Structures of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with substrates and products: atomic resolution insights into mechanisms of dihydroorotate oxidation and fumarate reduction

Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-reactions. These ligands bind to the same active site of TcDHOD, which is consistent with the one-site ping-pong Bi-Bi mechanism demonstrated by kinetic studies for family 1A DHODs. The binding of ligands to TcDHOD does not cause any significant structural changes to TcDHOD, and both reduced and oxidized FMN cofactors are in planar conformation, which indicates that the reduction of the FMN cofactor with dihydroorotate produces anionic reduced FMN. Therefore, they should be good models for the enzymatic reaction pathway of TcDHOD, although orotate and fumarate bind to TcDHOD with the oxidized FMN and dihydroorotate with the reduced FMN in the structures determined here. Cys130, which was identified as the active site base for family 1A DHOD (Fagan, R. L., Jensen, K. F., Bjornberg, O., and Palfey, B. A. (2007) Biochemistry 46, 4028-4036.), is well located for abstracting a proton from dihydroorotate C5 and transferring it to outside water molecules. The bound fumarate is in a twisted conformation, which induces partial charge separation represented as C 2 (delta-) and C 3 (delta+). Because of this partial charge separation, the thermodynamically favorable reduction of fumarate with reduced FMN seems to proceed in the way that C 2 (delta-) accepts a proton from Cys130 and C 3 (delta+) a hydride (or a hydride equivalent) from reduced FMN N 5 in TcDHOD.     

Novel tracking function of moving target using chaotic dynamics in a recurrent neural network model

Chaotic dynamics introduced in a recurrent neural network model is applied to controlling an object to track a moving target in two-dimensional space, which is set as an ill-posed problem. The motion increments of the object are determined by a group of motion functions calculated in real time with firing states of the neurons in the network. Several cyclic memory attractors that correspond to several simple motions of the object in two-dimensional space are embedded. Chaotic dynamics introduced in the network causes corresponding complex motions of the object in two-dimensional space. Adaptively real-time switching of control parameter results in constrained chaos (chaotic itinerancy) in the state space of the network and enables the object to track a moving target along a certain trajectory successfully. The performance of tracking is evaluated by calculating the success rate over 100 trials with respect to nine kinds of trajectories along which the target moves respectively. Computer experiments show that chaotic dynamics is useful to track a moving target. To understand the relations between these cases and chaotic dynamics, dynamical structure of chaotic dynamics is investigated from dynamical viewpoint.     

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium.

Abbreviations: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion     

A pulsed field gradient NMR study of the aggregation and hydration of parvalbumin

Pulsed field gradient NMR is a convenient alternative to traditional methods for measuring diffusion of biological macromolecules. In the present study, pulsed field gradient NMR was used to study the effects of calcium binding and hydration on carp parvalbumin. Carp parvalbumin is known to undergo large changes in tertiary structure with calcium loading. The diffusion coefficient is a sensitive guide to changes in molecular shape and in the present study the large changes in tertiary structure were clearly reflected in the measured diffusion coefficient upon calcium loading. The (monomeric) calcium-loaded form had a diffusion coefficient of 1.4 x 10(-10) m(2) s(-1) at 298 K, which conforms with the structure being a nearly spherical prolate ellipsoid from X-ray studies. The calcium-free form had a significantly lower diffusion coefficient of 1.1 x 10(-10) m(2) s(-1). The simplest explanation consistent with the change in diffusion coefficient is that the parvalbumin molecules form dimers upon the removal of Ca(2+) at the protein concentration studied (1 mM).     

The effect of salt or various cryoprotective agents on frog sciatic nerves

Desheathed frog sciatic nerves were tested for membrane damage by noting changes in their conduction velocity, externally recorded action potential, and absolute refractory period. Nerves were exposed for 45–60 min depending on the treatment to 1.5 osmolar environments consisting of concentrated Ringer's solution or Ringer's solution plus glycerol, sucrose, or dimethyl sulfoxide (DMSO). The nerves were exposed to these hypertonic solutions in one of four ways: (1) Gradual exposure plus gradual de-exposure, (2) Gradual exposure (plus direct de-exposure), (3) (direct exposure plus) plus gradual de-exposure, (4) Direct exposure (plus direct de-exposure). After treatment the nerves were allowed to recover for 45–60 min depending on the type of treatment in normal Ringer's solution. The results suggest the following: (1) Concentrated salt solutions damage nerve axons due to increased ion concentration rather than increased tonicity. (2) The effect of osmotic shock on sciatic nerves is relatively minor and completely reversible. (3) Axon membranes behave as simple permeability barriers with respect to sucrose and DMSO, but they appear to behave as dynamic entities which alter their metastable states with respect to concentrated salt or concentrated glycerol in Ringer's solution. (4) Freeze-thaw damage to sciatic nerve axon membranes—and perhaps to any cell membrane—is probably due to increased ionic concentration as well as increased osmolarity.     

Landscape‐scale lidar analysis of aboveground biomass distribution in secondary Brazilian Atlantic Forest

Secondary forests account for more than half of tropical forests and represent a growing carbon sink, but rates of biomass accumulation vary by a factor of two or more even among plots in the same landscape. To better understand the drivers of this variability, we used airborne lidar to measure forest canopy height and estimate biomass over 4529 ha at Serra do Conduru Park in Southern Bahia, Brazil. We measured trees in 30 georeferenced field plots (0.25‐ha each) to estimate biomass using allometry. Then we estimated aboveground biomass density (ABD) across the lidar study area using a statistical model developed from our field plots. This model related the 95th percentile of the distribution of lidar return heights to ABD. We overlaid this map of ABD on a Landsat‐derived forest age map to determine rates of biomass accumulation. We found rapid initial biomass regeneration (~6 Mg/ha yr), which slowed as forests aged. We also observed high variability in both height and biomass across the landscape within forests of similar age. Nevertheless, a regression model that accounted for spatial autocorrelation and included forest age, slope, and distance to roads or open areas explained 62 and 77 percent of the landscape variation in ABD and canopy height, respectively. Thus, while there is high spatial heterogeneity in forest recovery, and the drivers of this heterogeneity warrant further investigation, we suggest that a relatively simple set of predictor variables is sufficient to explain the majority of variance in both height and ABD in this landscape.