Regio-selective preparation of vinyl adenosine ester catalyzed by alkaline protease

The transesterification of divinyladipate with adenosine in DMF containing 20% (v/v) DMSO was catalyzed by Streptomyces sp. alkaline protease and esterification occurred exclusively at the 3-position of hydroxyl group of ribofuranose in adenosine to give 3-O-vinyladipoyl adenosine without other products.     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

The rice retrotransposon Tos17 prefers low-copy-number sequences as integration targets

The rice retrotransposon Tos17 is highly activated by tissue culture. To evaluate the impact of transposition of Tos17 on the rice genome and examine its utility for insertional mutagenesis, more than 100 sequences flanking newly transposed Tos17 copies were characterised. The 5-bp target-site duplications flanking Tos17 did not show any consensus sequence, and preferred nucleotides, A/T and G/C, were only found at the second and third nucleotides from both ends of the target site duplications, respectively, indicating that Tos17 has relatively low target-site specificity at the nucleotide sequence level. Integration targets were widely distributed over the chromosomes; however, preferential integration into the sucrose synthase 2 gene and into Tos17 itself was demonstrated by PCR screening using pooled DNA prepared from the mutant population. Hybridisation studies indicated that Tos17 preferentially integrates into low-copy-number regions of the genome. In agreement with this result, about 30% of flanking sequences examined showed significant homology to known genes. Taken together, these results show that Tos17 can have a significant impact on the rice genome and can be used as a tool for efficient insertional mutagenesis.     

Recombinant Rhodococcus sp. strain T09 can desulfurize DBT in the presence of inorganic sulfate

The putative Rhodococcus rrn promoter region was cloned from the benzothiophene desulfurizing Rhodococcus sp. strain T09, and the dibenzothiophene desulfurizing gene, dsz, was expressed under the control of the putative rrn promoter in the strain T09 using a Rhodococcus–E.coli shuttle vector. Strain T09 harboring the expression vector, pNT, could desulfurize dibenzothiophene in the presence of inorganic sulfate, methionine, or cysteine, while the Dsz phenotype was completely repressed in recombinant cells carrying the gene under the control of the native dsz promoter under the same conditions. Among the sulfur sources examined, no intermediates were detected and only the final desulfurized product, 2-hydroxy-biphenyl, was produced using ammonium sulfate as the sulfur source. Received: 4 December 2001 / Accepted: 7 January 2002     

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 degrees C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N,N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.     

Purification and characterization of a novel chitinase from Burkholderia cepacia strain KH2 isolated from the bed log of Lentinus edodes,Shiitake mushroom

One of the chitinases secreted in the culture filtrate of a gram-negative bacteria, Burkholderia cepacia strain KH2, which was isolated from the bed log of Lentinus edodes, Shiitake mushrooms, was purified by DEAE Sepharose CL-6B chromatography, followed by Sephacryl S-100 HR gel filtration. The purified enzyme was homogenous, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with an estimated molecular weight of 34,000 and an isoelectric point (pI) of 5.9. The enzyme was stable at pH values of 4.0-6.0, and at temperatures up to 50 degrees C; the optimum pH and temperature were 4.5 and 50 degrees C, respectively. The enzyme exhibited higher activities toward chitosan 7B, a 62% deacetylated chitosan, than toward the highly deacetylated chitosan substrates. The enzyme was observed to drastically hydrolyze partially deacetylated chitin substrates, with the subsequent formation of N-acetylchitooligosaccharides [(GlcNAc) (n), n=2-7]. Separation and quantification of the hydrolysis products of (GlcNAc) (n), n52-6, by HPLC showed the splitting into (GlcNAc)(n), n=3-6. Activity toward N-acetylchitobiose was not detected. Oligomers with a higher number of units than the starting substrate were also detected, which indicate transglycosylation activity.     

Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases

Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.