Biochemical Characterization of a Novel Indole Prenyltransferase from Streptomyces sp. SN-593

Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (−)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min⁻¹ μM⁻¹) for l-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-l-Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, ΔiptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-l-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.Natural products have been an important resource for drug discovery and development. Actinomycetes have been a rich source of natural products, and a wide variety of these chemicals have been used as medicinal drugs (7, 40) and as bioprobes (56) for the elucidation of biological functions. Recently, the screening of bioactive compounds from microorganisms has often resulted in the identification of previously isolated compounds. The decreasing hit rate for new chemicals has reduced the advantage of natural product screening. However, genome sequencing of Streptomyces species highlighted numerous potential areas with metabolic diversity (4, 25, 42). The number of cryptic gene clusters was much larger than that of secondary metabolites identified from each strain. In addition, the cryptic gene clusters contained genes encoding plenty of unique modification enzymes that had the potential to expand the chemical diversity in drug seeds.To uncover cryptic gene clusters that might code for biosynthesis of secondary metabolites, genome sequence-guided metabolite identification has been performed in combination with heterologous expression, gene knockout, and complementation analyses and silent gene activation studies. Many microbial metabolites have been discovered through genome mining approaches (5, 8, 26, 29, 34, 41, 50). On the other hand, predictions of protein function are not always successful from BLAST searches, because the substrates or products of unknown enzyme reactions cannot be predicted correctly. Only the type of protein function can be annotated by a homology search. The major difficulty for the identification of cryptic gene clusters is a lack of chemical information. Most gene clusters remain dormant or less active if there are no specific chemicals or physiological signals. Therefore, the discovery of secondary metabolites that are normally expressed at very low levels opens up a strategy for addressing the functions of cryptic gene clusters or unique genes. We performed a metabolite profiling and genome draft sequence analysis of a reveromycin A-producing strain, Streptomyces sp. SN-593 (43). Based on systematic isolation of secondary metabolites, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. There are many isolation reports on 6-DMAI derivatives from Streptomyces sp. (39, 46, 48), fungi (17, 24, 49), and plants (2, 3). However, the gene responsible for dimethylallyl transfer to the C-6 indole ring has not been identified for all living organisms. Because the unique modification enzyme retains a high potential to expand the diversity of natural products, we started a homology search and cloning of the target gene. Here we report the heterologous expression and biochemical characterization of a novel indole prenyltransferase (IptA) catalyzing the transfer of a dimethylallyl group to the C-6 indole ring.     

鹿药、四叶菜、山菠菜氨基酸和无机元素测定

鹿药、四叶菜和山菠菜都是长白山区常见的山野菜,经测定表明这3种野菜中都含有16种氨基酸,并且人体必需氨基酸含量都在总量的35%以上,对21种无机元素的测定结果显示鹿药、四叶菜和山菠菜分别含有17种、16种和15种无机元素.     

Recognition of oxidized thymine base on the single-stranded DNA by replication protein A

Replication protein A (RAP) is a eukaryotic single-stranded DNA binding protein involved in DNA replication, repair, and recombination. Recent studies indicate that RPA preferentially binds the damaged sites rather than the undamaged sites. Therefore, RPA is thought to be a member ofrepair factories or a sensor of lesion on DNA. To obtain further information of behavior of RPA against the oxidized lesion, we studied the binding affinity of RPA for the single-stranded DNA containing 5-formyluracil, a major lesion of thymine base yielded by the oxidation, using several synthetic oligonucleotides. The affinity of RPA for oligonucleotides was determined by gel shift assay. Results suggest that the surrounding sequence of 5-formyluracil may affect the affinity for RPA, and that the 5-formyluracil on the purine stretch but not the pyrimidine stretch increases the affinity for RPA. Results of affinity labeling experiment of RPA with the oligonucleotides containing 5-formyluracil indicate that RPA1 subunit may directly recognize and bind to the 5-formyluracil on the single-stranded DNA.     

