New insights about the monomer and homodimer structures of the human AOX1.

Human aldehyde oxidase (hAOX1) is a molybdenum dependent enzyme that plays an important role in the metabolism of various compounds either endogenous or xenobiotics. Due to its promiscuity, hAOX1 plays a major role in the pharmacokinetics of many drugs and therefore has gathered a lot of attention from the scientific community and, particularly, from the pharmaceutical industry. In this work, homology modelling, molecular docking and molecular dynamics simulations were used to study the structure of the monomer and dimer of human AOX. The results with the monomer of hAOX1 allowed to shed some light on the role played by thioridazine and two malonate ions that are co-crystalized in the recent X-ray structure of hAOX1. The results show that these molecules endorse several conformational rearrangements in the binding pocket of the enzyme and these changes have an impact in the active site topology as well as in the stability of the substrate (phthalazine). The results show that the presence of both molecules open two gates located at the entrance of the binding pocket, from which results the flooding of the active site. They also endorse several modifications in the shape of the binding pocket (namely the position of Lys893) that, together with the presence of the solvent molecules, favour the release of the substrate to the solvent. Further insights were also obtained with the assembled homodimer of hAOX1. The allosteric inhibitor (THI) binds closely to the region where the dimerization of both monomers occur. These findings suggest that THI can interfere with protein dimerization.

Protocol for Computational Enzymatic Reactivity Based on Geometry Optimisation.

Enzymes play a biologically essential role in performing and controlling an important share of the chemical processes occurring in life. However, despite their critical role in nature, attaining a clear understanding of the way an enzyme acts is still cumbersome. Computational enzymology is playing an increasingly important role in this field of research. It allows the elucidation of a complete and detailed mechanism of an enzymatic reaction, including the characterization of reaction intermediates and transition states from both structural and energetic points of view, which is something that no other single experiment can produce alone. In this review, we present a general computational strategy to study enzymatic mechanisms based on adiabatic mapping and free geometry optimization. These methods allow chemical reactions to be studied with high theoretical levels, and allow a more exhaustive exploration of the chemical reactional space than other available methods, albeit being limited to the extent that they explore the enzyme conformational space. Special attention is given to the choice of the theoretical levels, as well as describing the model systems that are currently used to study enzymatic reactions. With this, we aim to provide a good introduction for non-specialised users in this field of research. We also provide a selection of hand-picked examples from our own work that illustrate the power of computational enzymology to study catalytic mechanisms. Some of these studies constitute pioneering work in the field that were later validated by experimental means.

The mechanism of the Ser-(cis)Ser-Lys catalytic triad of peptide amidases.

In this paper, we report a theoretical investigation of the catalytic mechanism of peptide amidases that involve a Ser-(cis)Ser-Lys catalytic triad. Previous suggestions propose that these enzymes should follow a distinct catalytic mechanism from the one that is present in the classic Ser-His-Asp catalytic triad. The theoretical and computational results obtained in this work indicate the opposite idea, showing that both mechanisms are very similar and only few differences are observed between both reactions. The results reveal that the different alignment of the Ser-(cis)Ser-Lys catalytic triad in relation to the classical Ser-His-Asp triad may provide a better stabilisation of the reaction intermediates, and therefore make these enzymes catalytically more efficient. The catalytic mechanism has been determined at the M06-2X/6-311++G**//B3LYP/6-31G* level of theory and requires five sequential steps instead of the two that are generally proposed: (i) nucleophilic attack of serine on the carbonyl group of the substrate, forming the first tetrahedral intermediate, (ii) formation of an acyl-enzyme complex, (ii) release of an ammonia product, (iv) nucleophilic attack of a water molecule forming the second tetrahedral intermediate, and (iv) the release of the product of the reaction, the carboxylic acid. The computational results suggest that the rate-limiting step is the first one that requires an activation free energy of 15.93 kcal mol-1. This result agrees very well with the available experimental data that predict a reaction rate of 2200 s-1, which corresponds to a free energy barrier of 14 kcal mol-1.

Cholesterol Biosynthesis: A Mechanistic Overview.

