The nerve of ovulation-inducing factor in semen.

A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered.

Structure of a highly NADP+-specific isocitrate dehydrogenase.

Isocitrate dehydrogenase catalyzes the first oxidative and decarboxylation steps in the citric acid cycle. It also lies at a crucial bifurcation point between CO2-generating steps in the cycle and carbon-conserving steps in the glyoxylate bypass. Hence, the enzyme is a focus of regulation.

Biochemical isolation and purification of ovulation-inducing factor (OIF) in seminal plasma of llamas.

Background: The objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate.

Methods: Semen collected from male llamas by artificial vagina was centrifuged and the seminal plasma was harvested and stored frozen. Seminal plasma was thawed and loaded onto a Type 1 macro-prep ceramic hydroxylapatite column and elution was carried out using a lineal gradient with 350 mM sodium phosphate.

Crystallization and preliminary X-ray diffraction analysis of the Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant.

The Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant is stereospecific for (R)-alcohols instead of (S)-alcohols. Pyramidal crystals grown in the presence of (R)-phenylethanol via the hanging-drop vapour-diffusion method diffracted to 3.2 A resolution at the Canadian Light Source.

Crystallization and preliminary X-ray studies of the N-domain of the Wilson disease associated protein.

Wilson disease associated protein (ATP7B) is essential for copper transport in human cells. Mutations that affect ATP7B function result in Wilson's disease, a chronic copper toxicosis. Disease-causing mutations within the N-domain of ATP7B (WND) are known to disrupt ATP binding, but a high-resolution X-ray structure of the ATP-binding site has not been reported.

Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase.

Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase.

Structural investigation of a phosphorylation-catalyzed, isoaspartate-free, protein succinimide: crystallographic structure of post-succinimide His15Asp histidine-containing protein.

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation.

Structural, functional and calorimetric investigation of MosA, a dihydrodipicolinate synthase from Sinorhizobium meliloti l5-30, does not support involvement in rhizopine biosynthesis.

MosA is an enzyme from Sinorhizobium meliloti L5-30, a beneficial soil bacterium that forms a symbiotic relationship with leguminous plants. MosA was proposed to catalyze the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine (compounds known as rhizopines), despite the MosA sequence showing a strong resemblance to dihydrodipicolinate synthase (DHDPS) sequences rather than to methyltransferases. Our laboratory has already shown that MosA is an efficient catalyst of the DHDPS reaction.

Structure of a GTP-dependent bacterial PEP-carboxykinase from Corynebacterium glutamicum.

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2(1) with four molecules per asymmetric unit.

Histidine phosphorylation in biological systems.

Histidine phosphorylation is important in prokaryotes and occurs to the extent of 6% of total phosphorylation in eukaryotes. Nevertheless phosphohistidine residues are not normally observed in proteins due to rapid hydrolysis of the phosphoryl group under acidic conditions. Many rapid processes employ phosphohistidines, including the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), the bacterial two-component systems and reactions catalyzed by enzymes such as nucleoside diphosphate kinase and succinyl-CoA synthetase.

How does an enzyme recognize CO2?

Phosphoenolpyruvate carboxykinase (PCK) reversibly catalyzes the carboxylation of phosphoenolpyruvate to oxaloacetate. Carbon dioxide, and not bicarbonate ion, is the substrate utilized. Assays of the carboxylation reaction show that initial velocities are 7.

Crystallization, preliminary X-ray diffraction and structure analysis of Thermotoga maritima mannitol dehydrogenase.

Diffraction data have been collected from a crystal of Thermotoga maritima mannitol dehydrogenase at the Canadian Light Source. The crystal diffracted to 3.3 A resolution and belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 83.

Phylogenetic study of the evolution of PEP-carboxykinase.

Phosphoenolpyruvate carboxykinase (PCK) is the key enzyme to initiate the gluconeogenic pathway in vertebrates, yeast, plants and most bacteria. Nucleotide specificity divided all PCKs into two groups. All the eukaryotic mammalian and most archaeal PCKs are GTP-specific.

The structure of an ancient conserved domain establishes a structural basis for stable histidine phosphorylation and identifies a new family of adenosine-specific kinases.

Phosphorylation of both small molecules and proteins plays a central role in many biological processes. In proteins, phosphorylation most commonly targets the oxygen atoms of Ser, Thr, and Tyr. In contrast, stably phosphorylated His residues are rarely found, due to the lability of the N-P bond, and histidine phosphorylation features most often in transient processes.

Crystallization, preliminary X-ray diffraction and structure solution of MosA, a dihydrodipicolinate synthase from Sinorhizobium meliloti L5-30.

The structure of MosA, a dihydrodipicolinate synthase and reported methyltransferase from Sinorhizobium meliloti, has been solved using molecular replacement with Escherichia coli dihydrodipicolinate synthase as the model. A crystal grown in the presence of pyruvate diffracted X-rays to 2.3 A resolution using synchrotron radiation and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 69.

Substrate-free structure of a monomeric NADP isocitrate dehydrogenase: an open conformation phylogenetic relationship of isocitrate dehydrogenase.

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved.

Structure of PEP carboxykinase from the succinate-producing Actinobacillus succinogenes: a new conserved active-site motif.

Actinobacillus succinogenes can produce, via fermentation, high concentrations of succinate, an important industrial commodity. A key enzyme in this pathway is phosphoenolpyruvate carboxykinase (PCK), which catalyzes the production of oxaloacetate from phosphoenolpyruvate and carbon dioxide, with the concomitant conversion of adenosine 5'-diphosphate to adenosine 5'-triphosphate. 1.

Crystal structure of Anaerobiospirillum succiniciproducens PEP carboxykinase reveals an important active site loop.

The 2.2 Angstroms resolution crystal structure of the enzyme phosphoenolpyruvate carboxykinase (PCK) from the bacterium Anaerobiospirillum succiniciproducens complexed with ATP, Mg(2+), Mn(2+) and the transition state analogue oxalate has been solved. The 2.

Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase (PCK) catalyzes the conversion of oxaloacetate (OAA) to PEP and carbon dioxide with the subsequent conversion of nucleoside triphosphate to nucleoside diphosphate (NDP). The 1.9 A resolution structure of Escherichia coli PCK consisted of a 275-residue N-terminal domain and a 265-residue C-terminal domain with the active site located in a cleft between these domains.

The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus.

V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges.

Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase.

The Corynebacterium glutamicum (C. glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form.

Cobalt(II), nickel(II) and zinc(II) do not bind to intra-helical N(7) guanine positions in the B-form crystal structure of d(GGCGCC).

Three novel X-ray crystal structures for the DNA hexamer d(GGCGCC) in the B-form complexed to divalent cobalt, nickel and zinc ions have been determined to a resolution of 2.9-3.0 A.

Crystallization and preliminary X-ray crystallographic studies of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum.

Phosphoenolpyruvate carboxykinase (PCK) is a key enzyme involved in the regulation of gluconeogenesis. PCKs from higher animals require guanosine nucleotide for activity. PCK from Corynebacterium glutamicum is also GTP specific.

Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin.

The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511).

Mapping protein: carbohydrate interactions.

Many biologically important interactions occur between proteins and carbohydrates. The examination of these interactions at the atomic level is critical not only in understanding the nature of these interactions and their biological role, but also in the design of effective modulators of these interactions. While experimentally obtained structural information is preferred, quite often this information is unavailable.