Selectivity of Ni(II) and Zn(II) binding to Sporosarcina pasteurii UreE, a metallochaperone in the urease assembly: a calorimetric and crystallographic study

Urease is a nickel-dependent enzyme that plays a critical role in the biogeochemical nitrogen cycle by catalyzing the hydrolysis of urea to ammonia and carbamate. This enzyme, initially synthesized in the apo form, needs to be activated by incorporation of two nickel ions into the active site, a process driven by the dimeric metallochaperone UreE. Previous studies reported that this protein can bind different metal ions in vitro, beside the cognate Ni(II). This study explores the metal selectivity and affinity of UreE from Sporosarcina pasteurii (Sp, formerly known as Bacillus pasteurii) for cognate [Ni(II)] and noncognate [Zn(II)] metal ions. In particular, the thermodynamic parameters of SpUreE Ni(II) and Zn(II) binding have been determined using isothermal titration calorimetry. These experiments show that two Ni(II) ions bind to the protein dimer with positive cooperativity. The high-affinity site involves the conserved solvent-exposed His¹⁰⁰ and the C-terminal His¹⁴⁵, whereas the low-affinity site comprises also the C-terminal His¹⁴⁷. Zn(II) binding to the protein, occurring in the same protein regions and with similar affinity as compared to Ni(II), causes metal-driven dimerization of the protein dimer. The crystal structure of the protein obtained in the presence of equimolar amounts of both metal ions indicates that the high-affinity metal binding site binds Ni(II) preferentially over Zn(II). The ability of the protein to select Ni(II) over Zn(II) was confirmed by competition experiments in solution as well as by analysis of X-ray anomalous dispersion data. Overall, the thermodynamics and structural parameters that modulate the metal ion specificity of the different binding sites on the protein surface of SpUreE have been established.     

The liver fatty acid binding protein - comparison of cavity properties of intracellular lipid-binding proteins

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common -barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited portal region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.     

X-ray crystallographic and kinetic correlation of a clinically observed human fumarase mutation

Fumarase catalyzes the reversible conversion of fumarate to S- malate during the operation of the ubiquitous Kreb's cycle. Previous studies have shown that the active site includes side chains from three of the four subunits within the tetrameric enzyme. We used a clinically observed human mutation to narrow our search for potential catalytic groups within the fumarase active site. Offspring homozygous for the missense mutation, a G-955-C transversion in the fumarase gene, results in the substitution of a glutamine at amino acid 319 for the normal glutamic acid. To more fully understand the implications of this mutation, a single-step site-directed mutagenesis method was used to generate the homologous substitution at position 315 within fumarase C from Escherichia coli. Subsequent kinetic and X-ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate.     

Exploring routes to stabilize a cationic pyridoxamine in an artificial transaminase: site-directed mutagenesis versus synthetic cofactors

Two artificial transaminases were assembled by linking a pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between alpha-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The k(cat) values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group.     

Sequestration of the active site by interdomain shifting. Crystallographic and spectroscopic evidence for distinct conformations of L-3-hydroxyacyl-CoA dehydrogenase

l-3-Hydroxyacyl-CoA dehydrogenase reversibly catalyzes the conversion of l-3-hydroxyacyl-CoA to 3-ketoacyl-CoA concomitant with the reduction of NAD(+) to NADH as part of the beta-oxidation spiral. In this report, crystal structures have been solved for the apoenzyme, binary complexes of the enzyme with reduced cofactor or 3-hydroxybutyryl-CoA substrate, and an abortive ternary complex of the enzyme with NAD(+) and acetoacetyl-CoA. The models illustrate positioning of cofactor and substrate within the active site of the enzyme. Comparison of these structures with the previous model of the enzyme-NAD(+) complex reveals that although significant shifting of the NAD(+)-binding domain relative to the C-terminal domain occurs in the ternary and substrate-bound complexes, there are few differences between the apoenzyme and cofactor-bound complexes. Analysis of these models clarifies the role of key amino acids implicated in catalysis and highlights additional critical residues. Furthermore, a novel charge transfer complex has been identified in the course of abortive ternary complex formation, and its characterization provides additional insight into aspects of the catalytic mechanism of l-3-hydroxyacyl-CoA dehydrogenase.     