Cholesterol is an essential component of cell membranes and the precursor for the synthesis of steroid hormones and bile acids. The synthesis of this molecule occurs partially in a membranous world (especially the last steps), where the enzymes, substrates, and products involved tend to be extremely hydrophobic. The importance of cholesterol has increased in the past half-century because of its association with cardiovascular diseases, which are considered one of the leading causes of death worldwide. In light of the current need for new drugs capable of controlling the levels of cholesterol in the bloodstream, it is important to understand how cholesterol is synthesized in the organism and identify the main enzymes involved in this process. Taking this into account, this review presents a detailed description of several enzymes involved in the biosynthesis of cholesterol. In this regard, the structure and catalytic mechanism of the enzymes involved in cholesterol biosynthesis, from the initial two-carbon acetyl-CoA building block, will be reviewed and their current pharmacological importance discussed. We believe that this review may contribute to a deeper level of understanding of cholesterol metabolism and that it will serve as a useful resource for future studies of the cholesterol biosynthesis pathway.

The catalytic mechanism of carboxylesterases: a computational study.

The catalytic mechanism of carboxylesterases (CEs, EC 3.1.1.1) is explored by computational means. CEs hydrolyze ester, amide, and carbamate bonds found in xenobiotics and endobiotics. They can also perform transesterification, a reaction important, for instance, in cholesterol homeostasis. The catalytic mechanisms with three different substrates (ester, thioester, and amide) have been established at the M06-2X/6-311++G**//B3LYP/6-31G* level of theory. It was found that the reactions proceed through a mechanism involving four steps instead of two as is generally proposed: (i) nucleophilic attack of serine to the substrate, forming the first tetrahedral intermediate, (ii) formation of the acyl-enzyme complex concomitant with the release of the alcohol product, (iii) nucleophilic attack of a water or alcohol molecule forming the second tetrahedral intermediate, and (iv) the release of the second product of the reaction. The results agree very well with the available experimental data and show that the hydrolytic and the transesterification reactions are competitive processes when the substrate is an ester. In all the other studied substrates (thioester or amide), the hydrolytic and transesterification process are less favorable and some of them might not even take place under in vivo conditions.

Understanding the importance of the aromatic amino-acid residues as hot-spots.

Protein-protein interactions (PPI) are crucial for the establishment of life. However, its basic principles are still elusive and the recognition process is yet to be understood. It is important to look at the biomolecular structural space as a whole, in order to understand the principles behind conformation-function relationships. Since the application of an alanine scanning mutagenesis (ASM) study to the growth hormone it was demonstrated that only a small subset of residues at a protein-protein interface is essential for binding - the hot-spots (HS). Aromatic residues are some of the most typical HS at a protein-protein interface. To investigate the structural role of the interfacial aromatic residues in protein-protein interactions, we performed Molecular Dynamic (MD) simulations of protein-protein complexes in a water environment and calculated a variety of physical-chemical characteristics. ASM studies of single residues and of dimers or high-order clusters were performed to check for cooperativity within aromatic residues. Major differences were found between the behavior of non-HS aromatic residues and HS aromatic residues that can be used to design drugs to block the critical interactions or to predict major interactions at protein-protein complexes.

Sub-Bandgap Sensitization of Perovskite Semiconductors via Colloidal Quantum Dots Incorporation.

By taking advantage of the outstanding intrinsic optoelectronic properties of perovskite-based photovoltaic materials, together with the strong near-infrared (NIR) absorption and electronic confinement in PbS quantum dots (QDs), sub-bandgap photocurrent generation is possible, opening the way for solar cell efficiencies surpassing the classical limits. The present study shows an effective methodology for the inclusion of high densities of colloidal PbS QDs in a MAPbI3 (methylammonium lead iodide) perovskite matrix as a means to enhance the spectral window of photon absorption of the perovskite host film and allow photocurrent production below its bandgap. The QDs were introduced in the perovskite matrix in different sizes and concentrations to study the formation of quantum-confined levels within the host bandgap and the potential formation of a delocalized intermediate mini-band (IB). Pronounced sub-bandgap (in NIR) absorption was optically confirmed with the introduction of QDs in the perovskite. The consequent photocurrent generation was demonstrated via photoconductivity measurements, which indicated IB establishment in the films. Despite verifying the reduced crystallinity of the MAPbI3 matrix with a higher concentration and size of the embedded QDs, the nanostructured films showed pronounced enhancement (above 10-fold) in NIR absorption and consequent photocurrent generation at photon energies below the perovskite bandgap.

Frequency of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma species in cervical samples.

We investigated the relative frequencies of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma sp. in cervical samples. PCR analyses were performed in ectocervical and endocervical samples from 224 patients attending public health services in Belo Horizonte and Contagem, Minas Gerais Brazil. A high prevalence of colonisation of the cervix (6.3% for C. trachomatis, 4.0% for N. gonorrhoeae, 0.9% for M. genitalium, 21.9% for M. hominis, 38.4% for Ureaplasma sp.) was demonstrated not only for pathogens classically associated to cervicitis (C. trachomatis and N. gonorrhoeae), but also for M. hominis and Ureaplasma sp. These findings may be useful to guide more adequate diagnosis to interrupt transmission and to avoid negative impacts on the female reproductive tract.

