Search

FeS clusters are versatile cofactors of a variety of proteins, but the mechanisms of their biosynthesis are still unknown. The cystine C-S lyase from Synechocystis has been identified as a participant in ferredoxin FeS cluster formation. Herein, we report on the crystal structure of the lyase and of a complex with the reaction products of cystine cleavage at 1.8- and 1.55-A resolution, respectively. The sulfur-containing product was unequivocally identified as cysteine persulfide. The reactive persulfide group is fixed by a hydrogen bond to His-114 in the center of a hydrophobic pocket and is thereby shielded from the solvent. Binding and stabilization of the cysteine persulfide represent an alternative to the generation of a protein-bound persulfide by NifS-like proteins and point to the general importance of persulfidic compounds for FeS cluster assembly.

Shearer, J., I. Marchand, P. Sathasivam, M. A. Tarnopolsky, and T. E. Graham. Glycogenin activity in human skeletal muscle is proportional to muscle glycogen concentration. Am. J. Physiol. Endocrinol. Metab. 278: E177-E180, 2000.--The de novo biosynthesis of glycogen is catalyzed by glycogenin, a self-glucosylating protein primer. To date, the role of glycogenin in regulating glycogen metabolism and the attainment of maximal glycogen levels in skeletal muscle are unknown. We measured glycogenin activity after enzymatic removal of glucose by [Alpha]-amylase, an indirect measure of glycogenin amount. Seven male subjects performed an exercise and dietary protocol that resulted in one high-carbohydrate leg (HL) and one low-carbohydrate leg (LL) before testing. Resting muscle biopsies were obtained and analyzed for total glycogen, proglycogen (PG), macroglycogen (MG), and glycogenin activity. Results showed differences (P [is less than] 0.05) between HL and LL for total glycogen (438.0 [+ or -] 69.5 vs. 305.7 [+ or -] 57.4 mmol glucosyl units/kg dry wt) and PG (311.4 [+ or -] 38.1 vs. 227.3 [+ or -] 33.1 mmol glucosyl units/kg dry wt). A positive correlation between total muscle glycogen content and glycogenin activity (r = 0.84, P [is less than] 0.001) was observed. Similar positive correlations (P [is less than] 0.05) were also evident between both PG and MG concentration and glycogenin activity (PG, r = 0.82; MG, r = 0.84). It can be concluded that glycogenin does display activity in human skeletal muscle and is proportional to glycogen concentration. Thus it must be considered as a potential regulator of glycogen synthesis in human skeletal muscle. glycogen metabolism; proglycogen; macroglycogen; carbohydrate loading; carbohydrate synthesis

The marine shrimp Penaeus vannamei produces about 200000 yolk laden eggs during each ovarian maturation cycle. Each egg contains about 50 ng of yolk protein about 10% of the total protein of the egg. Based on in vitro assays both ovarian tissue and the hepatopancreas are capable of yolk protein biosynthesis. Eyestalk ablation removes the endocrine centers which normally regulate reproduction and is used to induce ovarian maturation. After eyestalk ablation yolk biosynthesis proceeds rapidly in both the ovaries and the hepatopancreas. Yolk proteins are transported in the hemolymph (1 mg/ml) during this process. Eyestalk endocrines are capable of both inhibiting and stimulating yolk biosynthesis in vivo and in vitro. Steroid hormones may also be involved in shrimp yolk biosynthesis. Supported by Florida Sea Grant Florida Dept. Ag. and US Mexico Foundation.

