A Trade-off Between Thermostability and Binding Affinity of Anti-(4-hydroxy-3-nitrophenyl)Acetyl Antibodies During the Course of Affinity Maturation

The Protein Journal - Somatic hypermutation (SHM) is one of the driving forces that increases antibody (Ab) affinity. We studied the effects of SHM on thermostability and affinity using three...     

NAD-Dependent and NADP-Dependent Alcohol Dehydrogenases in Escherichia coli

The isomeric ratio and level of natural xanthoxin (XAN) in tomato plants (Lycopersicon esculentum) were examined by a more reliable analytical method than has been reported before. Efforts were made to avoid artificial isomerization between c-XAN and t-XAN throughout the isolation, derivatization and GC-MS procedures. Natural XAN was separated from contaminating chlorophylls before rev. HPLC purification, derivatized to abscisic acid methyl ester (MeABA) in four chemical steps, and quantified with the deuterium-labeled internal standards on clear and reproducible full GC-EI-MS. It was revealed that the isomeric composition of natural XAN was exclusively shifted to c-XAN. The level of c-XAN was higher and more significantly induced by water stress in older plants. The significant role of c-XAN as an ABA biosynthetic precursor is suggested.     

Distribution of an enzyme system producing cis-3-hexenal and n-hexanal from linolenic and linoleic acids in some plants

The activity of the enzyme system (E₂-I) producing C₆-aldehydes from C₁₈-unsaturated fatty acids was investigated using about 40 plants. Green leaves of dicotyledonous plants belonging to the Sphenopsida, Pteropsida Theaceae and Leguminosae showed a high enzyme (E₂-I) activity but edible leafy vegetables and fuits and monocotyledonous plants showed a low activity. Seasonal changes in the enzyme (E₂-I) activities were observed. The concentrations of cis-3-hexenol (leaf alcohol) and trans-2-hexenal (leaf aldehyde) and the enzyme (E₂-I) activities showed a correlation; high concentrations were observed in the summer but they were low in the winter.     

Blätteralkohol (XVI)Blätteralkohol (XVII)

2-Methyl-4, 6-cyclohexadienaldehyde and n-butyraldehyde were treated with sodium in p-xylene to yield the aromatized “leaf alcohol reaction” product, 2-methyl-benzylalcohol, in a better yield than that with the cyclohexadienaldehyde alone. n-Butyric acid isolated from the reaction mixture unequivocally showed the operation of the “crossed Cannizzaro disproportionation” in this reaction, aliphatic aldehyde serving as the hydride donor. 2-Propyl-5-ethyl-4, 6-cyclohexadienaldehyde was obtained by the NaOH/H₂O-EtOH Michael-Aldol condensation of leaf aldehyde, gave 2-propyl-5-ethyl-benzylalcohol along with caproic acid.

On the basis of “leaf alcohol-reaction” mechanism, it was obtained following benzyl-alkohols; 2-methyl-, 2-propyl-, 2-methyl-5-ethyl-, 2-propyl-5-ethyl-benzylalcohol, from leaf alcohol and crotylalcohol.     

Identification and characterization of a GDSL lipase-like protein that catalyzes the ester-forming reaction for pyrethrin biosynthesis in Tanacetum cinerariifolium- a new target for plant protection

Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium.     

Aleuria aurantia lectin exhibits antifungal activity against Mucor racemosus

Aleuria aurantia lectin (AAL) is an L-fucose-specific lectin produced in the mycelia and fruit-bodies of the widespread ascomycete fungus Aleuria aurantia. It is extensively used in the detection of fucose, but its physiological role remains unknown. To investigate this, we analyzed the interaction between AAL and, a zygomycete fungus Mucor racemosus, which is assumed to contain fucose in its cell wall. AAL specifically bound to the hyphae of M. racemosus, because binding was inhibited by L-fucose but not by D-fucose. It inhibited the growth of the fungus at 1 μM, and the M. racemosus cells were remarkably disrupted at 7.5 μM. In contrast, two other fucose-specific lectins, Anguilla anguilla agglutinin and Ulex europaeus agglutinin, did not inhibit the growth of M. racemosus. These results suggest that the growth inhibition activity is unique to AAL, and that AAL could act as an antifungal protein in natural ecosystems.     

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.