Structure-based function discovery of an enzyme for the hydrolysis of phosphorylated sugar lactones

Two enzymes of unknown function from the cog1735 subset of the amidohydrolase superfamily (AHS), LMOf2365_2620 (Lmo2620) from Listeria monocytogenes str. 4b F2365 and Bh0225 from Bacillus halodurans C-125, were cloned, expressed, and purified to homogeneity. The catalytic functions of these two enzymes were interrogated by an integrated strategy encompassing bioinformatics, computational docking to three-dimensional crystal structures, and library screening. The three-dimensional structure of Lmo2620 was determined at a resolution of 1.6 ? with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 ? from the distal phosphate. The distal phosphate hydrogen bonds with Lys-242, Lys-244, Arg-275, and Tyr-278. Enzymes within cog1735 of the AHS have previously been shown to catalyze the hydrolysis of substituted lactones. Computational docking of the high-energy intermediate form of the KEGG database to the three-dimensional structure of Lmo2620 highly enriched anionic lactones versus other candidate substrates. The active site structure and the computational docking results suggested that probable substrates would likely include phosphorylated sugar lactones. A small library of diacid sugar lactones and phosphorylated sugar lactones was synthesized and tested for substrate activity with Lmo2620 and Bh0225. Two substrates were identified for these enzymes, D-lyxono-1,4-lactone-5-phosphate and l-ribono-1,4-lactone-5-phosphate. The k(cat)/K(m) values for the cobalt-substituted enzymes with these substrates are ~10(5) M(-1) s(-1).     

Synchronous Conidiation of Piricularia oryzae by the Replacement Culture

For the purpose of studying the mechanism of conidiogenesis in Piricularia oryzae, methods were developed to separate the phase of mycelial growth from that of conidiation and also to evaluate them quantitatively.

When P. oryzae was grown on media with low carbon : nitrogen ratio and high nitrogen concentration, conidiation did not take place, in spite of its vegetative growth. Conidiation occurred in a very short period of time when the above mycelia were replaced on nutritionally poor media. Cellophane membrane was overlaid on solid medium and conidia were spread uniformly. Evenly grown mycelial mat which could be easily transferred onto the post-culture medium was obtained. As the preculture medium, MSA medium with carbon: nitrogen ratio of 6.3 and nitrogen concentration of 1.5 g/liter was used. The evenly grown mycelial mat was cut into small square mats of 1.44 cm² each and the small square mycelial mats were transferred onto the post-culture medium together with the cellophane membrane. The conidiation took place in the post-culture and the vegetative growth in the preculture and the conidiation in the post-culture could be observed separately and quantitatively.

Conidiation did not occur at all in the preculture and the degree of conidiation which took place in the post-culture varied according to the precultural conditions. This means that it is a certain state of physiological condition in the preculture which determines the degree of conidiation in the post-culture. The authers designated this state of physiological condition as the “latent activity of conidiation” (LAC). For the purpose of the quantitative estimation of this activity, we expressed LAC in terms of the degree of conidiation in the post-culture under a defined cultural condition. The LAC was subject to change very easily, declined rapidly and disappeared upon prolonged preculture. Only young mycelia showed to have this activity.

The influences of the precultural condition on the development of the LAC and vegetative growth were generally parallel. However, the LAC was generally more sensitive to the environmental condition than the vegetative growth, especially to the temperature change.

The conidia formed were uniform in size and had high rate of germination. Several strains of P. oryzae tested showed very similar behavior.     

Isolation of Tenuazonic Acid from Blast-diseased Rice Plants

dl-(1245/36)-2,3,4,5,6-Pentachlorocyclohexanecarbonitrile was synthesized from (1234/ 56)-1,4,5,6-tetrachloro-2,3-epoxycyclohexane (α-BTC cis-epoxide). dl-(1245/36)-2,3,4,5,6-Pentachloro-1-methylcyclohexane was synthesized from the nitrile via dl-(1245/36)-2,3,4,5,6-pentachlorocyclohexylmethanol, the structure of which was confirmed by PMR spectroscopy using spin decoupling techniques and the shift reagent, Eu(DPM)₃. This series of compounds was shown to have the same configuration as γ-BHC. The conformational equilibrium of these compounds is discussed. dl-(1245/36)-2,3,5,6-Tetrachloro-1,4-dimethyl-cyclohexane was synthesized by a stepwise route involving a Diels-Alder reaction of trans,trans-hexadiene-2,4 with maleic anhydride.     