Titos, Esther, Nan Chiang, Charles N. Serhan, Mario Romano, Joan Gaya, Gloria Pueyo, and Joan Claria. Hepatocytes are a rich source of novel aspirin-triggered 15-epi-lipoxin [A.sub.4]. Am. J. Physiol. 277 (Cell Physiol. 46): C870--C877, 1999.--Novel aspirin (ASA)-triggered 15-epilipoxins (ATL) comprise new potent bioactive eicosanoids that may contribute to the therapeutic effect of this drug. ATL biosynthesis is initiated by ASA acetylation of cyclooxygenase (COX)-2 and was originally identified during the interaction of leukocytes with either endothelial or epithelial cells. Here, we examined ATL biosynthesis in rat hepatocytes either alone or in coincubation with nonparenchymal liver cells (NPC) and in liver homogenates from ASA-treated rats. Rat hepatocytes and CC-1 cells, a rat hepatocyte cell line, displayed COX-1 but not COX-2 mRNA expression and predominantly produced thromboxane [A.sub.2] ([TXA.sub.2]) and 15-hydroxyeicosatetraenoic acid (15-HETE). In these cells, ASA shifted the arachidonic acid metabolism from [TXA.sub.2] to 15-HETE in a concentration-dependent manner. In contrast, neither indomethacin, ibuprofen, valeryl salicylate, nor nimesulide was able to trigger 15-HETE biosynthesis. SKF-525A, a cytochrome P-450 inhibitor, significantly reduced the effect of ASA on 15-HETE biosynthesis. Furthermore, phenobarbital, a potent inducer of cytochrome P-450 activity, further increased ASA-induced 15-HETE production. ASA treatment of hepatocyte-NPC coincubations resulted in the generation of significant amounts of ATL. In addition, in vivo experiments demonstrated augmented hepatic levels of 15-epi-lipoxin [A.sub.4] in ASA-treated rats. Taken together and considering that ASA is hydrolyzed on its first pass through the portal circulation, these data indicate that, during ASA's consumption, liver tissue generates biologically relevant amounts of ATL by COX-2-independent mechanisms. liver cells; acetylsalicylic acid; 15-hydroxyeicosatetraenoic acid; cytochrome P-450

The African dwarf crocodile, Osteolaemus tetraspis (Crocodilidae, Reptilia), possesses a pair of skin glands, the paracloacal glands, the secretion of which is thought to be used to mark nest sites or attract mates. Ten aromatic steroidal esters were isolated from this secretion and characterized on the basis of NMR spectroscopic investigations, electrospray ionization-MS analyses, and chemical degradation. These esters, which account for more than 90% of the paracloacal glandular secretion, are derived from either cholesterol or cholestanol, esterified with a C-20 or C-22 acid closely related to dianeackerone, the only significant volatile compound found in this secretion. Osteolaemus tetraspis | paracloacal gland | cholesterol | biosynthesis

Wick, Debra A., Bellur Seetharam, and Nancy M. Dahms. Biosynthesis and secretion of the mannose 6-phosphate receptor and its ligands in polarized Caco-2 cells. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G506-G514, 1999.--We have analyzed the transport of newly synthesized mannose 6-phosphate (Man-6-P)-bearing proteins (i.e., lysosomal enzymes) in the polarized human colon adenocarcinoma cell line, Caco-2, by subjecting filter-grown cells to a pulse-chase labeling protocol using [[sup.35]S]methionine, and the resulting cell lysate, apical medium, and basolateral medium were immunoprecipitated with insulin-like growth factor II/Man-6-P receptor (IGF-II/MPR)-specific antisera. The results showed that the majority of secreted lysosomal enzymes accumulated in the apical medium at [is greater than] 2 h of chase and that this polarized distribution was facilitated by the IGF-II/MPR selectively endocytosing lysosomal enzymes from the basolateral surface. Treatment with various agents known to affect vesicular transport events demonstrated that incubations at 16[degrees]C or incubations with brefeldin A inhibited the secretion of lysosomal enzymes from both the apical and basolateral surface, whereas treatment with nocodazole selectively blocked apical secretion. In contrast, incubation with [NH.sub.4]Cl or nocodazole had a stimulatory effect on basolateral secretion. Taken together, these results demonstrate that the sorting of Man-6-P-containing proteins into the apical and basolateral secretory pathways is regulated by distinct components of the intracellular trafficking machinery. lysosomal enzymes; intracellular trafficking; insulin-like growth factor II receptor

A breakthrough in artificial photosynthesis was achieved through a system that captures carbon dioxide emissions before they are vented into the atmosphere and then, powered by solar energy, converts the [...]

The in vitro characterization of the gene spoT1, which is located within the putative sporolide cluster and whose product has catalyzed the initial biochemical reaction in the pathway to the cyclohexenone epoxide moiety of sporolides A and B, is described. The unusual pathway has shown another role for hydroxymandelic acid synthase (HMAS), which is associated with the biosynthesis of (S)-hydroxypehnylglycine.

The gil gene cluster reveals all the genes whose corresponding enzymes could be involved in the intriguing steps of 1 biosynthesis and which are crucial for establishing structural moieties important for its unique biological activities.

