Various inositide phosphatases participate in the regulation of inositol polyphosphate signaling molecules. Plant phytases are phosphatases that hydrolyze phytate to less-phosphorylated myo-inositol derivatives and phosphate. The phytase from Selenomonas ruminantium shares no sequence homology with other microbial phytases. Its crystal structure revealed a phytase fold of the dual-specificity phosphatase type. The active site is located near a conserved cysteine-containing (Cys241) P loop. We also solved two other crystal forms in which an inhibitor, myo-inositol hexasulfate, is cocrystallized with the enzyme. In the "standby" and the "inhibited" crystal forms, the inhibitor is bound, respectively, in a pocket slightly away from Cys241 and at the substrate binding site where the phosphate group to be hydrolyzed is held close to the -SH group of Cys241. Our structural and mutagenesis studies allow us to visualize the way in which the P loop-containing phytase attracts and hydrolyzes the substrate (phytate) sequentially.

Mycolic acids and multimethyl-branched fatty acids are found uniquely in the cell envelope of pathogenic mycobacteria. These unusually long fatty acids are essential for the survival, virulence, and antibiotic resistance of Mycobacterium tuberculosis. Acyl-CoA carboxylases (ACCases) commit acyl-CoAs to the biosynthesis of these unique fatty acids. Unlike other organisms such as Escherichia coli or humans that have only one or two ACCases, M. tuberculosis contains six ACCase carboxyltransferase domains, AccD1-6, whose specific roles in the pathogen are not well defined. Previous studies indicate that AccD4, AccD5, and AccD6 are important for cell envelope lipid biosynthesis and that its disruption leads to pathogen death. We have determined the 2.9-Å crystal structure of AccD5, whose sequence, structure, and active site are highly conserved with respect to the carboxyltransferase domain of the Streptomyces coelicolor propionyl-CoA carboxylase. Contrary to the previous proposal that AccD4 -5 accept long-chain acyl-CoAs as their substrates, both crystal structure and kinetic assay indicate that AccD5 prefers propionyl-CoA as its substrate and produces methylmalonyl-CoA, the substrate for the biosyntheses of multimethyl-branched fatty acids such as mycocerosic, phthioceranic, hydroxyphthioceranic, mycosanoic, and mycolipenic acids. Extensive in silico screening of National Cancer Institute compounds and the University of California, Irvine, ChemDB database resulted in the identification of one inhibitor with a K i of 13.1 M. Our results pave the way toward understanding the biological roles of key ACCases that commit acyl-CoAs to the biosynthesis of cell envelope fatty acids, in addition to providing a target for structure-based development of antituberculosis therapeutics.cell wall lipid ͉ multimethyl-branched fatty acid ͉ mycolic acid ͉ tuberculosis ͉ mycocerosic acid

A simple method was developed for the direct and simultaneous determination of copper, chromium, aluminum, and manganese in urine using a multielement GFAAS (Perkin-Elmer SIMAA6000). Pd was used as the chemical modifier along with a special purge gas (5% H2 in Ar). A simple calibration curve method can be used (with 1:1 dilution). A standard reference material (Seronorm Trace Elements Urine) was used to find the optimal temperature program and to confirm the accuracy of the technique. The analyzed values were within 90-110% of the certified values. The relative standard deviations were 1.7, 1.5, 1.6, and 1.5% for these four elements and the detection limits were 0.08 microg L(-1) for Cu, 0.05 microg L(-1) for Cr, 0.06 microg L(-1) for Al, and 0.06 microg L(-1) for Mn. The recoveries of Cu, Cr, Al, and Mn from real urine samples were 100 +/- 5%, except for Cu (80%). The found values of Cu, Cr, Al, and Mn in a real urine sample were 14.3, 0.78, 18.9, and 0.06 microg L(-1), respectively. Scanning electron micrographs were used to investigate the physical form of Pd on the surface of the platform in the graphite furnace. Use of 5% H2 in Ar as the purge gas resulted in smaller and more uniformly distributed Pd particles (Pd particle diameters 0.4-0.6 microm using 5% H2 in Ar compared to 0.4-1.2 microm using pure Ar), increasing the effect of the Pd chemical modifier and promoting the efficiency of atomization.

