Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway in elicitor-treated, cultured tomato cells.

The accumulation of phenylalanine-derived phenolic compounds is a well-known element of a plant's defense in response to pathogen attack. Phenylalanine, as well as the other two aromatic amino acids, tyrosine and tryptophan, is synthesized by way of the shikimate pathway. The first seven steps of the shikimate pathway (the prechorismate pathway) are common for the biosynthesis of all three aromatic amino acids. We have studied transcript levels of six genes--i.e., two 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes, one shikimate kinase gene, one 5-enolpyruvylshikimate 3-phosphate synthase gene, and two chorismate synthase genes--corresponding to four steps of the prechorismate pathway, in cultured tomato cells exposed to fungal elicitors. The abundance of transcripts specific for some of these genes increased 10- to 20-fold within 6 h after elicitor treatment, as did the abundance of phenylalanine ammonialyase-specific transcripts and the synthesis of ethylene. Interestingly, transcript accumulation occurred more rapidly for shikimate kinase than for the enzymes preceding or following it in the prechorismate pathway. Neither the inhibition of ethylene biosynthesis by aminoethoxyvinylglycine nor inhibition of phenylalanine ammonia-lyase (EC 4.3.1.5) activity by 2-aminoindan-2-phosphonic acid affected the time course or extent of transcript accumulation. Thus, the increased demand for phenylalanine in the phenylpropanoid pathway required after elicitor treatment appears to be met by increased de novo synthesis of its biosynthetic enzymes.

Crystal structure of 2,5-diketo-d-gluconic acid reductase A complexed with NADPH at 2.1-Å resolution

The three-dimensional structure of Corynebacterium 2,5-diketo-d-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-Å resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-d-gluconate (2,5-DKG) to 2-keto-l-gulonate. Thus the three-dimensional structure has now been solved for a prokaryotic example of the aldo–keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of l-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-l-gulonate from d-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.

Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes

CD22 is a B cell-restricted glycoprotein involved in signal transduction and modulation of cellular activation. It is also an I-type lectin (now designated Siglec-2), whose extracellular domain can specifically recognize α2–6-linked sialic acid (Sia) residues. This activity is postulated to mediate intercellular adhesion and/or to act as a coreceptor in antigen-induced B cell activation. However, studies with recombinant CD22 indicate that the lectin function can be inactivated by expression of α2–6-linked Sia residues on the same cell surface. To explore whether this masking phenomenon affects native CD22 on B cells, we first developed a probe to detect the lectin activity of recombinant CD22 expressed on Chinese hamster ovary cells (which have no endogenous α2–6-linked Sia residues). This probe is inactive against CD22-positive B lymphoma cells and Epstein–Barr virus-transformed lymphoblasts which express high levels of α2–6-linked Sia residues. Enzymatic desialylation unmasks the CD22 lectin activity, indicating that endogenous Sia residues block the CD22 lectin-binding site. Truncation of the side chains of cell surface Sia residues by mild periodate oxidation (known to abrogate Sia recognition by CD22) also had this unmasking effect, indicating that the effects of desialylation are not due to a loss of negative charge. Normal resting B cells from human peripheral blood gave similar findings. However, the lectin is partially unmasked during in vitro activation of these cells. Thus, the lectin activity of CD22 is restricted by endogenous sialylation in resting B cells and may be transiently unmasked during in vivo activation, perhaps to modulate intercellular or intracellular interactions at this critical stage in the humoral response.

A point mutation in the γ2 subunit of γ-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate γ-aminobutyric acid (GABA) evoked chloride currents of γ-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat γ2 or γ3, in combination with α1 and β2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a γ3 subunit display a 178-fold reduced affinity to zolpidem as compared with γ2-containing receptors. Eight chimeras between γ2 and γ3 were constructed followed by nine different point mutations in γ2, each to the homologous amino acid residue found in γ3. Chimeric or mutant γ subunits were coexpressed with α1 and β2 in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of γ2 (γ2M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [3H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (γ2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and γ2M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose–response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the γ2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, γ2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.

Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase

The gene encoding 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO; EC 1.14.12.4) was cloned by using an oligonucleotide probe corresponding to the N terminus of the enzyme to screen a DNA library of Pseudomonas sp. MA-1. The gene encodes for a protein of 379 amino acid residues corresponding to a molecular mass of 41.7 kDa, the same as that previously estimated for MHPCO. MHPCO was expressed in Escherichia coli and found to have the same properties as the native enzyme from Pseudomonas sp. MA-1. This study shows that MHPCO is a homotetrameric protein with one flavin adenine dinucleotide bound per subunit. Sequence comparison of the enzyme with other hydroxylases reveals regions that are conserved among aromatic flavoprotein hydroxylases.

A σ32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity

σ32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of σ32 with other sigma factors in the σ70 family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant σ32 proteins, Q80R. This reduced core interaction is exacerbated in the presence of σ70, which competes with σ32 for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.

A single receptor encoded by vzg-1/lpA1/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid

Extracellular lysophosphatidic acid (LPA) produces diverse cellular responses in many cell types. Recent reports of several molecularly distinct G protein-coupled receptors have raised the possibility that the responses to LPA stimulation could be mediated by the combination of several uni-functional receptors. To address this issue, we analyzed one receptor encoded by ventricular zone gene-1 (vzg-1) (also referred to as lpA1/edg-2) by using heterologous expression in a neuronal and nonneuronal cell line. VZG-1 expression was necessary and sufficient in mediating multiple effects of LPA: [3H]-LPA binding, G protein activation, stress fiber formation, neurite retraction, serum response element activation, and increased DNA synthesis. These results demonstrate that a single receptor, encoded by vzg-1, can activate multiple LPA-dependent responses in cells from distinct tissue lineages.

