The cyclization of farnesyl diphosphate and nerolidyl diphosphate by a purified recombinant delta-cadinene synthase

The first step in the conversion of the isoprenoid intermediate, farnesyl diphosphate (FDP), to sesquiterpene phytoalexins in cotton (Gossypium barbadense) plants is catalyzed by delta-cadinene (CDN) synthase. CDN is the precursor of desoxyhemigossypol and hemigossypol defense sesquiterpenes. In this paper we have studied the mechanism for the cyclization of FDP and the putative intermediate, nerolidyl diphosphate, to CDN. A purified recombinant CDN synthase (CDN1-C1) expressed in Escherichia coli from CDN1-C1 cDNA isolated from Gossypium arboreum cyclizes (1RS)-[1-2H](E, E)-FDP to >98% [5-2H]and [11-2H]CDN. Enzyme reaction mixtures cyclize (3RS)-[4,4,13,13,13-2H5]-nerolidyl diphosphate to 62.1% [8,8,15,15,15-2H5]-CDN, 15.8% [6,6,15,15,15-2H5]-alpha-bisabolol, 8.1% [6,6,15,15,15-2H5]-(beta)-bisabolene, 9.8% [4,4,13,13-2H4]-(E)-beta-farnesene, and 4.2% unknowns. Competitive studies show that (3R)-nerolidyl diphosphate is the active enantiomer of (3RS)-nerolidyl diphosphate that cyclized to CDN. The kcat/Km values demonstrate that the synthase uses (E,E)-FDP as effectively as (3R)-nerolidyl diphosphate in the formation of CDN. Cyclization studies with (3R)-nerolidyl diphosphate show that the formation of CDN, (E)-beta-farnesene, and beta-bisabolene are enzyme dependent, but the formation of alpha-bisabolol in the reaction mixtures was a Mg2+-dependent solvolysis of nerolidyl diphosphate. Enzyme mechanisms are proposed for the formation of CDN from (E,E)-FDP and for the formation of CDN, (E)-beta-farnesene, and beta-bisabolene from (3RS)-nerolidyl diphosphate. The primary structures of cotton CDN synthase and tobacco epi-aristolochene synthase show 48% identity, suggesting similar three-dimensional structures. We used the SWISS-MODEL to test this. The two enzymes have the same overall structure consisting of two alpha-helical domains and epi-aristolochene synthase is a good model for the structure of CDN synthase. Several amino acids in the primary structures of both synthases superimpose. The amino acids having catalytic roles in epi-aristochene synthase are substituted in the CDN synthase and may be related to differences in catalytic properties.     

Crystal structures of Escherichia coli glycerol kinase variant S58-->W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion

Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site [Ormo, M., Bystrom, C., and Remington, S. J. (1998) Biochemistry 37, 16565-16572]. The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition. Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures. The conformational difference is produced by a approximately 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily. Two of the ATP analogues bound in nonproductive conformations in both subunits. However, beta, gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol. This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion.     

Bi-sensory, striped representations: comparative insights from owl and platypus.

Bi-sensory striped arrays are described in owl and platypus that share some similarities with the other variant of bi-sensory striped array found in primate and carnivore striate cortex: ocular dominance columns. Like ocular dominance columns, the owl and platypus striped systems each involve two different topographic arrays that are cut into parallel stripes, and interdigitated, so that higher-order neurons can integrate across both arrays. Unlike ocular dominance stripes, which have a separate array for each eye, the striped array in the middle third of the owl tectum has a separate array for each cerebral hemisphere. Binocular neurons send outputs from both hemispheres to the striped array where they are segregated into parallel stripes according to hemisphere of origin. In platypus primary somatosensory cortex (S1), the two arrays of interdigitated stripes are derived from separate sensory systems in the bill, 40,000 electroreceptors and 60,000 mechanoreceptors. The stripes in platypus S1 cortex produce bimodal electrosensory-mechanosensory neurons with specificity for the time-of-arrival difference between the two systems. This "thunder-and-lightning" system would allow the platypus to estimate the distance of the prey using time disparities generated at the bill between the earlier electrical wave and the later mechanical wave caused by the motion of benthic prey. The functional significance of parallel, striped arrays is not clear, even for the highly-studied ocular dominance system, but a general strategy is proposed here that is based on the detection of temporal disparities between the two arrays that can be used to estimate distance.     

