Continuous counter current extraction, isolation and determination of solanesol in Nicotiana tobacum L. by non-aqueous reversed phase high performance liquid chromatography.

A method of continuous counter current extraction in a large-scale production of solanesol from tobacco leaves was developed. The crude extract containing 15-20% solanesol was subjected to a series of steps, viz., saponification, solvent recrystallization and column chromatography. The pure material was characterized by FT-IR, ESI-MS, 1H NMR and 13C NMR spectrometry. The analysis was carried out by a simple and rapid non-aqueous reversed-phase high performance liquid chromatographic method on a Hypersil BDS C18 column (250 mm x 4.6mm, particle size 5 microm) with isopropyl alcohol-methanol (60:40, v/v) as mobile phase and detection at 215 nm. The product purity was between 95 and 98% (w/w) as determined by HPLC.

Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA.

The crystal structure of the processivity factor required by eukaryotic DNA polymerase delta, proliferating cell nuclear antigen (PCNA) from S. cerevisiae, has been determined at 2.3 A resolution. Three PCNA molecules, each containing two topologically identical domains, are tightly associated to form a closed ring. The dimensions and electrostatic properties of the ring suggest that PCNA encircles duplex DNA, providing a DNA-bound platform for the attachment of the polymerase. The trimeric PCNA ring is strikingly similar to the dimeric ring formed by the beta subunit (processivity factor) of E. coli DNA polymerase III holoenzyme, with which it shares no significant sequence identity. This structural correspondence further substantiates the mechanistic connection between eukaryotic and prokaryotic DNA replication that has been suggested on biochemical grounds.

Crystallization of proliferating cell nuclear antigen (PCNA) from Saccharomyces cerevisiae.

Proliferating cell nuclear antigen (PCNA) is the component of the chromosomal DNA replication machinery in eukaryotic cells that confers high processivity upon DNA polymerase delta and epsilon. It has been proposed that PCNA functions by forming a trimeric complex with a ring-like structure through which DNA is threaded. PCNA from the yeast Saccharomyces cerevisiae has been crystallized in a cubic space group (P2(1)3, a = 121.1 A). Unexpectedly, a mercury derivative of PCNA yields crystals that diffract significantly better than crystals of the unmodified protein (2.4 A and 3.0 A resolution, respectively). Mass spectrometry reveals that the derivative results from the addition of two mercury atoms to the protein. Although crystals of the mercurated protein show evidence of non-isomorphism, the anomalous diffraction signal is strong and phases may be determined by multi-wavelength anomalous diffraction (MAD phasing).

Crystal structure of Escherichia coli thioredoxin reductase refined at 2 A resolution. Implications for a large conformational change during catalysis.

The crystal structures of three forms of Escherichia coli thioredoxin reductase have been refined: the oxidized form of the wild-type enzyme at 2.1 A resolution, a variant containing a cysteine to serine mutation at the active site (Cys138Ser) at 2.0 A resolution, and a complex of this variant with nicotinamide adenine dinucleotide phosphate (NADP+) at 2.3 A resolution. The enzyme mechanism involves the transfer of reducing equivalents from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to a disulfide bond in the enzyme, via a flavin adenine dinucleotide (FAD). Thioredoxin reductase contains FAD and NADPH binding domains that are structurally similar to the corresponding domains of the related enzyme glutathione reductase. The relative orientation of these domains is, however, very different in the two enzymes: when the FAD domains of thioredoxin and glutathione reductases are superimposed, the NADPH domain of one is rotated by 66 degrees with respect to the other. The observed binding mode of NADP+ in thioredoxin reductase is non-productive in that the nicotinamide ring is more than 17 A from the flavin ring system. While in glutathione reductase the redox active disulfide is located in the FAD domain, in thioredoxin reductase it is in the NADPH domain and is part of a four-residue sequence (Cys-Ala-Thr-Cys) that is close in structure to the corresponding region of thioredoxin (Cys-Gly-Pro-Cys), with a root-mean-square deviation of 0.22 A for atoms in the disulfide bonded ring. There are no significant conformational differences between the structure of the wild-type enzyme and that of the Cys138Ser mutant, except that a disulfide bond is not present in the latter. The disulfide bond is positioned productively in this conformation of the enzyme, i.e. it stacks against the flavin ring system in a position that would facilitate its reduction by the flavin. However, the cysteine residues are relatively inaccessible for interaction with the substrate, thioredoxin. These results suggest that thioredoxin reductase must undergo conformational changes during enzyme catalysis. All three structures reported here are for the same conformation of the enzyme and no direct evidence is available as yet for such conformational changes. The simplest possibility is that the NADPH domain rotates between the conformation observed here and an orientation similar to that seen in glutathione reductase. This would alternately place the nicotinamide ring and the disulfide bond near the flavin ring, and expose the cysteine residues for reaction with thioredoxin in the hypothetical conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

X-ray structure of trypanothione reductase from Crithidia fasciculata at 2.4-A resolution.

Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidine conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. We report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from -2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4 degrees in the domains that form the site, with relative shifts of as much as 2-3 A in residue positions. These results provide a detailed view of the residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

Convergent evolution of similar function in two structurally divergent enzymes.

An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase. Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin. Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site. Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other. We have determined the crystal structure of E. coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure. The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system. This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently.