微生物来源的黑色素生物合成抑制剂H7264 A和B的分离和鉴别

利用直接抑制产黑色素菌株产生黑色素的方法，从链霉素sp.H7264的次生代谢产物中分离到两个黑色素生物合成抑制剂H7264 A和B，经理化性质和光谱分析，它们与Enopeptin A和B同质。     

Cloning, characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin

The gene cluster encoding the deoxyoleandolide polyketide synthase (OlePKS) was isolated from the oleandomycin producing strain Streptomnyces antibioticus. Sequencing of the first two genes encoding OlePKS, together with the previously identified third gene revealed an overall genetic and protein architecture similar to that of the erythromycin gene cluster encoding the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. When the entire OlePKS (10,487 amino acids) was expressed in the heterologous host Streptomyces lividans, it produced 8,8a-deoxyoleandolide, an aglycone precursor of oleandomycin. The role of the P-450 monooxygenase, OleP, in oleandomycin biosynthesis was also examined in vivo by co-expression with DEBS in S. lividans. The production of 8,8a-dihydroxy-6-deoxyerythronolide B and other derivatives indicates that OleP is involved in the epoxidation pathway of oleandomycin biosynthesis. Since there are currently no genetic systems available for manipulation of the natural oleandomycin producing strain, the heterologous expression system reported here provides a useful tool for studying this important macrolide antibiotic.     

真菌Drechslera catenaria聚酮类代谢产物及聚酮合成酶基因的初步研究

目的 分离分析内脐蠕孢真菌(Drchslera catenaria)的次级聚酮类代谢产物,初步分析其非还原型Ⅰ型聚酮合成酶基因组成,为该菌株中大黄酚生物合成机制的阐明奠定理论基础.方法 应用Czapek Dox培养基发酵培养,酸化乙酸乙酯提取,硅胶柱层析及薄层制备等方法,对该菌株产生的聚酮类代谢产物进行分离纯化;利用紫外(UV),液相-质谱联用(LC-MS)和核磁共振谱(1H-NMR)鉴定化学结构;基于真菌聚酮合成酶保守序列设计引物,克隆与代谢产物合成相关的聚酮合成酶基因.结果与结论 从试验菌株菌丝体及发酵液中分离获得4个芳香聚酮类化合物,其结构分别为大黄酚(chrysophanol),长蠕孢素(helminthosporin),冰岛青霉素(islandicin),链蠕孢素(catenarin),其中后3个为首次从该真菌中得到.从基因组DNA中得到一段非还原性聚酮合成酶(PKS)基因序列(约5.3kb),与Pyrenophora tritici-repentis Pt-1C-BFP中黄色色素合成相关的基因片段有较高类似度,很有可能与内脐蠕孢真菌中大黄酚的生物合成有关;在距离该基因2.5kb处克隆到一段基因序列(0.79kb),与P.triticirepentis Pt-1C-BFP中β-酮酯酰基-ACP-还原酶(KR)基因完全相似,其很可能负责大黄素的6位羰基经还原形成大黄酚.     

微生物来源的黑色素生物合成抑制剂H7264的研究

通过建立的用链霉菌X59为鉴定菌的黑色素生物合成抑制剂筛选模型，从4000余个微生物代谢产物中找到一株活性化合物产生菌。该菌从四川省宜 地区土壤中分离，编号SIIA－H7264。该菌种经鉴定为链霉菌，其代谢产物H7264A和H7264B结构论证与化合物Enopeptin A,B同质，但Enopeptin尚未发现其有抑制黑色素生物合成活性。     

