Double electron-electron resonance shows cytochrome P450cam undergoes a conformational change in solution upon binding substrate.

Although cytochrome P450cam from Pseudomonas putida, the archetype for all heme monooxygenases, has long been known to have a closed active site, recent reports show that the enzyme can also be crystallized in at least two clusters of open conformations. This suggests that the enzyme may undergo significant conformational changes during substrate binding and catalytic turnover. However, these conformations were observed in the crystalline state, and information is needed about the conformations that are populated in solution. In this study, double electron-electron resonance experiments were performed to observe substrate-induced changes in distance as measured by the dipolar coupling between spin labels introduced onto the surface of the enzyme on opposite sides of the substrate access channel. The double electron-electron resonance data show a decrease of 0.8 nm in the distance between spin labels placed at S48C and S190C upon binding the substrate camphor. A rotamer distribution model based on the crystal structures adequately describes the observed distance distributions. These results demonstrate conclusively that, in the physiologically relevant solution state, the substrate-free enzyme exists in the open P450cam-O conformation and that camphor binding results in conversion to the closed P450cam-C form. This approach should be useful for investigating many other P450s, including mammalian forms, in which the role of conformational change is of central importance but not well understood.

The role of genomics and biotechnology in achieving global food security for high-oleic vegetable oil.

Health related concerns for dietary 'trans-fat' in the U.S. have mediated a significant decline in the use of hydrogenated vegetable oils in edible applications. Oils having a natural abundance of oleic acid provide many functional properties that are derived from partial hydrogenation of polyunsaturated oils. However, the long term agronomic production capacity of existing high-oleic oil crops to replace hydrogenated oil ingredients is not sustainable. Although improvements are expected in processing technology, genetic modification of seed composition offers the most promising tactic to increase the overall supply of high-oleic commodity oils. Genetic enhancement of oleic acid concentration has been demonstrated experimentally in nearly every oilseed. Private companies have launched production of genetically enhanced oleic acid cultivars such as: Nexera™ Omega-9 canola and Omega-9 sunflower oils. The E. I. du Pont de Nemours and Company plans commercial production of Plenish™ high-oleic soybeans in 2012. The Monsanto Co. plans commercial production of Vistive-Gold™ low-saturated high-oleic soybeans possibly as early as 2013. These 'new' high-oleic oilseeds must not only exhibit superior oil quality but also sequentially improved yield potential. Genetic maps that help breeders identify, locate and track useful genes will facilitate accomplishment of that goal. However, a reference sequence map in soybean is the only available chromosome scale assembly of an oilseed genome. Knowledge of genome structure enables technological advances that help increase soybean yielding ability, improve crop protection against biotic stresses, and reveal alleles for genes that mediate expression of quality traits. Led by soybean, genetically enhanced high-oleic vegetable oils that now are becoming commercially available may capture greater than 40% of the domestic consumption of vegetable oil in the U.S. by 2020. This innovation in oilseed technology is a positive step toward ensuring global food security for high-oleic vegetable oils.

Three clusters of conformational states in p450cam reveal a multistep pathway for closing of the substrate access channel.

Conformational changes in the substrate access channel have been observed for several forms of cytochrome P450, but the extent of conformational plasticity exhibited by a given isozyme has not been completely characterized. Here we present crystal structures of P450cam bound to a library of 12 active site probes containing a substrate analogue tethered to a variable linker. The structures provide a unique view of the range of protein conformations accessible during substrate binding. Principal component analysis of a total of 30 structures reveals three discrete clusters of conformations: closed (P450cam-C), intermediate (P450cam-I), and fully open (P450cam-O). Relative to P450cam-C, the P450cam-I state results predominantly from a retraction of helix F, while both helices F and G move in concert to reach the fully open P450cam-O state. Both P450cam-C and P450cam-I are well-defined states, while P450cam-O shows evidence of a somewhat broader distribution of conformations and includes the open form recently seen in the absence of substrate. The observed clustering of protein conformations over a wide range of ligand variants suggests a multistep closure of the enzyme around the substrate that begins by conformational selection from an ensemble of open conformations and proceeds through a well-defined intermediate, P450cam-I, before full closure to the P450cam-C state in the presence of small substrates. This multistep pathway may have significant implications for a full understanding of substrate specificity, kinetics, and coupling of substrate binding to P450 function.

P450cam visits an open conformation in the absence of substrate.

