The Active Site Sulfenic Acid Ligand in Nitrile Hydratases Can Function as a Nucleophile

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes; however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp2) form to a tetrahedral form (sp3). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid (BuBA) and phenylboronic acid (PBA) function as potent competitive inhibitors of PtNHase. X-ray crystal structures for BuBA and PBA complexed to PtNHase were solved and refined at 1.5, 1.6, and 1.2 Å resolution. The resulting PtNHase–boronic acid complexes represent a “snapshot” of reaction intermediates and implicate the cysteine-sulfenic acid ligand as the catalytic nucleophile, a heretofore unknown role for the αCys113–OH sulfenic acid ligand. Based on these data, a new mechanism of action for the hydration of nitriles by NHase is presented

Analyzing the Catalytic Role of Active Site Residues in the Fe-type Nitrile Hydratase from \u3cem\u3eComamonas testosteroni\u3c/em\u3e Ni1

A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (kcat = 10 ± 2 s−1) accounts for less than 1 % of the wild-type activity (kcat = 1100 ± 30 s−1) while the Km value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with kcat values of 220 ± 40 and 77 ± 13 s−1, respectively, and Km values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a kcat value of 132 ± 3 s−1 and a Km value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys104-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion

Noise Analysis and Measurement for Current Mode and Voltage Mode Active Pixel Sensor Readout Methods

A detailed experimental and theoretical investigation of noise in both current mode and voltage mode amorphous silicon (a-Si) active pixel sensors (APS) has been performed in this study. Both flicker (1/f) and thermal noise are considered. The experimental result in this study emphasizes the computation of the output noise variance, and not the output noise spectrum. This study determines which mode of operation is superior in term of output noise. The current noise power spectral density of a single a-Si TFT is also measured in order to find the suitable model for calculating the flicker noise. This experimental result matches Hooge’s model. The theoretical analysis shows that the voltage mode APS has an advantage over the current mode APS in terms of the flicker noise due to the operation of the readout process. The experimental data are compared to the theoretical analysis and are in good agreement. The results obtained in this study apply equally well to APS circuits made using polycrystalline silicon (poly-Si) and single crystal silicon

Involuntary saccades and binocular coordination during visual pursuit in Parkinson's disease

Prior studies of oculomotor function in Parkinson's disease (PD) have either focused on saccades while smooth pursuit eye movements were not involved, or tested smooth pursuit without considering the effect of any involuntary saccades. The present study investigated whether these involuntary saccades could serve as a useful biomarker for PD. Ten observers with PD participated in the study along with 10 age-matched normal control (NC) and 10 young control participants (YC). Observers fixated on a central cross while a disk (target) moved toward it from either side of the screen. Once the target reached the fixation cross, observers began to pursue the moving target until the target reached to the other side. To vary the difficulty of fixation and pursuit, the moving target was presented on a blank or a moving background. The moving background consisted of uniformly distributed dots moved in either the same or the opposite direction of the target once the target reached the central fixation cross. To investigate binocular coordination, each background condition was presented under a binocular condition, in which both eyes saw the same stimulus, and under a dichoptic condition, in which one eye saw only the target and the other eye only saw the background. The results showed that in both background conditions, observers with PD made more involuntary saccades than NC and YC during both fixation and pursuit periods while YC and NC showed no difference. Moreover, the difference between left and right eye positions increased over time during the pursuit period for PD group but not for the other two groups. This suggests that individuals with PD may be impaired not only in saccade inhibition, but also in binocular coordination during pursuit. [Meeting abstract presented at VSS 2016.]Accepted manuscrip

Parkinson Disease-Associated Mutation R1441H in LRRK2 Prolongs the “Active State” of its GTPase Domain

Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (RocR1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis

Eye movement control during visual pursuit in Parkinson's disease

BACKGROUND: Prior studies of oculomotor function in Parkinson’s disease (PD) have either focused on saccades without considering smooth pursuit, or tested smooth pursuit while excluding saccades. The present study investigated the control of saccadic eye movements during pursuit tasksand assessed the quality of binocular coordinationas potential sensitive markers of PD. METHODS: Observers fixated on a central cross while a target moved toward it. Once the target reached the fixation cross, observers began to pursue the moving target. To further investigate binocular coordination, the moving target was presented on both eyes (binocular condition), or on one eye only (dichoptic condition). RESULTS: The PD group made more saccades than age-matched normal control adults (NC) both during fixation and pursuit. The difference between left and right gaze positions increased over time during the pursuit period for PD but not for NC. The findings were not related to age, as NC and young-adult control group (YC) performed similarly on most of the eye movement measures, and were not correlated with classical measures of PD severity (e.g., Unified Parkinson’s Disease Rating Scale (UPDRS) score). DISCUSSION: Our results suggest that PD may be associated with impairment not only in saccade inhibition, but also in binocular coordination during pursuit, and these aspects of dysfunction may be useful in PD diagnosis or tracking of disease course.This work was supported in part by grants from the National Science Foundation (NSF SBE-0354378 to Arash Yazdanbakhsh and Bo Cao) and Office of Naval Research (ONR N00014-11-1-0535 to Bo Cao, Chia-Chien Wu, and Arash Yazdanbakhsh). There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. (SBE-0354378 - National Science Foundation (NSF); ONR N00014-11-1-0535 - Office of Naval Research)Published versio

