Effects of cyclization on conformational dynamics and binding properties of Lys48-linked di-ubiquitin.
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In solution, Lys48-linked di-ubiquitin exists in dynamic equilibrium between closed and open conformations. To understand the effect of interdomain motion in polyubiquitin chains on their ability to bind ligands, we cyclized di-ubiquitin by cross-linking the free C terminus of the proximal ubiquitin with the side chain of residue 48 in the distal ubiquitin, using a chemical cross-linker, 1,6-Hexane-bis-vinylsulfone. Our NMR studies confirm that the cyclization affects conformational dynamics in di-ubiquitin by restricting opening of the interface and shifting the conformational equilibrium toward closed conformations. The cyclization, however, did not rigidly lock di-ubiquitin in a single closed conformation: The chain undergoes slow exchange between at least two closed conformations, characterized by interdomain contacts involving the same hydrophobic patch residues (Leu8-Ile44-Val70) as in the uncyclized di-ubiquitin. Lowering the pH changes the relative populations of these conformations, but in contrast with the uncyclized di-ubiquitin, does not lead to opening of the interface. This restriction of domain motions inhibits direct access of protein molecules to the hydrophobic patch residues located at the very center of the interdomain interface in di-ubiquitin, although the residual motions are sufficient to allow access of small molecules to the interface. This renders di-ubiquitin unable to bind protein molecules (e.g., UBA2 domain) in the normal manner, and thus could interfere with Ub(2) recognition by various downstream effectors. These results emphasize the importance of the opening/closing domain motions for the recognition and function of di-ubiquitin and possibly longer polyubiquitin chains. (+info)
Carbonic anhydrase in Escherichia coli. A product of the cyn operon.
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The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase. The cyn operon also includes the gene cynS, encoding the enzyme cyanase. Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized. The purified enzyme was shown to contain 1 Zn2+/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme. The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate. The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases. but not with animal and algal carbonic anhydrases. Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate. (+info)
Foundations for directed alkaloid biosynthesis.
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In this issue of Chemistry & Biology, Bernhardt and coworkers [1] assay the functional plasticity of strictosidine synthase, a gateway enzyme in the biosynthetic pathways of monoterpene indole alklaloids, and the downstream operability of the products of strictosidine synthase variants in the larger context of the plant biosynthetic pathways. (+info)
Structure-based engineering of strictosidine synthase: auxiliary for alkaloid libraries.
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The highly substrate-specific strictosidine synthase (EC 4.3.3.2) catalyzes the biological Pictet-Spengler condensation between tryptamine and secologanin, leading to the synthesis of about 2000 monoterpenoid indole alkaloids in higher plants. The crystal structure of Rauvolfia serpentina strictosidine synthase (STR1) in complex with strictosidine has been elucidated here, allowing the rational site-directed mutation of the active center of STR1 and resulting in modulation of its substrate acceptance. Here, we report on the rational redesign of STR1 by generation of a Val208Ala mutant, further describing the influence on substrate acceptance and the enzyme-catalyzed synthesis of 10-methyl- and 10-methoxystrictosidines. Based on the addition of strictosidine to a crude strictosidine glucosidase preparation from Catharanthus cells, a combined chemoenzymatic approach to generating large alkaloid libraries for future pharmacological screenings is presented. (+info)
Thiocyanate hydrolase, the primary enzyme initiating thiocyanate degradation in the novel obligately chemolithoautotrophic halophilic sulfur-oxidizing bacterium Thiohalophilus thiocyanoxidans.
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Thiohalophilus thiocyanoxidans is a first halophilic sulfur-oxidizing chemolithoautotrophic bacterium capable of growth with thiocyanate as an electron donor at salinity up to 4 M NaCl. The cells, grown with thiocyanate, but not with thiosulfate, contained an enzyme complex hydrolyzing thiocyanate to sulfide and ammonia under anaerobic conditions with carbonyl sulfide as an intermediate. Despite the fact of utilization of the <>, high cyanase activity was also detected in thiocyanate-induced cells. Three-stage column chromotography resulted in a highly purified thiocyanate-hydrolyzing protein with an apparent molecular mass of 140 kDa that consists of three subunits with masses 17, 19 and 29 kDa. The enzyme is a Co,Fe-containing protein resembling on its function and subunit composition the enzyme thiocyanate hydrolase from the Betaproteobacterium Thiobacillus thioparus. Cyanase, copurified with thiocyanate hydrolase, is a bisubstrate multisubunit enzyme with an apparent subunit molecular mass of 14 kDa. A possible role of cyanase in thiocyanate degradation by T. thiocyanoxidans is discussed. (+info)
UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures.
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BACKGROUND: Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. RESULTS: Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3-4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. CONCLUSION: Our results demonstrate that cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed. (+info)
Strictosidine synthase: mechanism of a Pictet-Spengler catalyzing enzyme.
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The Pictet-Spengler reaction, which yields either a beta-carboline or a tetrahydroquinoline product from an aromatic amine and an aldehyde, is widely utilized in plant alkaloid biosynthesis. Here we deconvolute the role that the biosynthetic enzyme strictosidine synthase plays in catalyzing the stereoselective synthesis of a beta-carboline product. Notably, the rate-controlling step of the enzyme mechanism, as identified by the appearance of a primary kinetic isotope effect (KIE), is the rearomatization of a positively charged intermediate. The KIE of a nonenzymatic Pictet-Spengler reaction indicates that rearomatization is also rate-controlling in solution, suggesting that the enzyme does not significantly change the mechanism of the reaction. Additionally, the pH dependence of the solution and enzymatic reactions provides evidence for a sequence of acid-base catalysis steps that catalyze the Pictet-Spengler reaction. An additional acid-catalyzed step, most likely protonation of a carbinolamine intermediate, is also significantly rate controlling. We propose that this step is efficiently catalyzed by the enzyme. Structural analysis of a bisubstrate inhibitor bound to the enzyme suggests that the active site is exquisitely tuned to correctly orient the iminium intermediate for productive cyclization to form the diastereoselective product. Furthermore, ab initio calculations suggest the structures of possible productive transition states involved in the mechanism. Importantly, these calculations suggest that a spiroindolenine intermediate, often invoked in the Pictet-Spengler mechanism, does not occur. A detailed mechanism for enzymatic catalysis of the beta-carboline product is proposed from these data. (+info)
CSN5 isopeptidase activity links COP9 signalosome activation to breast cancer progression.
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