Advances in polyketide synthase structure and function

Recent progress in the understanding of polyketide synthase (PKS) continues to fuel the growth of combinatorial biosynthesis for natural product structural diversity. The structural analysis of many components of PKS, in particular for the modular type I 6-deoxyerythronilide B synthase (DEBS) involved in erythromycin biosynthesis, has provided structural imperatives for the observed biochemistry of DEBS and has enabled the generation of a working structural model of the entire DEBS system. New functions for PKS domains continue to be defined, such as the general control nonderepressible 5 (GCN5) N-acyltransferase strategy for polyketide chain initiation and the true identity of the elusive precursor for the methoxymalonylate extender unit. Novel molecular architectures have been continuously uncovered, including the 'AT-less' PKS and enediyne PKS, thereby expanding the known bacterial PKS paradigms beyond the prototypical type I, II and III PKSs. Finally, the genetic characterization of PKS in vivo and biochemical studies of PKS in vitro have also been greatly facilitated by the application of emerging technologies, such as RNA-mediated gene silencing, reconstitution of an entire polyketide biosynthetic pathway in a model heterologous host and Fourier-transform mass spectroscopy. The application of these technologies is discussed.     

聚酮合成酶底物专一性的研究进展

聚酮合成酶能够合成包括许多大环内酯类抗生索在内的聚酮类天然产物，它的结构和功能的模块性为组合生物合成的产生和发展奠定了基础。聚酮合成酶的酰基转移酶功能域可以特异性地决定所选择酰基辅酶A的种类．决定产物的结构。近年来，针对许多来源不同、结构各异的聚酮合成酶的底物专一性已经从氨基酸序列、结构和功能等方面进行了大量的研究．为更有效地应用聚酮合成酶开发新型抗生索奠定了基础。     

Folate analogues as inhibitors of thymidylate synthase

Recent demonstrations that deazafolate analogues may act as potent inhibitors of thymidylate synthase (TS) provided a firm rationale for the synthesis of N10-propargyl derivatives of 8-deazafolate and 8-deazaaminopterin (4). A complete assignment of the 1H NMR spectra of these compounds was made possible through application of 2D (COSY) techniques at 200 MHz. Data describing the inhibition of TS derived from human leukemia (K562) cells are presented. IC50 values of 2.25 and 1.26 microM were determined for 8-deaza-10-propargylfolate (3) and 8-deaza-10-propargylaminopterin, respectively. Comparison of the data for various folate analogues reveals a striking dependence of TS inhibitory potency upon the number of nitrogens in the folate pyrazine ring.     

Mevalonic acid analogs as inhibitors of cholesterol biosynthesis

A series of 20 mevalonic acid analogs was synthesized and tested for their ability to inhibit cholesterol biosynthesis from [2-14C]-mevalonate in rat liver homogenates. Removal of the 5-hydroxyl group from mevalonic acid produced an active inhibitor, 3-hydroxy-3-methylpentanoic acid. Removal of the 3-hydroxyl group, addition of an aromatic group in the 3-position, or insertion of a double bond reduced inhibitory activity. Compounds with an aromatic group or halide on the 5-position were active inhibitors. The most active inhibitor was 5-phenylpentanoic acid, with 50% inhibition at 0.064 mM.     

Nitrosoureido nucleosides as potential inhibitors of nucleotide biosynthesis

Several nitrosoureido nucleosides (3a, 3b, 5a, 7a, 7c, and 10a) designed as inhibitors of enzymes that metabolize pyrimidine nucleotides have been prepared and their chemical and biological properties studied. The methylnitrosoureas 3a and 3b were not significantly cytotoxic to H.Ep.-2 and L1210 cells in vitro but showed moderate activity in the P388 mouse leukemia screen (79% ILS for 3a and 56% ILS for 3b). The (chloroethyl)nitrosoureas 7a and 7c inhibited proliferation of L1210 cells, were cytotoxic to H.Ep.-2 cells, and demonstrated good activity against P388 in vivo (135% ILS with one 30-day survivor for 7a and 191% ILS with two 30-day survivors for 7c). Overnight exposure of L1210 cells to 7a and 7c resulted in cell enlargement accompanied by cell lysis. Macromolecular synthesis in enlarged cells, particularly RNA and protein synthesis, was markedly increased relative to that in untreated control cells. The half-lives of each of the nitrosoureas in pH 7 buffer was determined and compared with biological activity.     

