amino acids in sediments were found to be many orders of magnitude faster than those predicted from elevated temperature experiments using free amino acids in aqueous solution. The racemization rate enhancement of free amino acids in sediments may be due to the catalysis of the reaction by trace metals. Reversible first-order kinetics are followed for amino acids in the residue fraction isolated from sediments; the rate of racemization in this fraction is slower than that predicted for protein-bound amino acids. Various applications of amino acid diagenetic reactions are discussed. Racemization and the decomposition reaction of serine and threonine can both be used, with certain limitations, to make rough age estimates of deep-sea sediments back to several million years. The extent of racemization in foraminiferal tests which have been dated by some other independent technique can be used to estimate geothermal gradients, and thus heat flows, and to evaluate the bottom water temperature history in certain oceanic areas.

Amino acid concentrations in interstitial waters from ODP Sites 113-689 and 113-690

Biogenic calcareous and siliceous sediments were drilled at ODP Sites 689 and 690 on the Maud Rise, Antarctic Ocean. We analyzed dissolved combined amino acids (DCAA) and dissolved free amino acids (DFAA) in interstitial waters in order to characterize the amino acids in dissolved organic matter. The DFAA was predominant over the DCAA in interstitial waters at Sites 689 and 690, which contradicted the previous results from interstitial water and seawater studies. The DCAA in the interstitial waters probably originated from calcareous biogenic debris with less amounts of siliceous debris. Although glutamic acid constituted 41% of the total concentration of DCAA, it accounted for only 1% of the total concentration of DFAA due to the adsorption and/or reaction with biogenic carbonate. Ornithine, a nonprotein amino acid, is a decomposed product of arginine and made up 17 mol% of the total DFAA and. The total hydrolyzable amino acids (=DCAA + DFAA) accounted for 5 to 28% of the dissolved organic carbon (DOC) concentration, which implied that high molecular weight organic matter was a major contributor for the DOM (dissolved organic matter) in interstitial waters. Fairly positive correlation between the dissolved manganese and the total DCAA values suggested that the redox condition plays a significant role in controlling the total DCAA content. A small decrease in the sulfate concentration in the interstitial waters from both sites suggested fairly low microbial activity by sulfate-reducing bacteria.

Highly stereoselective decarboxylation of (+)-1-Bromo-1-chloro-2,2,2-trifluoropropanoic acid gives (+)-1-Bromo-1-chloro-2,2,2-trifluoroethane ((+)-Halothane) with retention of configuration

The absolute configuration of the title acid (2) has been determined to be S by X-ray crystallography. Thus, decarboxylation of 2 produces (S)-(+)-halothane with 99% retention of configuration. This behavior is compared to other stereoselective decarboxylation reactions of ?-haloacids from the literature that also give high degrees of retention of configuration when in the form of their quaternary ammonium salts, which contain one proton. The proton of the ammonium salt is necessary to protonate the anionic intermediate formed from decarboxylation. In the absence of this relatively acidic proton, we had previously found that using triethylene glycol (TEG) as both solvent and proton source for the decarboxylation reaction of acid 2 caused poor stereoselectivity. This was in contrast to 1,2,2,2-tetrafluoro-1-methoxypropionic acid (6), which showed a high degree of retention of configuration in TEG. To rationalize this differing behavior we report DFT studies at PCM-B3LYP/6-31++G** level of theory (the results were additionally confirmed with 6-311++G** and aug-cc-pVDZ basis sets). The energy barrier to inversion of configuration of the anionic reaction intermediate of acid 2 (11) is 10.23 kcal/mol. However, we find that the anionic intermediate from acid 6 (10) would rather undergo ?-elimination instead of inversion of configuration. Thus the planar transition state required for inversion of configuration is never reached, regardless of the rate of proton transfer to the anion.

Hyperspectral images and polymerase chain reaction (PCR) for the detection of allergen (peanuts traces) in powder foods.

The addition of preservatives to some kind of foods may be a potential risk of spoilage, as it is the transformation of sorbate into the off-odour 1-3-pentadiene by certain microbial species. This is the case of the capacity of some strains of moulds and yeasts that are able to decarboxylate sorbic acid and transform it into 1-3 pentadiene, a volatile compound with an unpleasant petroleum odour. (Casas et al. 2004).