Embryo-Specific Gene Expression in Microspore-Derived Embryos of Brassica napus. An Interaction between Abscisic Acid and Jasmonic Acid1, 2

The induction of napin and oleosin gene expression in Brassica napus microspore-derived embryos (MDEs) was studied to assess the possible interaction between abscisic acid (ABA) and jasmonic acid (JA). Napin and oleosin transcripts were detected sooner following treatment with ABA than JA. Treatment of MDEs with ABA plus JA gave an additive accumulation of both napin and oleosin mRNA, the absolute amount being dependent on the concentration of each hormone. Endogenous ABA levels were reduced by 10-fold after treatment with JA, negating the possibility that the observed additive interaction was due to JA-induced ABA biosynthesis. Also, JA did not significantly increase the uptake of [3H-ABA] from the medium into MDEs. This suggests that the additive interaction was not due to an enhanced carrier-mediated ABA uptake by JA. Finally, when JA was added to MDEs that had been treated with the ABA biosynthesis inhibitor fluridone, napin mRNA did not increase. Based on these results with the MDE system, it is possible that embryos of B. napus use endogenous JA to modulate ABA effects on expression of both napin and oleosin. In addition, JA could play a causal role in the reduction of ABA that occurs during late stages of seed development.

Acid-Growth Response and α-Expansins in Suspension Cultures of Bright Yellow 2 Tobacco1

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of α-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple α-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

Expression of Ornithine Decarboxylase Is Transiently Increased by Pollination, 2,4-Dichlorophenoxyacetic Acid, and Gibberellic Acid in Tomato Ovaries1

A cDNA encoding for a functional ornithine decarboxylase has been isolated from a cDNA library of carpels of tomato (Lycopersicon esculentum Mill.). Ornithine decarboxylase in tomato is represented by a single-copy gene that we show to be up-regulated during early fruit growth induced by 2,4-dichlorophenoxyacetic acid and gibberellic acid.

Poly (alpha 2,8-deaminoneuraminic acid) is expressed in lung on a single 150-kDa glycoprotein and is an oncodevelopmental antigen.

Homopolymers of alpha 2,8-linked N-acetylneuraminic acid [poly(alpha 2,8-Neu5Ac)] of the neural cell adhesion molecule NCAM have been shown to be temporally expressed during lung development and represent a marker for small cell lung carcinoma. We report the presence of a further polysialic acid in lung that consists of oligo/polymers of alpha 2,8-linked deaminoneuraminic acid residues [poly (alpha 2,8-KDN)], as detected with a monoclonal antibody in conjunction with a specific sialidase. Although the various cell types forming the bronchi, alveolar septs, and blood vessels were positive for poly (alpha 2,8-KDN) by immunohistochemistry, this polysialic acid was found on a single 150-kDa glycoprotein by immunoblot analysis. The poly(alpha 2,8-KDN)-bearing glycoprotein was not related to an NCAM protein based on immunochemical criteria. The expression of the poly (alpha 2,8-KDN) was developmentally regulated as evidenced by its gradual disappearance in the rat lung parenchyma commencing 1 week after birth. In adult lung the blood vessel endothelia and the smooth muscle fibers of both blood vessels and bronchi were positive but not the bronchial and alveolar epithelium. The poly (alpha 2,8-KDN)-bearing 150-kDa glycoprotein became reexpressed in various histological types of lung carcinomas and cell lines derived from them and represents a new oncodevelopmental antigen in lung.

Cell-type and promoter-context dependent retinoic acid receptor (RAR) redundancies for RAR beta 2 and Hoxa-1 activation in F9 and P19 cells can be artefactually generated by gene knockouts.

By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.

Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses.

In recent years, it has become apparent that salicylic acid (SA) plays an important role in plant defense responses to pathogen attack. Previous studies have suggested that one of SA's mechanisms of action is the inhibition of catalase, resulting in elevated levels of H2O2, which activate defense-related genes. Here we demonstrate that SA also inhibits ascorbate peroxoidase (APX), the other key enzyme for scavenging H2O2. The synthetic inducer of defense responses, 2,6-dichloroisonicotinic acid (INA), was also found to be an effective inhibitor of APX. In the presence of 750 microM ascorbic acid (AsA), substrate-dependent IC50 values of 78 microM and 95 microM were obtained for SA and INA, respectively. Furthermore, the ability of SA analogues to block APX activity correlated with their ability to induce defense-related genes in tobacco and enhance resistance to tobacco mosaic virus. Inhibition of APX by SA appears to be reversible, thus differing from the time-dependent, irreversible inactivation by suicide substrates such as p-aminophenol. In contrast to APX, the guaiacol-utilizing peroxidases, which participate in the synthesis and crosslinking of cell wall components as part of the defense response, are not inhibited by SA or INA. The inhibition of both catalase and APX, but not guaiacol peroxidases, supports the hypothesis that SA-induced defense responses are mediated, in part, through elevated H2O2 levels or coupled perturbations of the cellular redox state.