Given activity training data from Hight-Throughput Screening (HTS) experiments, virtual HighThroughput Screening (vHTS) methods aim to predict in silico the activity of untested chemicals. We present a novel method, the Influence Relevance Voter (IRV), specifically tailored for the vHTS task. The IRV is a low-parameter neural network which refines a k-nearest neighbor classifier by non-linearly combining the influences of a chemical's neighbors in the training set. Influences are decomposed, also non-linearly, into a relevance component and a vote component.The IRV is benchmarked using the data and rules of two large, open, competitions, and its performance compared to the performance of other participating methods, as well as of an in-house Support Vector Machine (SVM) method. On these benchmark datasets, IRV achieves state-of-theart results, comparable to the SVM in one case, and significantly better than the SVM in the other, retrieving three times as many actives in the top 1% of its prediction-sorted list.The IRV presents several other important advantages over SVMs and other methods: (1) the output predictions have a probabilistic semantic; (2) the underlying inferences are interpretable; (3) the training time is very short, on the order of minutes even for very large data sets; (4) the risk of overfitting is minimal, due to the small number of free parameters; and (5) additional information can easily be incorporated into the IRV architecture. Combined with its performance, these qualities make the IRV particularly well suited for vHTS.Virtual High-Throughput Screening (vHTS) is the cost-effective, in silico complement of experimental HTS. A vHTS algorithm uses data from HTS experiments to predict the activity of new sets of compounds in silico. Although vHTS is sometimes cast as a classification task, it is more appropriately described as a ranking task, where the goal is to rank additional compounds, such that active compounds are close to the the top of the prediction-sorted list as possible. The experiments required to verify a hit are expensive, so it is critical that true actives be recognized as early as possible. Accurately ordering actives by their degree of activity, however, is not critical. The vHTS task, therefore, differs from the `ranking' task of the machine learning literature, in that the goal is not to precisely order the chemicals in relation to each other, but rather to globally rank as many actives as possible above the bulk of the inactives. Furthermore, proper vHTS training data for the ranking task, is often unavailable. E-mail: sswamida@ics.uci NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAn important algorithm proposed for vHTS is the k-Nearest Neighbor (kNN) classifier, a nonparametric method which has been shown effective in a number of other problems. 1,2 In the kNN approach, each new data point is classified by integrating information from its neighborhood in the training set in a very simple way. Specifically, a new data point is assigne...

Anionic and cationic defects are considered as one of the crucial factors that affect carrier transport property and degradation of perovskite photovoltaic materials. Herein, we demonstrate a simple passivation of hot casted perovskite film in air by a dipolar ion, 2-thiophene ethylammonium chloride (TEACl), showing enhanced power conversion efficiency (PCE) of solar cell from 15.44% to 18.84% with increased open circuit voltage (V oc) by 50 mV. The dipolar ion of TEACl can simultaneously passivate both cationic and anionic defects. Space charge limited current model, Urbach energy analysis, and photoluminescence spectroscopy were conducted and revealed that the defects passivated by TEACl reduced the defects density of perovskite films, nonradiative recombination, and electronic disorder. In addition, the device with TEACl passivation exhibited outstanding stability of power output (<0.1% decay) as compared with the device without passivation (>8% decay) from the 300 s measurement of current verse time plots (J–T plots) at 65% relative humidity and 50 °C in air. The 80% of initial PCE was maintained after 700 h storage. As compared to conventional passivation approaches which are typically carried out at complicated crystallization step of perovskite, this post-treatment process can be easily done on the crystallized perovskite film. This facile approach is upscale and compatible with conventional coating techniques such as slot-die coating, spray, etc. for high-quality perovskite film.

The crystal structure of a small, basic DNA binding protein, Sso10b2, from the thermoacidophilic archaeon Sulfolobus solfataricus was determined by the Zn multiwavelength anomalous diffraction method and refined to 1.85 Å resolution. The 89-amino-acid protein adopts a ␤␣␤␣␤␤ topology. The structure is similar to that of Sso10b1 (also called Alba) from the same organism. However, Sso10b2 contains an arginine-rich loop RDRRR motif, which may play an important role in nucleic acid binding. There are two independent Sso10b2 proteins in the asymmetric unit, and a plausible stable dimer could be deduced from the crystal structure. Topology comparison revealed that Sso10b2 is similar to several RNA-binding proteins, including IF3-C, YhhP, and DNase I. Models of the Sso10b2 dimer bound to either B-DNA or A-DNA have been constructed.

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The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologues multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities towards these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456 and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity towards acetyl-, propionyl- and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456 and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.