P450cam from Pseudomonas putida is the best characterized member of the vast family of cytochrome P450s, and it has long been believed to have a more rigid and closed active site relative to other P450s. Here we report X-ray structures of P450cam crystallized in the absence of substrate and at high and low [K(+)]. The camphor-free structures are observed in a distinct open conformation characterized by a water-filled channel created by the retraction of the F and G helices, disorder of the B' helix, and loss of the K(+) binding site. Crystallization in the presence of K(+) alone does not alter the open conformation, while crystallization with camphor alone is sufficient for closure of the channel. Soaking crystals of the open conformation in excess camphor does not promote camphor binding or closure, suggesting resistance to conformational change by the crystal lattice. This open conformation is remarkably similar to that seen upon binding large tethered substrates, showing that it is not the result of a perturbation by the ligand. Redissolved crystals of the open conformation are observed as a mixture of P420 and P450 forms, which is converted to the P450 form upon addition of camphor and K(+). These data reveal that P450cam can dynamically visit an open conformation that allows access to the deeply buried active site without being induced by substrate or ligand.

Effects of elevated atmospheric CO2 on soil microbial biomass, activity, and diversity in a chaparral ecosystem.

This study reports the effects of long-term elevated atmospheric CO2 on root production and microbial activity, biomass, and diversity in a chaparral ecosystem in southern California. The free air CO2 enrichment (FACE) ring was located in a stand dominated by the woody shrub Adenostoma fasciculatum. Between 1995 and 2003, the FACE ring maintained an average daytime atmospheric CO2 concentration of 550 ppm. During the last two years of operation, observations were made on soil cores collected from the FACE ring and adjacent areas of chaparral with ambient CO2 levels. Root biomass roughly doubled in the FACE plot. Microbial biomass and activity were related to soil organic matter (OM) content, and so analysis of covariance was used to detect CO2 effects while controlling for variation across the landscape. Extracellular enzymatic activity (cellulase and amylase) and microbial biomass C (chloroform fumigation-extraction) increased more rapidly with OM in the FACE plot than in controls, but glucose substrate-induced respiration (SIR) rates did not. The metabolic quotient (field respiration over potential respiration) was significantly higher in FACE samples, possibly indicating that microbial respiration was less C limited under high CO2. The treatments also differed in the ratio of SIR to microbial biomass C, indicating a metabolic difference between the microbial communities. Bacterial diversity, described by 16S rRNA clone libraries, was unaffected by the CO2 treatment, but fungal biomass was stimulated. Furthermore, fungal biomass was correlated with cellulase and amylase activities, indicating that fungi were responsible for the stimulation of enzymatic activity in the FACE treatment.

Sicklepod (Senna obtusifolia) seed processing and potential utilization.

Sicklepod (Senna obtusifolia) is a leguminous plant that infests soybean fields in the southeastern United States. Its seeds contain a variety of toxic, highly colored compounds, mainly anthraquinones together with a small amount of fat. These compounds contaminate and lower the quality of soybean oil when inadequately cleaned soybean seed from this area is processed. The sorting of sicklepod seed from a soybean harvest is an additional economic burden on the farmer beyond the cost of proper disposal of the weed seed to avoid worsening field infestation. Fortunately, sicklepod seed also contains substantial amounts of carbohydrates and proteins. These edible components when freed from anthraquinones have a market in pet food as well as potential in human foods because of the high galactomannan ratio of the polysaccharides. Sicklepod seed was dehulled, and the ground endosperm was defatted, followed by sequential solvent extraction of the defatted seed meal to isolate the anthraquinones, carbohydrates, and protein components into their respective classes. Each class of isolate was spectroscopically identified.

A new form of crystalline rubisco and the conversion to its common dodecahedral form.

In this paper, we present a new purification procedure that yields a new crystalline form of rubisco and has enabled us to completely remove this most abundant protein from tobacco leaf extract. The crystals formed within 48 h after refrigeration at 4 degrees C at pH 5.6. However, these crystals were not well-ordered crystals and lacked well-defined facets or edges. The remaining leaf extract (fraction 2 protein) was void of rubisco. Conversion of this new crystalline form of rubisco to its common dodecahedral form was achieved by dialysing the protein solution in Tris buffer at pH 8.0 or purified water. Since the molecular size of its large subunit of rubisco (55 kD) is similar to that of the papillomavirus capsid protein, L1 (57 kD), its complete removal from fraction 2-protein may facilitate the detection, purification, and recovery of the Li protein.