In vitro biosynthesis of [Thr2,Leu5,D-Hiv8,Leu10]-cyclosporin, a cyclosporin-related peptolide, with immunosuppressive activity by a multienzyme polypeptide

A new cyclic peptolide (SDZ 214-103), which is produced by the fungus Cylindrotrichumoligospermum (Corda) BONORDEN (Dreyfuss, M. M., Schreier, M. H., Tscherter, H., and Wenger, R. (June 15, 1988) European Patent Application 0 296 123 A2) and is closely related to cyclosporin A (CyA), has as the main structural difference D-2-hydroxyisovaleric acid in ester linkage at position 8 instead of D-alanine in the cyclosporins. This peptolide exerts similar biological activities to CyA. We were able to prepare an enzyme fraction of crude extracts of the mycelium, which is capable of synthesizing the peptolide with consumption of the constitutive amino acids, D-2-hydroxyisovaleric acid, ATP, and S-adenosyl-L-methionine. The in vitro product co-chromatographs with authentic peptolide on thin layer chromatography and high performance liquid chromatography and shows similar immunosuppressive activity in vitro. The enzyme does not synthesize CyA, whereas cyclosporin synthetase does not synthesize the peptolide. Peptolide synthetase has a high molecular weight (in the same range as cyclosporin synthetase) and also does not appear to be glycosylated. The enzyme cross-reacts with antibodies directed specifically against cyclosporin synthetase.     

An improved purification procedure for cyclosporin synthetase

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation-->gel filtration-->hydrophobic interaction chromatography-->anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24-29 degrees C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.     

Enzymatic synthesis of cyclosporin A

An enzyme fraction, isolated from crude extracts of the fungus Tolypocladium inflatum, strain 7939/F, is able to synthesize the undecapeptide cyclosporin A. The formation of cyclosporin A was monitored by incorporation of the radiolabeled constituent amino acids of cyclosporin A or by using S-adenosyl-L-[14C-methyl] methionine. The structure of cyclosporin A, synthesized enzymatically in vitro, was confirmed by chromatographic comparison with the authentic compound and by amino acid analyses. Replacement of L-2-aminobutyric acid in the reaction mixture by L-alanine, L-threonine, L-valine, or L-norvaline yields the naturally occurring cyclosporins B, C, D, and G. Also, D-alanine could be replaced by D-serine to yield [D-Ser8]cyclosporin A.     

Cyclosporin synthetase. The most complex peptide synthesizing multienzyme polypeptide so far described

Cyclosporin A and its homologues are synthesized by a single multifunctional enzyme from their precursor amino acids. Cyclosporin synthetase is a polypeptide chain with a molecular mass of approximately 800 kDa. In 3% polyacrylamide-sodium dodecyl sulfate gels it shows a single band of approximately 650 kDa, which appears to not be glycosylated. The enzyme could be purified to near-homogeneity in five steps. A 72-fold purification was obtained. All constitutive amino acids of cyclosporins are activated as thioesters via aminoadenylation by the same enzyme. Then N-methylation of the thioester-bound amino acids which are present in methylated form in the cyclosporin molecule takes place, whereby S-adenosyl-L-methionine serves as the methyl group donor. Methyltransferase activity is an integral entity of the enzyme; this could be shown by a photoaffinity labeling method. 4'-Phosphopantetheine is a prosthetic group of cyclosporin synthetase similar to other peptide and depsipeptide synthetases. Cyclosporin synthetase shows cross-reactions with monoclonal antibodies directed against enniatin synthetase.     

Targeted gene disruption and functional complementation of cytochrome P450 Hydroyxlase involved in cyclosporin A hydroxylation in Sebekia benihana

A cyclic undecapeptide-family natural product, cyclosporin A (CyA), which is one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex in a filamentous fungal strain named Tolypocladium niveum. Previously, structural modifications of cyclosporins such as a regionspecific hydroxylation at the 4th N-methyl leucine in a rare actinomycetes called Sebekia benihana were reported to lead to dramatic changes in their bioactive spectra. However, the reason behind this change could not be determined since a system to genetically manipulate S. benihana has not yet been developed. To address this limitation, in this study, we utilized the most commonly practiced gene manipulation techniques including conjugation-based foreign gene transfer-and-expression as well as targeted gene disruption to genetically manipulate S. benihana. Using these optimized genetic manipulation systems, a putative cytochrome P450 hydroxylase (CYP) gene named CYP506, which is involved in CyA hydroxylation in S. benihana, was specifically disrupted and genetically complemented. The S. benihana deltaCYP506 exhibited a significantly reduced CyA hydroxylation yield as well as considerable yield restoration by functional complementation of the S. benihana CYP506 gene, suggesting that the genetically manipulated S. benihana CYP mutant strains may serve as a more efficient bioconversion host for various valuable metabolites including CyA.     