The Incorporation of Ciliatine (2-Aminoethylphosphonic Acid) into Lipids of the Rat Liver

Four alcohols, 1-penten-3-ol, n-amylalcohol, trans-2-hexenol and one of the linalool oxides, were newly identified as the components of carbonyl-free neutral fraction of the essential oil of black tea.

On the gas chromatogram of carbonyl fraction three unknown peaks were identified with those of n-valeraldehyde, n-heptanal and trans-2-octenal.

From these results almost all main components of carbonyl and carbonyl free fractions were clarified.

Flavor change during the manufacture of black tea was investigated by gas chromatography. During withering, hexylalcohol, nerol, trans-2-hexenoic acid, trans-2-heхenol, linalool oxide (cis, furanoid), n-valeraldehyde, capronaldehyde, n-heptanal, trans-2-hexenal, trans-2-octenal, benzaldehyde, phenylacetaldehyde, n-butyric, isovaleric, n-caproic, cis-3-hexenoic and salicylic acids and o-cresol were increased, especially the former three greatly increased, while cis-2-pentenol, linalool, geraniol, benzylalcohol, phenylethanol and acetic acid diminished markedly. In the process of fermentation almost all constituents increased, especially, 1-penten-3-ol, cis-2-pentenol, benzylalcohol, trans-2-hexenal, benzaldehyde, n-caproic, cis-3-hexenoic and salicylic acids were remarkable.

On firing, most alcohols, carbonyl and phenolic compounds decreased remarkably whereas acetic, propionic and isobutyric acids greatly increased.     

Genome-Wide Association Study for Levels of Total Serum IgE Identifies HLA-C in a Japanese Population

Most of the previously reported loci for total immunoglobulin E (IgE) levels are related to Th2 cell-dependent pathways. We undertook a genome-wide association study (GWAS) to identify genetic loci responsible for IgE regulation. A total of 479,940 single nucleotide polymorphisms (SNPs) were tested for association with total serum IgE levels in 1180 Japanese adults. Fine-mapping with SNP imputation demonstrated 6 candidate regions: the PYHIN1/IFI16, MHC classes I and II, LEMD2, GRAMD1B, and chr13∶60576338 regions. Replication of these candidate loci in each region was assessed in 2 independent Japanese cohorts (n = 1110 and 1364, respectively). SNP rs3130941 in the HLA-C region was consistently associated with total IgE levels in 3 independent populations, and the meta-analysis yielded genome-wide significance (P = 1.07×10⁻¹⁰). Using our GWAS results, we also assessed the reproducibility of previously reported gene associations with total IgE levels. Nine of 32 candidate genes identified by a literature search were associated with total IgE levels after correction for multiple testing. Our findings demonstrate that SNPs in the HLA-C region are strongly associated with total serum IgE levels in the Japanese population and that some of the previously reported genetic associations are replicated across ethnic groups.     

ORAI1 genetic polymorphisms associated with the susceptibility of atopic dermatitis in Japanese and Taiwanese populations

Atopic dermatitis is a chronic inflammatory skin disease. Multiple genetic and environmental factors are thought to be responsible for susceptibility to AD. In this study, we collected 2,478 DNA samples including 209 AD patients and 729 control subjects from Taiwanese population and 513 AD patients and 1027 control subject from Japanese population for sequencing and genotyping ORAI1. A total of 14 genetic variants including 3 novel single-nucleotide polymorphisms (SNPs) in the ORAI1 gene were identified. Our results indicated that a non-synonymous SNP (rs3741596, Ser218Gly) associated with the susceptibility of AD in the Japanese population but not in the Taiwanese population. However, there is another SNP of ORAI1 (rs3741595) associated with the risk of AD in the Taiwanese population but not in the Japanese population. Taken together, our results indicated that genetic polymorphisms of ORAI1 are very likely to be involved in the susceptibility of AD.     