Identification of a shared gene signature and biological mechanism between diabetic foot ulcers and cutaneous lupus erythemnatosus by transcriptomic analysis

Diabetic foot ulcers (DFU) and cutaneous lupus erythematosus (CLE) are both diseases that can seriously affect a patient’s quality of life and generate economic pressure in society. Symptomatically, both DLU and CLE exhibit delayed healing and excessive inflammation; however, there is little evidence to support a molecular and cellular connection between these two diseases. In this study, we investigated potential common characteristics between DFU and CLE at the molecular level to provide new insights into skin diseases and regeneration, and identify potential targets for the development of new therapies. The gene expression profiles of DFU and CLE were obtained from the Gene Expression Omnibus (GEO) database and used for analysis. A total of 41 common differentially expressed genes (DEGs), 16 upregulated genes and 25 downregulated genes, were identified between DFU and CLE. GO and KEGG analysis showed that abnormalities in epidermal cells and the activation of inflammatory factors were both involved in the occurrence and development of DFU and CLE. Protein-protein interaction network (PPI) and sub-module analysis identified enrichment in seven common key genes which is KRT16, S100A7, KRT77, OASL, S100A9, EPGN and SAMD9. Based on these seven key genes, we further identified five miRNAs(has-mir-532-5p, has-mir-324-3p,has-mir-106a-5p,has-mir-20a-5p,has-mir-93-5p) and7 transcription factors including CEBPA, CEBPB, GLI1, EP30D, JUN,SP1, NFE2L2 as potential upstream molecules. Functional immune infiltration assays showed that these genes were related to immune cells. The CIBERSORT algorithm and Pearson method were used to determine the correlations between key genes and immune cells, and reverse key gene-immune cell correlations were found between DFU and CLE. Finally, the DGIbd database demonstrated that Paquinimod and Tasquinimod could be used to target S100A9 and Ribavirin could be used to target OASL. Our findings highlight common gene expression characteristics and signaling pathways between DFU and CLE, indicating a close association between these two diseases. This provides guidance for the development of targeted therapies and mutual interactions

Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase With Atypical Selectivity

Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 106 M–1 s–1 for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) compared to those of typical AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a “kinked” α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. Implications for the catalytic mechanism of AHL lactonases are discussed

The Crystal Structure of Nitrosomonas Europaea Sucrose Synthase Reveals Critical Conformational Changes and Insights into the Sucrose Metabolism in Prokaryotes

In this paper we report the first crystal structure of a prokaryotic sucrose synthase from the non-photosynthetic bacterium Nitrosomonas europaea. The obtained structure was in an open form, whereas the only other available structure from the plant Arabidopsis thaliana was in a closed conformation. Comparative structural analysis revealed a “hinge-latch” combination, which is critical to transition between the open and closed forms of the enzyme. The N. europaea sucrose synthase shares the same fold as the GT-B family of the retaining glycosyltransferases. In addition, a triad of conserved homologous catalytic residues in the family showed to be functionally critical in the N. europaea sucrose synthase (Arg567, Lys572, Glu663). This implies that sucrose synthase shares not only a common origin with the GT-B family, but also a similar catalytic mechanism. The enzyme preferred transferring glucose from ADP-glucose rather than UDP-glucose like the eukaryotic counterparts. This predicts that these prokaryotic organisms have a different sucrose metabolic scenario from plants. Nucleotide preference determines where the glucose moiety is targeted after sucrose is degraded

Construction and verification of a novel prognostic risk model for kidney renal clear cell carcinoma based on immunity-related genes

Background: Currently, there are no useful biomarkers or prognostic risk markers for the diagnosis of kidney renal clear cell carcinoma (KIRC), although recent research has shown that both, the onset and progression of KIRC, are substantially influenced by immune-associated genes (IAGs).Objective: This work aims to create and verify the prognostic value of an immune risk score signature (IRSS) based on IAGs for KIRC using bioinformatics and public databases.Methods: Differentially expressed genes (DEGs) related to the immune systems (IAGs) in KIRC tissues were identified from The Cancer Genome Atlas (TCGA) databases. The DEGs between the tumor and normal tissues were identified using gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses. Furthermore, a prognostic IRSS model was constructed and its prognostic and predictive performance was analyzed using survival analyses and nomograms. Kidney renal papillary cell carcinoma (KIRP) sets were utilized to further validate this model.Results: Six independent immunity-related genes (PAEP, PI3, SAA2, SAA1, IL20RB, and IFI30) correlated with prognosis were identified and used to construct an IRSS model. According to the Kaplan-Meier curve, patients in the high-risk group had significantly poorer prognoses than those of patients in the low-risk group in both, the verification set (p <0.049; HR = 1.84; 95% CI = 1.02–3.32) and the training set (p < 0.001; HR = 3.12, 95% CI = 2.23–4.37). The numbers of regulatory T cells (Tregs) were significantly positively correlated with the six immunity-related genes identified, with correlation coefficients were 0.385, 0.415, 0.399, 0.451, 0.485, and 0.333, respectively (p <0.001).Conclusion: This work investigated the association between immune infiltration, immunity-related gene expression, and severity of KIRC to construct and verify a prognostic risk model for KIRC and KIRP