Determination of minor flavonol-glycosides and sugar-free flavonols in the tepals of several species of cereoideae (Cactaceae)

Eight kinds of flavonoids were isolated by crystallization or paper-chromatography from the tepals of several cactaceous plants, i.e.,Astrophytum ornatum Web.,Notocactus apricus A. Berg.,Echinopsis huotii Lab.,Aylostera pseudodeminuta Backbg. andNeochilenia napina Backbg. The structures of six flavonols were determined by UV spectral means and co-PC comparison as quercetin and its 7-O-galactoside (coptiside II), kaempferol and its 3-O-rhamnosylglucoside (nicotiflorin), and isorhamnetin and its 3-O-rhamnosylglucoside (narcissin). The remaining two flavonoids were partially characterized as kaempferol 3, 7-O-diglycoside and 5-hydroxy-3,4′-oxygenated flavonol derivative.     

Protective role of HSP27 against UVC-induced cell death in human cells

It is an intriguing problem whether heat shock proteins (HSPs) play a protective role in UVC-induced cell death in human cells, and the problem has not been solved. To search for the HSPs involved in UVC resistance, gene expression profiles using cDNA array were compared between UVC-sensitive human RSa cells and their UVC-resistant variant AP(r)-1 cells. The expression levels of heat shock protein 27 (HSP27) were lower in RSa cells than in AP(r)-1 cells. RSa cells transfected with sense HSP27 cDNA showed slightly lower sensitivity to UVC-induced cell death than the control cells transfected with a vector alone and much lower sensitivity than RSa cells transfected with the antisense HSP27 cDNA. Furthermore, the removal capacities of the two major types of UVC-damaged DNA (thymine dimers and (6-4)photoproducts) in the cells with the up-regulation of HSP27 were moderately elevated compared with those in the control cells, while those in the cells with down-regulation were remarkably suppressed. These results suggest that HSP27 is involved in the UVC-resistance of human cells, at least those tested, possibly via functioning in nucleotide excision repair.     

Binding between azurocidin and calreticulin: its involvement in the activation of peripheral monocytes

We found that azurocidin, a secretory protein in neutrophils, binds to calreticulin, a multifunctional chaperone of the endoplasmic reticulum. Azurocidin is known to induce cytokine production in monocytes, but the mechanism of monocyte activation by azurocidin remains unknown. On the other hand, an antibacterial peptide, KLKLLLLLKLK-NH(2) (L5), is known to bind to cell surface calreticulin of human neutrophils, resulting in their activation to produce O(2)(-). Therefore, we examined whether cell surface calreticulin is involved in the activation of human monocytes by azurocidin to produce IL-6. We found that carlreticulin is in fact located on the surface of monocytes and that the IL-6 production stimulated by an azurucidin is inhibited by anti-calreticulin antibody. Possibly, binding between cell surface calreticulin and azurocidin is prerequisite for the activation of monocytes by azurocidin to produce IL-6.     

Genetic polymorphims in promoter region of osteopontin gene may be a marker reflecting hepatitis activity in chronic hepatitis C patients

BACKGROUND AND AIMS: Osteopontin, an extracellular matrix protein with RGD motif, is shown to be a cytokine essential for Th1 immune response initiation. Genetic polymorphisms in the osteopontin gene (OPN) determine the magnitude of immunity against rickettsial infection in mice. Similar polymorphisms, if present also in human beings, might affect hepatitis activity in those infected with HCV. METHODS: Blood was collected from 176 patients with chronic hepatitis C. SNPs in the promoter region of OPN were analyzed in 20 patients by direct sequencing of DNA fragments amplified by PCR and in 156 patients by Invader assay. Ninety-five patients compatible to evaluation criteria were classified into three groups depending on maximal serum ALT levels during the observation periods at least for 2 years as follows; lower than 30IU/L (low-activity group), between 30 and 80IU/L with no hepatoprotective treatment (medium-activity group), and higher than 80IU/L irrespective of hepatoprotective treatment (high-activity group). RESULTS: There were 16, 19, and 60 patients in the low-, medium-, and high-activity groups, respectively. Four SNPs (nt -155, -443, -616, and -1748) were detected in the promoter region of OPN. Among them, the SNP at nt -443 (C or T) was a novel one and showed an association with hepatitis activity in our patients: T/T homozygosity was found in 2 (13%), 8 (42%), and 25 (44%), and C/T heterozygosity in 12 (75%), 8 (42%), and 23 (40%), in the low-, medium-, and high-activity groups, respectively. The other 3 SNPs already known showed linkage disequilibrium with D(') and r(2) greater than 0.937 to each other without correlation to disease activity. CONCLUSIONS. OPN promoter region SNP at nt -433 may be a useful marker reflecting hepatitis activity in chronic hepatitis C patients.