Monosaccharide-based water-soluble fluorescent tags

Monosaccharides are fluorescently labeled under microwave irradiation by N-(coumarin-3-carbonyl)benzotriazole 4. 1,2:3,4-di- O-isopropylidene-alpha- d-galactopyranose 9 gives 12 (90%), 1,2:5,6-di- O-isopropylidene- d-glucose 10 gives 13 (89%), 2,3:5,6-di- O-isopropylidene-alpha- d-mannofuranose 11 gives 14 (65%) (all by O-acylation) and 2,3,4,5-tetra- O-pivaloyl-beta- d-galactopyranosylamine 15 gives 16 (60%) (by N-acylation). Similarly, the coumarin-containing activated lysine derivatives 7 and 8 afford the l-lysine-scaffold based coumarin labeled sugars 17, 18a, b, and 19 (67-85%) which, after removal of the diisopropylidene groups, provide water-soluble fluorescent derivatives.     

Frequency distribution of catch per tow by the danish trawlers in the waters off the Bristol Bay,with some considerations on the relation of total catch to the weight limit of hauling

1. The type of the frequency distribution of catch per tow observed by the Danish trawlers of a fish-meal fleet in the waters off the Bristol Bay during the period from May 1 to 9 in 1960 in discussed.
2. Catch per tow varied considerably from as much as one ton to ten tons and the tow-time also did markedly tow by tow from one to thirty minutes or longer. Therefore, as a preliminary step to discuss the type of the frequency distribution, we examined whether catch varied depending on the tow-time or not; and we found the catch deviated almost independently of the tow-time.
3. Here, however much fish might be caught by a two, the part over ten tons was released; consequently, the frequency increased gradually from zero to nine tons and increased abruptly at ten tons but none of the haul could get a better catch than ten tons. And examining the relation between Student's t and the grade of catch in tons (n) in the range from zero to nine tons, we found that such a peculiar type of the frequency distribution as observed actually was derived from the series of the catch well fitted for the normal distribution by the modification due to releasing of a part of catch. This fact evolved, the possible series of the frequencies and the total of the possible catch when no part of catch is released, are estimated.
4. Then some considerations are given to the problem (1) how high percentage of the catch on the total of the possible catch is obliged to release because of the low weight limit of hauling and (2) to what percentage of the catch we can successfully make into available when we increase the weight limit of hauling to a certain value; and the results is shown in Fig. 3, which tells that (1) 70 to 95 percents of the catch were available in the actual operation and (2) scarecely any increase in catch is expected even if the weight limit of hauling is increased higher than fifteen tons.

     

Genotype-Specific Interaction of Latent TGFβ Binding Protein 4 with TGFβ

Latent TGFβ binding proteins are extracellular matrix proteins that bind latent TGFβ to form the large latent complex. Nonsynonymous polymorphisms in LTBP4, a member of the latent TGFβ binding protein gene family, have been linked to several human diseases, underscoring the importance of TGFβ regulation for a range of phenotypes. Because of strong linkage disequilibrium across the LTBP4 gene, humans have two main LTBP4 alleles that differ at four amino acid positions, referred to as IAAM and VTTT for the encoded residues. VTTT is considered the “risk” allele and associates with increased intracellular TGFβ signaling and more deleterious phenotypes in muscular dystrophy and other diseases. We now evaluated LTBP4 nsSNPs in dilated cardiomyopathy, a distinct disorder associated with TGFβ signaling. We stratified based on self-identified ethnicity and found that the LTBP4 VTTT allele is associated with increased risk of dilated cardiomyopathy in European Americans extending the diseases that associate with LTBP4 genotype. However, the association of LTBP4 SNPs with dilated cardiomyopathy was not observed in African Americans. To elucidate the mechanism by which LTBP4 genotype exerts this differential effect, TGFβ’s association with LTBP4 protein was examined. LTBP4 protein with the IAAM residues bound more latent TGFβ compared to the LTBP4 VTTT protein. Together these data provide support that LTBP4 genotype exerts its effect through differential avidity for TGFβ accounting for the differences in TGFβ signaling attributed to these two alleles.