Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.     

Morphological analysis of three equivocal sibling species of Deprea (Solanaceae)

Taxonomic confusion among closely related and morphologically similar Deprea species has persisted in the literature and in the identification of species. Morphological variation among three closely related, monophyletic Deprea species was studied to determine if and how they can be distinguished. Their sympatric occurrence in Venezuela afforded an opportunity to couple field study with analysis of herbarium specimens representing their entire geographic range. An analysis of 94 morphological characters resulted in five vegetative and 13 reproductive taxonomically informative traits. Canonical variates analysis clearly separated the three species using six quantitative traits. We conclude that these taxa, although quite variable and similar morphologically, are taxonomically distinct. Results of character analysis indicated that D. orinocensis is morphologically more similar to D. bitteriana than either are to D. paneroi. In D. paneroi, small, sterile anthers on fruit-bearing plants and the absence of fruits on plants possessing large, plllen-bearing anthers, suggest cryptic dioecy. Based on these data, D. granulosa is considered to be a synonym of D. orinocensis: Athenaea bitteriana, a misapplied synonym, is the correct basionym and is applicable to many specimens identified as D. granulosa. We submit a new combination, D. bitteriana (Werderm.) Sawyer & Benítez, and designate a lectotype to accommodate these findings.     

Complete assignment of 1H, 13C and 15N chemical shifts for bovine β-lactoglobulin: Secondary structure and topology of the native state is retained in a partially unfolded form

Although -lactoglobulin (-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet understood fully in terms of its three-dimensional (3D) structure. For example, despite a recent high-resolution crystal structure, it is still not clear why the two common variants of bovine -LG which differ by just two residues have different aggregation properties during milk processing. We have conducted solution-state NMR studies on a recombinant form of the A variant of -LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a dimer. Using a¹³ C,¹⁵N-labelled sample, expressed in Pichia pastoris, we have employed the standard combination of 3D heteronuclear NMR techniques to obtain near complete assignments of proton, carbon and nitrogen resonances. Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residues preceding proline within the sequence. From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary structure and topology of monomeric -LG A. It includes eight antiparallel -strands arranged in a barrel, flanked by an -helix, which is typical of a member of the lipocalin family. A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary structure and overall topology. Both forms have a ninth -strand which, at the higher pH, forms part of the dimer interface. These studies represent the first full NMR assignment of -LG and will form the basis for a complete characterisation of the solution structure and dynamics of this protein and its variants.     

A biphasic model for micro-indentation of a hydrogel-based contact lens

The stiffness and hydraulic permeability of soft contact lenses may influence its clinical performance, e.g., on-eye movement, fitting, and wettability, and may be related to the occurrence of complications; e.g., lesions. It is therefore important to determine these properties in the design of comfortable contact lenses. Micro-indentation provides a nondestructive means of measuring mechanical properties of soft, hydrated contact lenses. However, certain geometrical and material considerations must be taken into account when analyzing output force-displacement (F-D) data. Rather than solely having a solid response, mechanical behavior of hydrogel contact lenses can be described as the coupled interaction between fluid transport through pores and solid matrix deformation. In addition, indentation of thin membranes ( approximately 100 microm) requires special consideration of boundary conditions at lens surfaces and at the indenter contact region. In this study, a biphasic finite element model was developed to simulate the micro-indentation of a hydrogel contact lens. The model accounts for a curved, thin hydrogel membrane supported on an impermeable mold. A time-varying boundary condition was implemented to model the contact interface between the impermeable spherical indenter and the lens. Parametric studies varying the indentation velocities and hydraulic permeability show F-D curves have a sensitive region outside of which the force response reaches asymptotic limits governed by either the solid matrix (slow indentation velocity, large permeability) or the fluid transport (high indentation velocity, low permeability). Using these results, biphasic properties (Young's modulus and hydraulic permeability) were estimated by fitting model results to F-D curves obtained at multiple indentation velocities (1.2 and 20 microm/s). Fitting to micro-indentation tests of Etafilcon A resulted in an estimated permeability range of 1.0 x 10(-15) to 5.0 x 10(-15) m(4)N s and Young's modulus range of 130 to 170 kPa.     