A convergent synthesis of (+)-duocarmycin A, whose strategy is readily amenable to the synthesis of its congener, (+)-duocarmycin SA, is reported. A convergent synthesis is achieved and the novel copper-mediated aryl amination reaction is used for forming all of the aryl-nitrogen bonds present in the duocarmycins, thereby providing a superior alternative to the existing palladium-catalyzed protocols.

A new approach to obtain hybrid nonribosomal peptide synthetases (NRPSs) is used to show that the deletion of an entire module in an NRPS assembly line caused the secretion of the predicted peptide antibiotic variant with a decreased ring size. It is concluded that the surgery in the linkers between entire modules seems to be a robust approach to rationally design NRPSs in nonribosomal peptide antibiotic producer strains.

Thymidylate synthase (TS) is a homodimer of approximately 72 kDa which catalyzes the reductive methylation of 2-deoxyuridine monophosphate (dUMP) using 5,10-methylenetetrahydrofolate ([CH.sub.2][H.sub.4]folate) to form deoxythymidine monophosphate (dTMP) and dihydrofolate ([H.sub.2]folate). [...]

A study investigated the initial rates of the synthesis of the enzyme acetyl-coenzyme A (CoA) from Clostridium thermoaceticum to determine whether CO2 affects the acetyl-CoA synthase activity. Findings indicate that CO2 is a substrate for the synthesis of acetyl-CoA and that under these conditions, CO is an intermediate that travels through a tunnel from the C- to the A-cluster. A low-potential one-electron redox site on either acetyl-CoA synthase or methylated corrinoid-iron-sulfur protein must be reduced at, or prior to, the rate-limiting step of catalysis.

Hkd motif belongs to the new superfamily of proteins with a conserved motif that includes a HxK(3/4U)D that indicates a stretch of four protein residues. The residues are mainly hydrophobic proteins that are detected in phospholipases D. These superfamily of proteins demonstrate enzymatic activity and catalyse the formation of phosphoester bonds. The conserved motiff of aspartate, lysine and histidine form a catalytic triad. These amino acids hydrolyze the phosphoester bonds by a mechanism that is similar to that of the HAD superfamily.

The preparation of macromolecules that are almost identical to the natural products is now possible, using living polymerization methods. The method consists of addition of monomers to an initiator molecule that is stabilized. The formation of DNA polymers using photolithographic techniques and the supply of oligonucleotides in the market to compare sequences to prepare the exact duplicates are new methods that have been developed.

The structural elucidation of antillatoxin, an ichthyotoxic cyclic lipopeptide from the tropical cyanobacterium Lyngbya majuscula, reveals the spectacular adaptive and biosynthetic capabilities of cyanobacteria. Antillatoxin is isolated as an amorphous powder from the ichthyotoxic crude extract by successive chromatography on silica and RP-18 gels, followed by final purification using RP-18 HPLC.

Cloning and characterization of a group of five genes from Escherichia coli (thiCEFGH), which help the biosynthesis of thiamin, enables a mechanistic study of these genes present in small amounts in wild-type E. coli. An analysis of E. coli mutated in these genes reveals the necessity of the thiE gene for cleaving the p-hydroxybenzyl group from a thiamin precursor and for catalyzing the coupling between a pyrimidine and a thiazole during the biosynthesis.

The total synthesis of biosynthetic precursor of swinholide A, preswinholide A, is achieved for the first time. A boron-mediated anti aldol reaction for the coupling of complex fragments is used. Final elaboration of secoacid derivative into swinholide A is possible because all the 15 stereogenic centers and appropriate protecting groups are in position.

Investigations of the functions of biosynthetic genes of the vitamin B12 pathway in aerobic bacterium Pseudomonas denitrificans reveal the structure and synthesize genetically engineered pentamethylated intermediate of vitamin B12 biosynthesis, precorrin-5. This has enhanced available information on the stages between precorrin-3 and precorrin-6x and the intermediates between (4-C)-5-aminolevulinic acid and hydrogenobyrinic acid.

Investigations of the biosynthesis of structurally linked metabolites, brevianamides, helped accomplish the first total synthesis of (+)-paraherquamide B. A regioselective indole-to-oxindole oxidative pinacol-type realignment, a chemoselective 3 degree amide reduction, a Pd(II)-mediated indole cyclization, a completely stereocontrolled intramolecular SN2' reaction and the use of Somei/Kametani reaction were part of this synthesis. Paraherquamides are intricate, hetracyclic toxic mold metabolites.