以苯丙氨酰-tRNA合成酶为靶点的新型抗结核药物高通量筛选模型的建立和应用

【目的】建立结核分枝杆菌PheRS抑制剂高通量模型,并运用此模型筛选化合物和发酵液样品。【方法】克隆和表达结核分枝杆菌PheRS蛋白并优化其酶活测定方法,在此基础上建立结核分枝杆菌PheRS抑制剂高通量筛选模型,并通过耻垢分枝杆菌作为检定菌对筛选到的样品进行抗菌活性测定及细胞毒性评价。【结果】运用此模型筛选了化合物样品11 600个,发酵液样品5 200个,筛选得到阳性化合物9个,阳性发酵液37个。而后通过耻垢分枝杆菌作为检定菌的抗菌活性测定及细胞毒性评价后,得到了6个发酵液阳性样品。【结论】建立的PheRS抑制剂模型可成功用于化合物和微生物发酵液的高效筛选,得到的6个发酵液阳性样品在酶水平和抗分枝杆菌方面均具有良好活性且毒性较低,值得进一步研究。     

An unusual side chain C-C cleavage at the MeBmt amino acid in cyclosporin A

Summary The mixture of products obtained by alkaline treatment of cyclosporin A was analyzed by HPLC-continuous-flow-FAB/MS. The changes involve the atypical amino acid (4R)-4-((E)-2-butenyl)-4,N-dimethyl-L-threonine (MeBmt) without affecting the cyclic structure. The main degradation pathway is dehydration producing all four possible anhydro-MeBmt containing cyclosporins. A new cyclosporin, [Sar¹]CS, resulting from the side chain cleavage of MeBmt has been isolated and characterized.     

Spore inoculum optimization to maximize cyclosporin A production in Tolypocladium niveum

The cyclic undecapeptide, cyclosporin A (CyA), is one of the most commonly prescribed immunosuppressive drugs. It is generated nonribosomally from a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. In order to maximize the production of CyA by wild-type T. niveum (ATCC 34921), each of three culture stages (sporulation culture, growth culture, and production culture) were sequentially optimized. Among the three potential sporulation media, the SSMA medium generated the highest numbers of T. niveum spores. The SSM and SM media were then selected as the optimal growth and production culture media, respectively. The addition of valine and fructose to the SM production medium was also determined to be crucial for CyA biosynthesis. In this optimized three-stage culture system, 3% of the spore inoculum generated the highest level of CyA productivity in a 15-day T. niveum production culture, thereby implying that the determination of an appropriate size of T. niveum spore inoculum plays a critical role in the maximization of CyA production.     

Occurrence of cyclosporins and cyclosporin-like peptolides in fungi

Apart from 17 previously listed fungal taxa producing cyclosporin A and its natural congeners B to Z, several additional and taxonomically diverse strains producing the single novel component [Thr², Leu⁵, Leu¹⁰]cyclosporin or cyclosporin-like peptolides (eg SDZ 214-103=[Thr², Leu⁵, D-Hiv⁸, Leu¹⁰]cyclosporin) have recently been described. We report here the isolation of two further and novel cyclosporins, [Thr², Leu⁵, Ala¹⁰]cyclosporin (2) and [Thr², Ile⁵] cyclosporin (3), from strains classified asAcremonium luzulae (Fuckel) W Gams andLeptostroma anamorph ofHypoderma eucalyptii Cooke & Harkn, respectively. In both new strains the usual pattern of cyclosporins A to Z is not found. The structure elucidations of2 and3 are based on NMR spectroscopy, and biological data (immunosuppressive activity, cyclophilin-binding affinity and antifungal effects) are presented.     

Isolation and covalent structure of the aspirin-modified, active-site region of prostaglandin synthetase

Aspirin (acetylsalicylic acid) inhibits prostaglandin synthesis by acetylating a single internal serine residue of the initial enzyme in the biosynthetic pathway, prostaglandin synthetase. In this study, the region of the enzyme that is modified by aspirin has been isolated, and its amino acid sequence has been determined. Sheep vesicular gland [acetyl-3H]prostaglandin synthetase was purified following treatment with [acetyl-3H]aspirin and digest with pepsin. An acetyl-3H-labeled peptic peptide of approximately 25 residues was isolated by high-pressure liquid chromatography, and its amino acid sequence was determined to be Ile-Glu-Met-Gly-Ala-Pro-Phe-Ser-Leu-Lys-Gly-Leu-Gly-Asn-Pro-Ile-Glu-Ser-Pro-Glu-Tyr. The acetylated serine residue was located at position 8 in this sequence. The current study marks this polypeptide sequence as a region related to an active site of the enzyme.     