Generation of new epothilones by genetic engineering of a polyketide synthase in Myxococcus xanthus

Epothilones, potent cytotoxic agents and potential anticancer drugs, are complex polyketides produced by a modular polyketide synthase (PKS). The epothilone PKS genes were introduced and expressed in Myxococcus xanthus and engineered to generate novel unnatural natural products which can be used as new scaffolds for chemical modification. Inactivation of the KR domain in module 6 of the epo PKS resulted in accumulation of 9-oxoepothilone D and its isomer 8-epi-9-oxoepothilone D as the major products. Modification of the KR domain in module 4 resulted in the production of the expected compound 12,13-dihydro-13-oxoepothilone C in trace amounts, and the unexpected compound 11,12-dehydro-12,13-dihydro-13-oxoepothilone D as the major product. The other expected compound, 12,13-dihydro-13-oxoepothilone D, was not detected. The unexpected 13-oxo derivative produced indicates that the ER domain of module 5 has substrate-specificity requirements and suggests a second enzymatic role for the domain.     

Phylogenomic analysis of polyketide synthase genes in actinomycetes: structural analysis of KS domains and modules of polyketide synthases

Polyketides are complex and diverse secondary metabolites, synthesised by large multifunctional enzymes, Polyketide Synthases (PKS). The phylogenomic analysis of β-ketosynthase (KS) domains and PKSs within actinomycetes suggests the contribution of point mutations, gene duplications, horizontal gene transfer and homologous recombination in the evolution of PKSs. PKS genealogy suggested the ancestral module structure with KS-AT-ACP domain composition. KS domains showed similar core and highly variable loop regions at the dimer interface, which seems to affect the selectivity of the primer unit. In PKS modules, the linker regions comprise a significant fraction of the module. The reducing domains (ketoreductase and dehydrogenase) protrude out from the central axis of the module and also responsible for extreme variability in the final products. Thus, phylogenomic and structural analysis of PKSs can assist in the artificial reprogramming of PKSs.     

Epoxy-acetogenins and other polyketide epoxy derivatives as inhibitors of the mitochondrial respiratory chain complex I

Annonaceous acetogenins (ACG), an extensive group of cytotoxic natural products, are antitumor agents whose main mode of action is inhibition of the mammalian mitochondrial complex I. Herein we describe the importance of the different chemical groups along the alkyl chain for optimal inhibitory potency, discussing the structurally relevant factors present in these compounds. For this purpose, a series of epoxide derivatives from alpha-linolenic acid were prepared and their activity compared with that of epoxy-acetogenins and tetrahydrofuranic (THF) acetogenins isolated from Rollinia membranacea.     

Inhibitory effects of cinnamic acid on melanin biosynthesis in skin

Cinnamic acid is a wildly distributed phenylpropanoid component naturally occurring in plants, and is mainly found in Cinnamomum cassia BLUME and Panax ginseng. Cinnamic acid was recently reported to exert a tyrosinase inhibitory effect. However, research on melanocytes and animal bodies was not reported until now. In this study, we examined the effects of cinnamic acid on melanin biosynthesis within the melanocytes and brown guinea pigs. Melan-a cells were used to examine the effects of cinnamic acid in the melanocytes. Treatment with 100 ppm of cinnamic acid resulted in a significant reduction of melanin production in the melan-a cells at 29.0%. This compound also exhibited a potent inhibitory effect on tyrosinase activity and reduced tyrosinase expression in the melan-a cells. Moreover, cinnamic acid exhibited depigmenting activity on the UV-B-induced hyperpigmentation of brown guinea pig skin. Our results suggest that cinnamic acid might act as a skin whitening agent via inhibition of tyrosinase activity and expression within melanocytes.     

Some pyrazoles as inhibitors of purine biosynthesis de novo.

The inhibitory effect of some pyrazole derivatives on purine biosynthesis was studied in a pigeon liver cell-free system. It was demonstrated that 3-amino-4-carbethoxypyrazole, 3-amino-4-carboxypyrazole and 3-(3′3′-bis-β-chloroethyltriazenyl-1′)-4-carbethoxypyrazole were inhibitors, while N-β-hydroxyethyl-3-amino-4-carbethoxypyrazole was almost inactive. A possible mechanism of action is discussed.