MT1 and MT2 melatonin receptors play opposite roles in brain cancer progression.

Primary brain tumors remain among the deadliest of all cancers. Glioma grade IV (glioblastoma), the most common and malignant type of brain cancer, is associated with a 5-year survival rate of < 5%. Melatonin has been widely reported as an anticancer molecule, and we have recently demonstrated that the ability of gliomas to synthesize and accumulate this indolamine in the surrounding microenvironment negatively correlates with tumor malignancy. However, our understanding of the specific effects mediated through the activation of melatonin membrane receptors remains limited. Thus, here we investigated the specific roles of MT1 and MT2 in gliomas and medulloblastomas. Using the MT2 antagonist DH97, we showed that MT1 activation has a negative impact on the proliferation of human glioma and medulloblastoma cell lines, while MT2 activation has an opposite effect. Accordingly, gliomas have a decreased mRNA expression of MT1 (also known as MTNR1A) and an increased mRNA expression of MT2 (also known as MTNR1B) compared to the normal brain cortex. The MT1/MT2 expression ratio negatively correlates with the expression of cell cycle-related genes and is a positive prognostic factor in gliomas. Notably, we showed that functional selective drugs that simultaneously activate MT1 and inhibit MT2 exert robust anti-tumor effects in vitro and in vivo, downregulating the expression of cell cycle and energy metabolism genes in glioma stem-like cells. Overall, we provided the first evidence regarding the differential roles of MT1 and MT2 in brain tumor progression, highlighting their relevance as druggable targets. KEY MESSAGES: • MT1 impairs while MT2 promotes the proliferation of glioma and medulloblastoma cell lines. • Gliomas have a decreased expression of MT1 and an increased expression of MT2 compared to normal brain cortex. • Tumors with a high MT1/MT2 expression ratio have significantly better survival rates. • Functional selective drugs that simultaneously activate MT1 and inhibit MT2 downregulate the expression of cell cycle and energy metabolism genes in glioma stem-like cells and exert robust anti-tumor effects in vivo.

MADAMM: a multistaged docking with an automated molecular modeling protocol.

Dealing with receptor flexibility in docking methodology is still a problem. The main reason behind this difficulty is the large number of degrees of freedom that have to be considered in this kind of calculations. In this paper, we present an automated procedure, called MADAMM, that allows flexibilization of both the receptor and the ligand during a multistaged docking with an automated molecular modeling protocol. We show that the orientation of particular residues at the interface between the protein and the ligand have a crucial influence on the way they interact during the docking process, and the standard docking methodologies failed to predict their correct mode of binding. We present some examples that demonstrate the capabilities of this approach when compared with traditional docking methodologies.

ABAD: a potential therapeutic target for Abeta-induced mitochondrial dysfunction in Alzheimer's disease.

Amyloid-beta-peptide (Abetabinding to mitochondrial Abeta-binding alcohol dehydrogenase (ABAD) enzyme triggers a series of events leading to mitochondrial dysfunction characteristic of Alzheimer's disease (AD). Thus this interaction may represent a novel target for treatment strategy against AD. In this review we summarize current findings regarding the ABAD-Abeta interaction, namely structural and biophysical data, available inhibitors and more recent data from proteomic studies.

Prevalence and incidence of surgical site infections in the European Union/European Economic Area: how do these measures relate?

BACKGROUND: In 2011-2012, the European Centre for Disease Prevention and Control (ECDC) initiated the first European point prevalence survey (PPS) of healthcare-associated infections (HCAIs) in addition to targeted surveillance of the incidence of specific types of HCAI such as surgical site infections (SSIs). AIM: To investigate whether national and multi-country SSI incidence can be estimated from ECDC PPS data. METHODS: In all, 159 hospitals were included from 15 countries that participated in both ECDC surveillance modules, aligning surgical procedures in the incidence surveillance to corresponding specialties from the PPS. National daily prevalence of SSIs was simulated from the incidence surveillance data, the Rhame and Sudderth (R&S) formula was used to estimate national and multi-country SSI incidence from the PPS data, and national incidence per specialty was predicted using a linear model including data from the PPS. FINDINGS: The simulation of daily SSI prevalence from incidence surveillance of SSIs showed that prevalence fluctuated randomly depending on the day of measurement. The correlation between the national aggregated incidence estimated with R&S formula and observed SSI incidence was low (correlation coefficient = 0.24), but specialty-specific incidence results were more reliable, especially when the number of included patients was large (correlation coefficients ranging from 0.40 to 1.00). The linear prediction model including PPS data had low proportion of explained variance (0.40). CONCLUSION: Due to a lack of accuracy, use of PPS data to estimate SSI incidence is recommended only in situations where incidence surveillance of SSIs is not performed, and where sufficiently large samples of PPS data are available.