Role of a critical water in scytalone dehydratase-catalyzed reaction

Scytalone dehydratase (EC 4.2.1.94) catalyzes the dehydration of two important intermediates in the biosynthesis of melanin, and it functions without metal ions or any cofactors. Using molecular orbital theory, we have examined the role of a critical water molecule in the mechanism of scytalone dehydratase. The water, together with an internal hydrogen bonding, contributes significantly to the stabilization of the transition state (or the enolate intermediate). The role of two active site tyrosines (Tyr-50 and Tyr-30) is (i) to hold the critical water in place so that it may stabilize the transition state without much structural rearrangement during the catalytic reaction, and (ii) to polarize the water, making it a better general acid. The stereochemistry of the scytalone dehydratase-catalyzed dehydration is also discussed.

Non-enzymatic and enzymatic hydrolysis of alkyl halides: A haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an SN2 displacement reaction

The semiempirical PM3 method, calibrated against ab initio HF/6–31+G(d) theory, has been used to elucidate the reaction of 1,2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of Xanthobacter autothropicus. Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations. The three most significant observations of the present study are that: (i) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of AcO− with DCE; (ii) the structures of the transition states in the gas-phase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (iii) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal/mol. The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.

Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis

Rhodopsin is a prototypical G protein-coupled receptor that is activated by photoisomerization of its 11-cis-retinal chromophore. Mutant forms of rhodopsin were prepared in which the carboxylic acid counterion was moved relative to the positively charged chromophore Schiff base. Nanosecond time-resolved laser photolysis measurements of wild-type recombinant rhodopsin and two mutant pigments then were used to determine reaction schemes and spectra of their early photolysis intermediates. These results, together with linear dichroism data, yielded detailed structural information concerning chromophore movements during the first microsecond after photolysis. These chromophore structural changes provide a basis for understanding the relative movement of rhodopsin’s transmembrane helices 3 and 6 required for activation of rhodopsin. Thus, early structural changes following isomerization of retinal are linked to the activation of this G protein-coupled receptor. Such rapid structural changes lie at the heart of the pharmacologically important signal transduction mechanisms in a large variety of receptors, which use extrinsic activators, but are impossible to study in receptors using diffusible agonist ligands.

Biosynthetic origin of conjugated double bonds: Production of fatty acid components of high-value drying oils in transgenic soybean embryos

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of α-eleostearic (18:3Δ9cis,11trans,13trans) and α-parinaric (18:4Δ9cis,11trans,13trans,15cis) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of α-eleostearic and α-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Δ12-oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of α-eleostearic and α-parinaric acids, neither of which is present in untransformed soybean embryos. α-Eleostearic and α-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Δ12-oleic acid desaturase-type enzymes that we have termed “conjugase.”

Glutamic acid 286 in subunit I of cytochrome bo3 is involved in proton translocation

Glutamic acid 286 (E286; Escherichia coli cytochrome bo3 numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome bo3 vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O2. We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.

Genetic control of abscisic acid biosynthesis in maize

Abscisic acid (ABA), an apocarotenoid synthesized from cleavage of carotenoids, regulates seed maturation and stress responses in plants. The viviparous seed mutants of maize identify genes involved in synthesis and perception of ABA. Two alleles of a new mutant, viviparous14 (vp14), were identified by transposon mutagenesis. Mutant embryos had normal sensitivity to ABA, and detached leaves of mutant seedlings showed markedly higher rates of water loss than those of wild type. The ABA content of developing mutant embryos was 70% lower than that of wild type, indicating a defect in ABA biosynthesis. vp14 embryos were not deficient in epoxy-carotenoids, and extracts of vp14 embryos efficiently converted the carotenoid cleavage product, xanthoxin, to ABA, suggesting a lesion in the cleavage reaction. vp14 was cloned by transposon tagging. The VP14 protein sequence is similar to bacterial lignostilbene dioxygenases (LSD). LSD catalyzes a double-bond cleavage reaction that is closely analogous to the carotenoid cleavage reaction of ABA biosynthesis. Southern blots indicated a family of four to six related genes in maize. The Vp14 mRNA is expressed in embryos and roots and is strongly induced in leaves by water stress. A family of Vp14-related genes evidently controls the first committed step of ABA biosynthesis. These genes are likely to play a key role in the developmental and environmental control of ABA synthesis in plants.

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1–1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)—this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain

A series of chimeral genes, consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cDNA, and yeast ACC1 3′-tail, was used to complement a yeast ACC1 mutation. These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins. Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5′-end, complement the yeast mutation. Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 μM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases. This inhibition resembles the inhibition of wheat plastid ACCase observed in vitro and in vivo. The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase. Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors. The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors.