Two Diterpene Synthases from Chryseobacterium: Chryseodiene Synthase and Wanjudiene Synthase

Two bacterial diterpene synthases (DTSs) from Chryseobacterium were characterised. The first enzyme yielded the new compound chryseodiene that closely resembles the known fusicoccane diterpenes from fungi, but its experimentally and computationally studied cyclisation mechanism is fundamentally different to the mechanism of fusicoccadiene synthase. The second enzyme produced wanjudiene, a diterpene hydrocarbon with a new skeleton, besides traces of the enantiomer of bonnadiene that was recently discovered from Allokutzneria albata.     

Diterpene Biosynthesis in Actinomycetes: Studies on Cattleyene Synthase and Phomopsene Synthase

Three diterpene synthases from actinomycetes have been studied. The first enzyme from Streptomyces cattleya produced the novel compound cattleyene. The other two enzymes from Nocardia testacea and Nocardia rhamnosiphila were identified as phomopsene synthases. The cyclisation mechanism of cattleyene synthase and the EIMS fragmentation mechanism of its product were extensively studied by incubation experiments with isotopically labelled precursors. Oxidative transformations expanded the chemical space of these unique diterpenes.     

Investigation of an Allosteric Deoxyhypusine Synthase Inhibitor in P. falciparum

The treatment of a variety of protozoal infections, in particular those causing disabling human diseases, is still hampered by a lack of drugs or increasing resistance to registered drugs. However, in recent years, remarkable progress has been achieved to combat neglected tropical diseases by sequencing the parasites’ genomes or the validation of new targets in the parasites by novel genetic manipulation techniques, leading to loss of function. The novel amino acid hypusine is a posttranslational modification (PTM) that occurs in eukaryotic initiation factor 5A (EIF5A) at a specific lysine residue. This modification occurs by two steps catalyzed by deoxyhypusine synthase (dhs) and deoxyhypusine hydroxylase (DOHH) enzymes. dhs from Plasmodium has been validated as a druggable target by small molecules and reverse genetics. Recently, the synthesis of a series of human dhs inhibitors led to 6-bromo-N-(1H-indol-4yl)-1-benzothiophene-2-carboxamide, a potent allosteric inhibitor with an IC₅₀ value of 0.062 µM. We investigated this allosteric dhs inhibitor in Plasmodium. In vitro P. falciparum growth assays showed weak inhibition activity, with IC₅₀ values of 46.1 µM for the Dd2 strain and 51.5 µM for the 3D7 strain, respectively. The antimalarial activity could not be attributed to the targeting of the Pfdhs gene, as shown by chemogenomic profiling with transgenically modified P. falciparum lines. Moreover, in dose-dependent enzymatic assays with purified recombinant P. falciparum dhs protein, only 45% inhibition was observed at an inhibitor dose of 0.4 µM. These data are in agreement with a homology-modeled Pfdhs, suggesting significant structural differences in the allosteric site between the human and parasite enzymes. Virtual screening of the allosteric database identified candidate ligand binding to novel binding pockets identified in P. falciparum dhs, which might foster the development of parasite-specific inhibitors.     

Mechanistic Investigations of Two Bacterial Diterpene Cyclases: Spiroviolene Synthase and Tsukubadiene Synthase

The mechanisms of two diterpene cyclases from streptomycetes—one with an unknown product that was identified as the spirocyclic hydrocarbon spiroviolene and one with the known product tsukubadiene—were investigated in detail by isotope labeling experiments. Although the structures of the products were very different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions, before they diverge towards the individual products, which is reflected in the close phylogenetic relationship of the enzymes.     

Phomoxanthone A Targets ATP Synthase

Phomoxanthone A is a naturally occurring molecule and a powerful anti-cancer agent, although its mechanism of action is unknown. To facilitate the determination of its biological target(s), we used affinity-based labelling using a phomoxanthone A probe. Labelled proteins were pulled down, subjected to chemoproteomics analysis using LC-MS/MS and ATP synthase was identified as a likely target. Mitochondrial ATP synthase was validated in cultured cells lysates and in live intact cells. Our studies show sixty percent inhibition of ATP synthase by 260 μM phomoxanthone A.     