Monounsaturated fatty acyl-coenzyme A is predictive of atherosclerosis in human apoB-100 transgenic, LDLr-/- mice

ACAT2, the enzyme responsible for the formation of cholesteryl esters incorporated into apolipoprotein B-containing lipoproteins by the small intestine and liver, forms predominantly cholesteryl oleate from acyl-CoA and free cholesterol. The accumulation of cholesteryl oleate in plasma lipoproteins has been found to be predictive of atherosclerosis. Accordingly, a method was developed in which fatty acyl-CoA subspecies could be extracted from mouse liver and quantified. Analyses were performed on liver tissue from mice fed one of four diets enriched with one particular type of dietary fatty acid: saturated, monounsaturated, n-3 polyunsaturated, or n-6 polyunsaturated. We found that the hepatic fatty acyl-CoA pools reflected the fatty acid composition of the diet fed. The highest percentage of fatty acyl-CoAs across all diet groups was in monoacyl-CoAs, and values were 36% and 46% for the n-3 and n-6 polyunsaturated diet groups and 55% and 62% in the saturated and monounsaturated diet groups, respectively. The percentage of hepatic acyl-CoA as oleoyl-CoA was also highly correlated to liver cholesteryl ester, plasma cholesterol, LDL molecular weight, and atherosclerosis extent. These data suggest that replacing monounsaturated with polyunsaturated fat can benefit coronary heart disease by reducing the availability of oleoyl-CoA in the substrate pool of hepatic ACAT2, thereby reducing cholesteryl oleate secretion and accumulation in plasma lipoproteins.     

25-Hydroxyvitamin D requirement for maintaining skeletal health utilising a Sprague-Dawley rat model

To study the role of vitamin D to optimise bone architecture, we have developed an animal model to investigate the effects of frank vitamin D-deficiency as well as graded depletion of circulating 25-hydroxyvitamin D(3) (25D) levels on the skeleton. Rats fed on dietary vitamin D levels from 0 to 500 ng/day achieved diet-dependent circulating levels of 25D ranging from 11 to 115 nmol/L. Levels of serum 1,25-dihydroxyvitamin D(3) (1,25D) increased as dietary vitamin D increased between 0 and 200 ng/day at which point a maximum level was achieved and retained with higher vitamin D intakes. The renal levels of 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1) mRNA were highest in animal groups fed on vitamin D between 0 and 300 ng/day. In contrast, renal 25-hydroxyvitamin D 24-hydroxylase (CYP24) mRNA levels increased as dietary vitamin D increased achieving maximum levels in animals receiving 500 ng vitamin D/day. This animal model of vitamin D depletion is suitable to provide invaluable information on the serum levels of 25D and dietary calcium intake necessary for optimal bone structure. Such information is essential for developing nutritional recommendations to reduce the incidence of osteoporotic hip fractures.     

Nowhere to run: semi-permeable barriers affect pronghorn space use

Animal movement can mediate the ecological consequences of fragmentation; however, barriers such as fences, roads, and railways are becoming a pervasive threat to wildlife. Pronghorn (Antilocapra americana) habitat in western North America has been fragmented by roads, railways, and fences. Although pronghorn are sensitive to barriers, neither the relative permeability of different barriers to crossing nor their influence on space use have been quantified. We used a large global positioning system (GPS)-collar dataset of pronghorn (n = 1,010 animal-years) in Wyoming, USA, to first quantify the likelihood that pronghorn cross each of 5 different anthropogenic barriers, including fences, county roads, railroads, state highways, and interstate highways (i.e., interstates). Next, we assessed how each barrier influenced pronghorn space use during the winter as indexed by the area occupied, and daily displacement relative to the density of barriers on an individual's winter range. The semi-permeability of the 5 barriers varied substantially, with the interstate being the most severe barrier to pronghorn movement. Pronghorn were >300 times less likely to cross interstates compared to state highways. Although pronghorn space use was rarely influenced by barriers within individual core winter ranges, pronghorn space use was constrained by barriers on the buffered periphery of individual winter ranges. Despite their different permeability to movement, the density of fences and combined interstates and railroads had similarly negative effects on pronghorn space use. Our results illustrate that the degree to which pronghorn avoid crossing barriers may scale up to affect access to habitat. Additionally, our results indicate that the effects of barriers on habitat access are not proportional to their permeability. Our results add to a growing consensus that effective management of mobile species depends on understanding how different kinds of semi-permeable barriers influence access and use of habitats.