Role of acetoacetyl-CoA synthetase in acetoacetate utilization by tumor cells

Tumors of peripheral tissues contain low levels of succinyl CoA-acetoacetate CoA transferase activity which is not induced in vitro by prolonged cultivation in 2.5 mM DL-3-hydroxybutyrate. Although this enzyme is considered to be the main agent controlling the extent to which ketone bodies serve as metabolic substrates such tumors metabolize D(-)-3-hydroxy[3(14)C]butyrate to 14CO2. Also addition of 3-hydroxybutyrate and/or acetoacetate reduces the amount of 14CO2 produced from D-[U-14C] glucose suggesting a common metabolic intermediate. These observations can be accounted for by the presence of acetoacetyl-CoA synthetase, an enzyme which is able to synthesize acetoacetyl-CoA directly from acetoacetate, ATP and coenzyme A. This is the first demonstration of this enzyme in tumor tissue. The rate of metabolism of acetoacetate by this enzyme is sufficient to account for the production of CO2 from 3-hydroxybutyrate.     

Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays.

A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.     

Characterization of theanine-forming enzyme from Methylovorus mays no. 9 in respect to utilization of theanine production

For development of theanine production from glutamic acid and ethylamine by coupling yeast sugar fermentation as an ATP-regenerating system, several strains were selected from among about 200 methylamine- and/or methanol-assimilating bacteria depending on the theanine-forming activity of their permeated cells. The amount of theanine formed by the cells of the selected strains was much larger than that by the cells of Escherichia coli AD494 (DE3) expressing Pseudomonas taetrolens Y-30 glutamine synthetase (GS), which has been found to be a usable enzyme for theanine production. A GS-like enzyme responsible for the theanine-forming reaction was obtained from an obligate methylotroph isolate, Methylovorus mays No. 9. The enzyme was induced by methylamine in the culture medium. A molecular mass of 410-470 kDa was obtained by gel filtration of the enzyme, and 51 kDa by SDS-PAGE analysis. The enzyme showed high activity toward methylamine rather than ammonia, which indicates that it is similar to known gamma-glutamylmethylamide synthetase. The isolated enzyme also had high reactivity to ethylamine in a neutral pH range, and formed theanine from glutamic acid and ethylamine in a reaction mixture containing a yeast sugar fermentation system for ATP-regeneration.     

Differential effects of cyclosporins A and G on functional activation of a T-helper-lymphocyte line mediating experimental autoimmune uveoretinitis

The effect and relative efficiency of cyclosporin A (CsA) and cyclosporin G (CsG) on suppressing the activation of primed autoimmune rat T-helper lymphocytes were assayed. The autoimmune T-helper cells (ThS) are a long-term line specific to the retinal soluble antigen (SAg) and can adoptively transfer experimental autoimmune uveoretinitis (EAU), after in vitro reactivation with antigen or mitogen, to naive syngeneic hosts. Antigen-driven production of interleukin-2 (IL-2) and antigen-driven proliferation were inhibited in a dose-dependent manner and to a similar extent at each of the respective cyclosporin concentrations. CsA was 8-10 times more potent than CsG, with ID50-CsA occurring at 0.5 to 2 ng/ml, and ID50-CsG at 5 to 20 ng/ml, depending on the experiment and the cyclosporin batch. Addition of exogenous lymphokines in the form of rat spleen concanavalin A (Con A)-conditioned medium (SCM) or recombinant IL-2 (but not recombinant IL-1) was able to reverse only about half of the inhibition, as measured along the linear part of the dose-response curve. Inhibition of IL-2 production was lost if a maximally inhibitory dose of cyclosporin was added to the cultures later than 8 hr after antigen stimulation, while proliferation was still suppressed to 50% by cyclosporin added as late as 12 hr and could not be restored by addition of SCM. Both cyclosporins at concentrations that blocked proliferation and IL-2 production significantly suppressed the generation of high-affinity and low-affinity IL-2 receptors by ThS in response to antigen (as assayed by direct binding of 125I-IL-2). These results suggest that CsA and CsG inhibit antigen-induced expansion of ThS by interfering with more than one activation step. In contrast, the in vitro activation of the uveitogenic potential of ThS cells, incubated with antigen in the presence of CsA or CsG and adoptively transferred into untreated recipients, was not affected by the cyclosporins. Thus, triggering of the pathogenic potential of primed autoimmune T-helper lymphocytes can take place in the presence of cyclosporin and in the absence of cellular proliferation.     

