IDENTITY OF THE PHOTORECEPTORS FOR THE PERCEPTION OF IRRADIANCE IN PHOTOSYNTHETIC LIGHT ACCLIMATION IN TOMATO*

The photoreceptors involved in the photosynthetic acclimation of tomato (Lycopersicon esculentum Mill.) to increased irradiance were investigated. Plants were transferred from 100 p.mol m^?2 s^?1 cool white fluorescent light to higher irradiances of white light or white light supplemented with blue, red, green or yellow light. In these experiements light of all wavelengths tested was capable of causing acclimation as measured by the rate of light-saturated photosynthesis. It was concluded that the photosynthetic system rather than the blue-absorbing photoreceptor or phytochrome system acts as the photoreceptor for increased irradiance. No acclimation was observed in response to increased CO₂ levels, but increasing light integral at a constant irradiance was effective in bringing about acclimation. We conclude that acclimation is a response to increased photosynthetic light capture rather than increased photosynthetic carbon fixation, and involves a photon counting mechanism.     

Elucidation of the mechanism of polysaccharide cleavage by chondroitin AC lyase from Flavobacterium heparinum

Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism resulting in disaccharides or oligosaccharides with delta4,5-unsaturated uronic acid residues at their nonreducing end. Mechanistic details concerning the ordering of the bond-breaking and -forming steps of this enzymatic reaction are nonexistent, mainly due to the inhomogeneous nature of the polymeric substrates. The creation of a new class of synthetic substrates for this enzyme has allowed the measurement of defined and reproducible k(cat) and K(m) values and has expanded the range of mechanistic studies that can be performed. The primary deuterium kinetic isotope effect upon k(cat)/K(m) for the abstraction of the proton alpha to the carboxylic acid was measured to be 1.67 +/- 0.07, showing that deprotonation occurs in a rate-limiting step. Using substrates with leaving groups of differing reactivity, a flat linear free energy relationship was produced, indicating that the C4-O4 bond is not broken in a rate-determining step. Taken together, these results strongly suggest a stepwise mechanism. Consistent with this was the measurement of a secondary deuterium kinetic isotope effect upon k(cat)/K(m) of 1.01 +/- 0.03 on a 4-[(2)H]-substrate, indicating that no sp2 character is developed at C4 during the rate-limiting step, thereby ruling out a concerted syn-elimination.     

Local crystal chemistry, induced strain and short range order in the cubic pyrochlore (Bi1.5−αZn0.5−β)(Zn0.5−γNb1.5−δ)O(7−1.5α−β−γ−2.5δ) (BZN)

Clear evidence (in the form of structured diffuse scattering) is found for short range ordering of metal ions and associated induced structural relaxation in two members of the cubic BZN pyrochlore (Bi_1.5−αZn_0.5−β)(Zn_0.5−γNb_1.5−δ)O_{(7−1.5α−β−γ−2.5δ)} solid solution. An average neutron powder diffraction structure refinement is carried out for one of these. Electron probe micro-analysis suggests that the primary mechanism for non-stoichiometry in cubic BZN is the removal of ZnO from the nominally fully occupied (Bi_1.5Zn_0.5)(Zn_0.5Nb_1.5)O₇ end-member. A detailed bond valence sum analysis of a recently reported average crystal structure is used to suggest possible local cation ordering schemes along with the induced displacive relaxation that is likely to accompany such local cation ordering. The observed diffuse distribution is qualitatively interpreted via Monte Carlo modelling.     

A combined diffraction (XRD, electron and neutron) and electrical study of Na3MoO3F3

Na₃MoO₃F₃, a member of the A₂BM^VIO₃F₃ family of elpasolite-related oxyfluorides, has been prepared by the reaction of NaF with MoO₃ at 650°C. It is shown by a combined X-ray, electron and neutron diffraction study, that the true symmetry of Na₃MoO₃F₃ is not monoclinic (pseudo-orthorhombic) as previously reported but instead triclinic (metrically rhombohedral) P1. The superstructure unit cell is given by a=−a_R+b_R, b=c_R, c=(a_R+b_R+c_R), when expressed with respect to the underlying rhombohedral parent structure. Neutron diffraction refinement of the rhombohedral sub-structure shows O and F to be fully ordered with alternate (111)_R planes being occupied by O and then F. The nature of the distortion away from the high temperature parent structure is consistent with a combination of φφφ type rotations of the octahedral framework together with ppp type displacements of the octahedral cations, which is not observed in any of the closely related A₂BM^VIO₃F₃ family members. Bond valence arguments are used to give a plausible explanation for this difference in behavior.     