The synthesis of mycosporine-like amino acids (MAAs) by cultured, symbiotic dinoflagellates

We tested the hypothesis that there is a relation between phylotypes (phylogenetic types, as determined by restriction fragment length polymorphism (RFLP) and partial sequence analysis of the small subunit ribosomal RNA gene (SSUrDNA)) and the synthesis of mycosporine-like amino acids (MAAs) by symbiotic dinoflagellates under the influence of ultraviolet radiation (UV-B/A) and photosynthetically active radiation (PAR). We exposed 27 isolates of symbiotic dinoflagellates simultaneously to UV-B/A and PAR, and subsequently determined the MAAs present in cell extracts and in the media. The algae used included 24 isolates of Symbiodinium spp. originating from jellyfishes, sea anemones, zoanthids, scleractinians, octocorals, and bivalves, and three others in the genera Gymnodinium, Gloeodinium and Amphidinium from a jellyfish, an hydrocoral and a flatworm, respectively. In this study, all of the phylotype A Symbiodinium spp. synthesized up to three identified MAAs. None of the 11 cultured phylotypes B and C Symbiodinium spp. synthesized MAAs. The three non-Symbiodinium symbionts also synthesized up to three MAAs. The results support a conclusion that phylotype A Symbiodinium spp. have a high predilection for the synthesis of MAAs, while phylotypes B and C do not. Synthesis of MAAs by symbiotic dinoflagellates in culture does not appear to relate directly to depths or to the UV exposure regimes from which the consortia were collected.     

Modeling <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> metabolism: from genome to metabolic fluxes

Background  

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years.     

Simulation of human atherosclerotic femoral plaque tissue: the influence of plaque material model on numerical results

Background

Due to the limited number of experimental studies that mechanically characterise human atherosclerotic plaque tissue from the femoral arteries, a recent trend has emerged in current literature whereby one set of material data based on aortic plaque tissue is employed to numerically represent diseased femoral artery tissue. This study aims to generate novel vessel-appropriate material models for femoral plaque tissue and assess the influence of using material models based on experimental data generated from aortic plaque testing to represent diseased femoral arterial tissue.

Methods

Novel material models based on experimental data generated from testing of atherosclerotic femoral artery tissue are developed and a computational analysis of the revascularisation of a quarter model idealised diseased femoral artery from a 90% diameter stenosis to a 10% diameter stenosis is performed using these novel material models. The simulation is also performed using material models based on experimental data obtained from aortic plaque testing in order to examine the effect of employing vessel appropriate material models versus those currently employed in literature to represent femoral plaque tissue.

Results

Simulations that employ material models based on atherosclerotic aortic tissue exhibit much higher maximum principal stresses within the plaque than simulations that employ material models based on atherosclerotic femoral tissue. Specifically, employing a material model based on calcified aortic tissue, instead of one based on heavily calcified femoral tissue, to represent diseased femoral arterial vessels results in a 487 fold increase in maximum principal stress within the plaque at a depth of 0.8 mm from the lumen.

Conclusions

Large differences are induced on numerical results as a consequence of employing material models based on aortic plaque, in place of material models based on femoral plaque, to represent a diseased femoral vessel. Due to these large discrepancies, future studies should seek to employ vessel-appropriate material models to simulate the response of diseased femoral tissue in order to obtain the most accurate numerical results.

     

Review and classification of variability analysis techniques with clinical applications

Background

Representation of independent biophysical sources using Fourier analysis can be inefficient because the basis is sinusoidal and general. When complex fractionated atrial electrograms (CFAE) are acquired during atrial fibrillation (AF), the electrogram morphology depends on the mix of distinct nonsinusoidal generators. Identification of these generators using efficient methods of representation and comparison would be useful for targeting catheter ablation sites to prevent arrhythmia reinduction.

Method

A data-driven basis and transform is described which utilizes the ensemble average of signal segments to identify and distinguish CFAE morphologic components and frequencies. Calculation of the dominant frequency (DF) of actual CFAE, and identification of simulated independent generator frequencies and morphologies embedded in CFAE, is done using a total of 216 recordings from 10 paroxysmal and 10 persistent AF patients. The transform is tested versus Fourier analysis to detect spectral components in the presence of phase noise and interference. Correspondence is shown between ensemble basis vectors of highest power and corresponding synthetic drivers embedded in CFAE.

Results

The ensemble basis is orthogonal, and efficient for representation of CFAE components as compared with Fourier analysis (p ≤ 0.002). When three synthetic drivers with additive phase noise and interference were decomposed, the top three peaks in the ensemble power spectrum corresponded to the driver frequencies more closely as compared with top Fourier power spectrum peaks (p ≤ 0.005). The synthesized drivers with phase noise and interference were extractable from their corresponding ensemble basis with a mean error of less than 10%.

Conclusions

The new transform is able to efficiently identify CFAE features using DF calculation and by discerning morphologic differences. Unlike the Fourier transform method, it does not distort CFAE signals prior to analysis, and is relatively robust to jitter in periodic events. Thus the ensemble method can provide a useful alternative for quantitative characterization of CFAE during clinical study.