Subcloning, expression, purification, and characterization of Haemophilus influenzae glycerol kinase

Glycerol kinase (EC 2.7.1.30) is a bacterial sugar kinase and a member of the sugar kinase/actin/hsc-70 superfamily of enzymes. The enzyme from Escherichia coli is an allosteric regulatory enzyme whose activity is inhibited by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (previously termed III(Glc)). Comparison of its primary structure with that of the highly similar Haemophilus influenzae glycerol kinase reveals that the amino acid sequence for the binding site for FBP is conserved while the amino acid sequence for the binding site for IIA(Glc) contains differences that are predicted to prevent its inhibition. To test this hypothesis, the H. influenzae glpK gene was assembled from DNA library fragments and subcloned into pUC18. The enzyme is expressed at high levels in E. coli. It was purified to greater than 90% homogeneity by taking advantage of its solubility behavior in a procedure that requires no column chromatography. The initial-velocity kinetic parameters of the purified enzyme are similar to those of the E. coli glycerol kinase. The H. influenzae glycerol kinase is inhibited by FBP but not by IIA(Glc), in agreement with the prediction based on sequence comparison. Sedimentation velocity experiments reveal that inhibition of HiGK by FBP is associated with oligomerization, behavior which is similar to EcGK. The possibility of utilizing mutagenesis studies to exploit the high degree of similarity of these two enzymes to elucidate the mechanism of allosteric regulation by IIA(Glc) is discussed.     

Leaf senescence-like characteristics contribute to cotton's premature photosynthetic decline

Leaf and canopy photosynthesis of cotton (Gossypium hirsutum L.) declines as the crop approaches cutout, just as the assimilate needs for reproductive growth are peaking. Our objective with this study was to determine whether this decline is due to remobilization of leaf components to support the reproductive growth or due to some cue from the changing environmental conditions during the growing season. Field studies were conducted in 1995–1996 at Stoneville, Mississippi, using six cotton genotypes and two planting dates (early and late), which produced two distinctly different cotton populations reaching cutout at different times. Among the six genotypes were a photoperiod sensitive line (non-flowering) and its counter part which had photoperiod insensitive genes backcrossed four times to the photoperiod sensitive line (flowering). This pair was used to assess the degree that the photosynthetic decline could be attributed to reproductive sink development. Leaf CO₂-exchange rate (CER) and chlorophyll (Chl) fluorescence measurements were taken in mid-August, a period corresponding to cutout for the early planted plots, and those leaves were collected. Leaf Chl level, soluble protein level, various soluble carbohydrate levels and Rubisco activities were assayed on those leaves. Averaged across years, leaf CER and soluble protein levels were reduced approximately 14% and 18%, respectively, for the early planted compared to the late planted cotton. Neither leaf Chl levels or Chl fluorescence Fv/Fm values for Photosystem II yield were altered by the planting date. In 1996, leaves from the non-flowering line had 12% greater Chl and 20% greater soluble protein levels than the flowering line. However, in 1996, the CER of the early planted non-flowering line was reduced 10% compared to the late planted. Although remobilization of leaf N to reproductive growth appears to be the principle component causing the cutout photosynthetic decline, the data also indicate that environmental factors can play a small role in causing the decline. This revised version was published online in June 2006 with corrections to the Cover Date.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Artefacts induced on <Emphasis Type="Italic">c</Emphasis>-type haem proteins by electrode surfaces

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met–His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c ₃ from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.     

The structure of echovirus type 12 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD 55)

Echovirus type 12 (EV12), an Enterovirus of the Picornaviridae family, uses the complement regulator decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF consisting of short consensus repeat domains 3 and 4 from cryo-negative stain electron microscopy data (EMD code 1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the 2-fold symmetry axes. Despite this general similarity our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF(34) and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB code 1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF binding phenotype in these viruses.     

Structural and mutagenesis studies on the cytochrome c peroxidase from Rhodobacter capsulatus provide new insights into structure-function relationships of bacterial di-heme peroxidases

Cytochrome c peroxidases (CCP) play a key role in cellular detoxification by catalyzing the reduction of hydrogen peroxide to water. The di-heme CCP from Rhodobacter capsulatus is the fastest enzyme (1060 s(-1)), when tested with its physiological cytochrome c substrate, among all di-heme CCPs characterized to date and has, therefore, been an attractive target to investigate structure-function relationships for this family of enzymes. Here, we combine for the first time structural studies with site-directed mutagenesis and spectroscopic studies of the mutant enzymes to investigate the roles of amino acid residues that have previously been suggested to be important for activity. The crystal structure of R. capsulatus at 2.7 Angstroms in the fully oxidized state confirms the overall molecular scaffold seen in other di-heme CCPs but further reveals that a segment of about 10 amino acids near the peroxide binding site is disordered in all four molecules in the asymmetric unit of the crystal. Structural and sequence comparisons with other structurally characterized CCPs suggest that flexibility in this part of the molecular scaffold is an inherent molecular property of the R. capsulatus CCP and of CCPs in general and that it correlates with the levels of activity seen in CCPs characterized, thus, far. Mutagenesis studies support the spin switch model and the roles that Met-118, Glu-117, and Trp-97 play in this model. Our results help to clarify a number of aspects of the debate on structure-function relationships in this family of bacterial CCPs and set the stage for future studies.