The cyclosporins inhibit lymphocyte activation at more than one site

Cyclosporin A (CsA), a potent immunosuppressive agent, acts primarily by inhibiting T cell function. Although several potential sites of action have been identified, the mechanisms whereby CsA mediates its immunosuppressive properties have not been fully delineated. We have examined the effects of the immunosuppressive cyclosporins, CsA, dihydrocyclosporin D, and cyclosporin G, and a nonimmunosuppressive analog, cyclosporin H, on early events associated with activation of human T cells. Interleukin 2 (IL 2) receptor expression, as measured by immunofluorescence, was unaffected by CsA. Despite this, in the continuous presence of CsA, exogenous IL 2 did not bypass CsA inhibition of phytohemagglutinin (PHA)-induced proliferation. Thus, one site of activity of CsA is on IL 2-induced proliferation of IL 2 receptor-expressing cells. In addition, several potential mechanisms for inhibiting IL 2 secretion were identified. Changes in cytosolic free Ca2+ ([Ca2+]i), an obligatory event for PHA-induced IL 2 secretion, were inhibited by a 30-min preincubation with the immunosuppressive cyclosporins but not the inactive analog. In this action, the drug effects cannot be distinguished from that of Ca2+ channel blockers. The active compounds also resulted in membrane depolarization, an effect which may, in part, explain the reduction in PHA-induced changes in [Ca2+]i. These results identify multiple sites of action of the immunosuppressive cyclosporins, the combination of which likely accounts for their selective inhibition of T cell function in vitro and in vivo.     

Glucose metabolism by Lactobacillus divergens

Earlier studies on the fermentation of D-[1-14C]- and D-[3,4-14C]glucose by Lactobacillus divergens showed that lactate was the major fermentation product and that it was probably produced by glycolysis. It was therefore recommend that L. divergens be reclassified as a homofermentative organism. In the present investigation, products of D-[1-14C]-,D-[2-14C]- and D-[3,4-14C]glucose fermented by L. divergens were isolated, and their specific radioactivities and the distribution patterns of radioactivity in their C-atoms were determined. The positional labelling patterns of the fermentation products, their specific radioactivities and their concentrations confirmed that glucose is degraded via the glycolytic pathway. Some secondary decarboxylation/dissimilation of pyruvate to acetate, formate and CO2 was also observed. These results provide conclusive proof that L. divergens is indeed a homofermentative organism. Results obtained with D-[U-14C]glucose showed that approximately three-quarters of the lactate but less than 10% each of the formate and acetate were produced from glucose. The remainder was presumably derived to a varying degree from endogenous non-glucose sources such as fructose and/or amino acids.     

Characterization of two members of a novel malic enzyme class.

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.     

高氨氮利用酵母菌的筛选及相关酶活性

【背景】蛋白饲料的缺乏,促进了蛋白含量高、安全性能好的酵母类单细胞蛋白的研究与应用。【目的】筛选氨氮利用能力强的菌株,为单细胞蛋白的发酵提供优良菌株。【方法】从土壤、奶制品、水果采集样品分离酵母菌,根据形态学和分子生物学鉴定菌株,然后以硫酸铵为唯一氮源培养基,测定菌落大小、菌体干重、蛋白质含量,复筛氨氮利用率高的酵母菌,并对复筛菌株氨同化相关酶活性进行测定。【结果】经过形态学、分子生物学鉴定和氨氮利用能力评价,获得3株高氨氮利用的酵母菌,分别是胶红酵母(Rhodotorula mucilaginosa)、酿酒酵母(Saccharomyces cerevisiae)和戴尔有孢圆酵母(Torulaspora delbrueckii)。通过比较3株酵母菌的谷氨酸脱氢酶、谷氨酸合成酶、谷氨酰胺合成酶活性,酿酒酵母的3种酶活性最高,其次是胶红酵母。【结论】从奶酪和西瓜中分离的胶红酵母N5和酿酒酵母J1具有较强的氨氮利用能力以及酶活性,可为单细胞蛋白发酵提供优良菌株。     

The chloroplast 32 kDa protein is synthesized on thylakoid-bound ribosomes in Chlamydomonas reinhardtii

A cloned cpDNA fragment containing a portion of the gene for the 32–36 kDa thylakoid protein of Chlamydomonas (polypeptide D-1) was isolated. Hybridization probing of RNA from soluble and membrane fractions of Chlamydomonas showed that the mRNA for D-1 is bound to thylakoid membranes. Run-off translation of thylakoid-bound polysomes (rough thylakoids) with [³⁵S]-methionine yields polypeptide D-1 as the major product. Peptide mapping with S. aureus V-8 protease of D-1 synthesized (1) in vivo, (2) in vitro by rough thylakoids and (3) in the reticulocyte lysate directed by non-polyadenylated RNA showed that D-1 is synthesized as a precursor in the reticulocyte lysate but as the mature polypeptide by rough thylakoids.