几丁质合成酶抑制剂

几丁质合成酶是生物合成几丁质的关键物质。几丁质是昆虫表皮和真菌细胞壁的特征成分,由于存在的特殊性而成为农药、医药研发的独特靶标。几丁质合成酶抑制剂由于具有安全、高效等特点,成为农用杀虫、杀螨、杀菌剂以及医药抗真菌药物的研发热点。本文综述了天然及人工合成的几丁质合成酶抑制剂的研究进展,并对其发展趋势进行了展望。     

拟南芥乙酰羟基酸合成酶与磺酰脲的相互作用以及CoMFA研究

在分子水平上较为详尽地研究了85个磺酰脲类化合物与植物源野生型拟南芥AHAS酶的离体相互作用, 测定了这些化合物对AHAS酶的抑制常数Kiapp. 采用比较分子力场方法(CoMFA)对这些化合物与AHAS酶的相互作用进行了三维构效关系研究, 用此模型预测了检验组10个化合物的pKiapp值, 模型的预测结果与测试结果一致.     

Conversion of Anthranilate Synthase into Isochorismate Synthase: Implications for the Evolution of Chorismate‐Utilizing Enzymes

Chorismate‐utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as well as free‐living and infectious microorganisms. Among these enzymes are the homologous primary metabolic anthranilate synthase (AS) and secondary metabolic isochorismate synthase (ICS). Both catalyze mechanistically related reactions by using ammonia and water as nucleophiles, respectively. We report that the nucleophile specificity of AS can be extended from ammonia to water by just two amino acid exchanges in a channel leading to the active site. The observed ICS/AS bifunctionality demonstrates that a secondary metabolic enzyme can readily evolve from a primary metabolic enzyme without requiring an initial gene duplication event. In a general sense, these findings add to our understanding how nature has used the structurally predetermined features of enzyme superfamilies to evolve new reactions.     

Towards Photochromic Azobenzene-Based Inhibitors for Tryptophan Synthase

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene-based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto-unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light.     

GSK-3抑制剂研究进展

糖原合成酶激酶-3 (Glycogen synthase kinase-3, GSK-3)是一个多功能的丝氨酸/苏氨酸蛋白激酶,不仅参与肝糖代谢过程,而且还参与Wnt和Hedgehog信号通路,通过磷酸化多种底物蛋白来调节细胞的生理过程。GSK-3抑制剂作为目前倍受关注的小分子抑制剂,对治疗神经退化性疾病,癌症,II型糖尿病具有潜在的疗效。本文针对已开发出的GSK-3抑制剂,对其结构,与蛋白的作用模式以及构效关系进行阐述,为进一步设计合理的药物先导化合物和特异性小分子化学探针提供有益的启示。     

An O-Acetylserine (thiol) Lyase from Leucaena leucocephala Is a Cysteine Synthase But Not a Mimosine Synthase

In plants, the final step of cysteine formation is catalyzed by O-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the K _m was calculated to be 1,850?±?414 μM sulfide and the V _max was 200.6?±?19.92 μM cysteine min^?1. The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases.     

A Model for the Cobalamin-Dependent Methionine Synthase

The acid-catalyzed transfer of a Me group from N,N-dimethylaniline ( 6 ) to vitamin-B₁₂-derived Co^I complexes 2a , b was realized (Scheme 3). Hexane-1-thiol ( 8 ) was methylated by the methylcobalt complexes 4a , b in the presence of pyridine. Conditions for the complete cycle, i.e., Me transfer from 6 to 8 with Co^I complexes acting as a nucleophile and a nucleofuge have been established. The importance of Zn²⁺ as activating agent and of the basicity of tertiary amines for the Me transfer has been investigated.     

An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping

Di‐ and sesterterpene synthases produce C₂₀ and C₂₅ isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene ( 1 ), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.     

Occurrence of Polypeptides in Other Organisms Cross-reacting with Antibodies Against A. Xylinum Cellulose Synthase

Antibodies (anti-83 and anti-93) against the cellulose synthase complex from A. xylinum ATCC 53582 have been employed to study the evolutionary conservation of this enzyme complex among various A. xylinum strains, selected species of other cellulose- producing bacteria, algae, and vascular plants. Of the 18 A. xylinum strains examined, the 83 Kd polypeptide clearly is detected only in 4 strains while the 93 Kd polypeptide is observed in all 18 strains. Assuming that the revised acsAB gene (Saxena et al., 1994) encoding the 83 and 93 Kd polypeptides as a single polypeptide holds true for all A. xylinum strains, it is proposed that the cellulose synthase is conserved in A. xylinum but with varying degrees of homology. An unknown regulatory mechanism causing the degradation of the 83 Kd polypeptide in response to agitated culturing conditions has been suggested to explain the absence of the 83 Kd polypeptide in most of the Acetobacter strains examined. A. xylinum cellulose synthase appears to be conserved in phylogenetically related Rhizobium and Agrobacterium species, but not in algae and plants.