Isolation of sterols with cyclopropyl-containing side chains from the cultured marine alga peridiniumfoliaceum

The sterol fraction of the cultured dinoflagellate $\text{Peridinium}$$\text{foliaceum}$ contains in addition to cholesterol, 24-methylcholesterol, 4,24-dimethylcholesterol and dinosterol ($\text{3c}$) small amounts of the cyclopropyl-containing sterols gorgosterol ($\text{1a}$) and 5α-gorgostanol ($\text{1b}$) as well as 4α-methylgorgostanol ($\text{1c}$) and E-4α,22ξ,23ξ (or 24ξ)-trimethylcholest-17(20)-en-3β-ol ($\text{6c}$ or $\text{7c}$). The biosynthetic implications of the first isolation of such sterols from a free-living alga are emphasized.     

Orthorhombic superstructures within the rare earth strontium-doped cobaltate perovskites: Ln1−xSrxCoO3−δ (Ln=Y, Dy-Yb; 0.750?x?0.875)

A combination of electron, synchrotron X-ray and neutron powder diffraction reveals a new orthorhombic structure type within the Sr-doped rare earth perovskite cobaltates Ln_1−xSr_xCoO_3−δ (Ln=Y³⁺, Dy³⁺, Ho³⁺, Er³⁺, Tm³⁺and Yb³⁺). Electron diffraction shows a C-centred cell based on a 2√2a_p×4a_p×4√2a_p superstructure of the basic perovskite unit. Not all of these very weak satellite reflections are evident in the synchrotron X-ray and neutron powder diffraction data and the average structure of each member of this series could only be refined based on Cmma symmetry and a 2√2a_p×4a_p×2√2a_p cell. The nature of structural and magnetic ordering in these phases relies on both oxygen vacancy and cation distribution. A small range of solid solution exists where this orthorhombic structure type is observed, centred roughly around the compositions Ln_0.2Sr_0.8CoO_3−δ. In the case of Yb³⁺ the pure orthorhombic phase was only observed for 0.850?x?0.875. Tetragonal (I4/mmm; 2a_p×2a_p×4a_p) superstructures were observed for compositions having higher or lower Sr-doping levels, or for compounds with rare earth ions larger than Dy³⁺. These orthorhombic phases show mixed valence (3+/4+) cobalt oxidation states between 3.2+ and 3.3+. DC magnetic susceptibility measurements show an additional magnetic transition for these orthorhombic phases compared to the associated tetragonal compounds with critical temperatures > 330 K.     

Synthesis and testing of 2-deoxy-2,2-dihaloglycosides as mechanism-based inhibitors of alpha-glycosidases

The synthesis of a series of 2-deoxy-2,2-dihaloglycosyl halides as potential alpha-glycosidase inactivators has been achieved via the halogenation of protected 2-fluoroglycal precursors. Direct chlorination of per-O-acetylated 2-fluoro-d-glucal and 2-fluoromaltal followed by basic deprotection yielded the corresponding 2-chloro-2-deoxy-2-fluoroglycosyl chlorides. Reaction of the per-O-acetylated 2-fluoroglycals with acetyl hypofluorite or Selectfluor yielded the 2-deoxy-2,2-difluoroglycosyl derivatives, which were converted to their alpha-chlorides using thionyl chloride and deprotected under basic conditions. Trinitrophenyl glycosides of the 2-deoxy-2,2-difluoro mono- and disaccharides were synthesized by arylation of the hemiacetals with picryl fluoride, then deprotected with HCl in methanol. All three monosaccharide derivatives caused active site-directed, time-dependent inactivation of yeast alpha-glucosidase via the trapping of covalent glycosyl-enzyme intermediates, and kinetic parameters for inactivation by each compound were determined. Surprisingly neither of the 2-deoxy-2,2-dihalomaltosyl chlorides caused time-dependent inactivation of human pancreatic alpha-amylase, despite the fact that the trinitrophenyl 2-deoxy-2,2-difluoromaltoside functioned in that mode. The trinitrophenyl glycosides appear to be approximately 1000-fold more reactive than the corresponding chlorides in the enzyme active sites.     

Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus

Immobilized biocatalysts, including particulate enzymes, represent an attractive tool for research and industrial applications because they combine the specificity of native enzymes with the advantage that they can be readily separated from end product and reused. We demonstrated the use of the Caulobacter crescentus surface (S)-layer protein (RsaA) secretion apparatus for the generation of particulate enzymes. Specifically, a candidate protein made previously by fusion of the beta-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi with the C-terminus of RsaA was evaluated. Cex/RsaA cleaved the glycosidic linkage in the artificial substrate p-nitrophenyl-beta-D-cellobioside with a KM similar to that of native Cex (1.1 mM for Cex/RsaA vs 0.60 mM for Cex), indicating that the particulate Cex enzyme was able to bind substrate with wild-type affinity. By contrast, the kcat value was significantly reduced (0.08 s-1 for Cex/RsaA vs 15.8 s-1 for Cex), likely owing to the fact that the RsaA C-terminus induces spontaneous unstructured aggregation of the recombinant protein. Here, we demonstrated that not only can an RsaA fusion protein be cheaply produced and purified to a high yield (76 mg/L of dry wt for Cex/RsaA), but it can also be efficiently recycled. The Caulobacter S-layer secretion system therefore offers an attractive new model system for the production of particulate biocatalysts.