Drug design: new inhibitors for HIV-1 protease based on Nelfinavir as lead.

Nelfinavir (Viracept) is a potent, non-peptidic inhibitor of HIV-1 Protease, which has been marketed for the treatment of HIV infected patients. However, HIV-1 develops drug-resistance which decreases the affinity of Nelfinavir for the binding pocket of Protease. We present here three new variants of Nelfinavir, which we have designed with computational tools, with greater affinity for HIV-1 Protease than Nelfinavir itself. Accordingly, we have introduced rational modifications in Nelfinavir, optimizing its affinity to the most conserved amino acids in Protease, in order to increase the efficiency of the three new inhibitors. Minimization and molecular dynamics simulations have been carried out on four complexes, HIV-1 Protease with Nelfinavir and subsequently with the new inhibitors, respectively, in order to analyze the behavior of the systems. Additionally, we have calculated the binding free energy differences Protease:inhibitor, which gave us a quantitative idea of the new molecules inhibitory efficiency in silico.

Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.

INTRODUCTION: Amino acid depletion in the blood serum is currently being exploited and explored for therapies in tumors or viral infections that are auxotrophic for a certain amino acid or have a metabolic defect and cannot produce it. The success of these treatments is because normal cells remain unaltered since they are less demanding and/or can synthesize these compounds in sufficient amounts for their needs by other mechanisms. Areas covered: This review is focused on amino acid depriving enzymes and their formulations that have been successfully used in the treatment of several types of cancer and viral infections. Particular attention will be given to the enzymes L-asparaginase, L-arginase, L-arginine deiminase, and L-methionine-γ-lyase. Expert opinion: The immunogenicity and other toxic effects are perhaps the major limitations of these therapies, but they have been successfully decreased either through the expression of these enzymes from other organisms, recombination processes, pegylation of the selected enzymes or by specific mutations in the proteins. In 2006, FDA has already approved the use of L-asparaginase in the treatment of acute lymphoblastic leukemia. Other enzymes and in particular L-arginase, L-arginine deiminase, and L-methioninase have been showing promising results in vitro and in vivo studies.

Detailed microscopic study of the full zipA:FtsZ interface.

Protein-protein interaction networks are very important for a wide range of biological processes. Crystallographic structures and mutational studies have generated a large number of information that allowed the discovery of energetically important determinants of specificity at intermolecular protein interfaces and the understanding of the structural and energetic characteristics of the binding hot spots. In this study we have used the improved MMPB/SA (molecular mechanics/Poisson-Boltzmann surface area) approach that combining molecular mechanics and continuum solvent permits to calculate the free energy differences upon alanine mutation. For a better understanding of the binding determinants of the complex formed between the FtsZ fragment and ZipA we extended the alanine scanning mutagenesis study to all interfacial residues of this complex. As a result, we present new mutations that allowed the discovery of residues for which the binding free energy differences upon alanine mutation are higher than 2.0 kcal/mol. We also observed the formation of a hydrophobic pocket with a high warm spot spatial complementarity between FtsZ and ZipA. Small molecules could be designed to bind to these amino acid residues hindering the binding of FtsZ to ZipA. Hence, these mutational data can be used to design new drugs to control more efficiently bacterial infections.

Molecular dynamics model of unliganded HIV-1 reverse transcriptase.

HIV-1 RT is one of the most important antiviral targets in the treatment of acquired immunodeficiency syndrome (AIDS). Several crystallographic structures are available for this enzyme, mostly with bound inhibitors. Despite their importance for structure based drug design towards new anti-HIV retrovirals, the X-ray structures of the unliganded enzyme could only be obtained incomplete, with a low resolution and until recently even the conformation of the p66 thumb was controversial. In this work we have aligned different X-ray RT structures, and built up a computational model of RT using homology modeling, which was afterwards refined and validated through MD simulations with explicit solvent. The model enzyme was structurally stable through the whole MD simulation, showing a RMSD of 2 Angstrom from the starting geometry. The Ramanchandram plot has improved along the simulation. Both intra-domain and interdomain movements were observed. The thumb kept its closed conformation through the whole simulation. A contact map, hydration sites study and a detailed analysis of the solvation of the nucleotide binding site are also presented.

Including sheath effects in the interpretation of planar retarding potential analyzer's low-energy ion data.

The interpretation of planar retarding potential analyzers (RPA) during ionospheric sounding rocket missions requires modeling the thick 3D plasma sheath. This paper overviews the theory of RPAs with an emphasis placed on the impact of the sheath on current-voltage (I-V) curves. It then describes the Petite Ion Probe (PIP) which has been designed to function in this difficult regime. The data analysis procedure for this instrument is discussed in detail. Data analysis begins by modeling the sheath with the Spacecraft Plasma Interaction System (SPIS), a particle-in-cell code. Test particles are traced through the sheath and detector to determine the detector's response. A training set is constructed from these simulated curves for a support vector regression analysis which relates the properties of the I-V curve to the properties of the plasma. The first in situ use of the PIPs occurred during the MICA sounding rocket mission which launched from Poker Flat, Alaska in February of 2012. These data are presented as a case study, providing valuable cross-instrument comparisons. A heritage top-hat thermal ion electrostatic analyzer, called the HT, and a multi-needle Langmuir probe have been used to validate both the PIPs and the data analysis method. Compared to the HT, the PIP ion temperature measurements agree with a root-mean-square error of 0.023 eV. These two instruments agree on the parallel-to-B plasma flow velocity with a root-mean-square error of 130 m/s. The PIP with its field of view aligned perpendicular-to-B provided a density measurement with an 11% error compared to the multi-needle Langmuir Probe. Higher error in the other PIP's density measurement is likely due to simplifications in the SPIS model geometry.

A protein homologous to glyceraldehyde-3-phosphate dehydrogenase is induced in the cell wall of a flocculent Kluyveromyces marxianus.

A protein with an apparent molecular weight of 37,000 (p37) is present in very large amounts in the cell wall of Kluyveromyces marxianus, after the induction of flocculation of the yeast. This protein was isolated by preparative gel electrophoresis and its purity checked by SDS-PAGE and reverse-phase HPLC. SDS-PAGE, endoglycosidase-H treatment and peptide sequencing indicated that p37 is a glycoprotein with a high identity to cytosolic glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae. Polyclonal antibodies were used for Western blot analysis and immunocytochemistry, which showed that p37 is present in the cell wall of non-flocculent K. marxianus and, therefore, is a constitutive protein of the cell wall.

Kluyveromyces marxianus flocculence and growth at high temperature is dependent on the presence of the protein p37.

A Kluyveromyces marxianus mutant deficient in p37, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-like protein, was obtained and characterized with respect to flocculation behaviour, resistance to temperatures above the optimum for growth, morphology, growth, calcofluor white sensitivity and GAPDH activity. In YPD media, the mutant cells were unable to flocculate and were thermosensitive. However, this thermosensitivity could be overcome by the presence of calcium. Calcofluor white was toxic to the mutant, indicating that the mutation affects cell wall structure. The contribution of p37 to total GAPDH activity was 25% when cells were using glucose as carbon source and 50% when cells were growing in 3% ethanol. These results indicate that p37 is likely to be involved in thermotolerance and flocculation, which can be related to its contribution to cell wall integrity.

Quantitative analysis of the effect of freezing on donor sperm motion kinetics.

One hundred ninety-two semen specimens from 14 donors were analyzed on a CellSoft Semen Analyzer (CRYO Resources, New York, NY) before and after freezing. Mean post-thaw motility decreased by 52%. Correlation of the percent decrease in post-thaw versus prefreeze motility was significant but of poor predictive value. Comparisons of the percent change in post-thaw motility and sperm motion kinetics between four discrete ranges of prefreeze motility (32% to 66%, 67% to 76%, 77% to 84%, 85% to 94%) revealed that the effect of freezing on sperm cell survival was equivalent between all ranges. However, significant differences occurred between these ranges for curvilinear velocity, straight line velocity, and mean amplitude of lateral head displacement but not for linearity nor beat cross frequency. All correlations between prefreeze and post-thaw motion variables were significant but closest for curvilinear velocity and straight line velocity. Furthermore, correlations of prefreeze versus post-thaw velocity measurements were strongest for those cells within the 65% to 85% range of prefreeze motility. We suggest that sperm survival is independent of prefreeze motility. However, velocity kinetics appear stable after freezing for those specimens that had an initial motility of 65% to 85%.