Nitrate- and Nitrite-Sensing Histidine Kinases: Function, Structure, and Natural Diversity

Under anaerobic conditions, bacteria may utilize nitrates and nitrites as electron acceptors. Sensitivity to nitrous compounds is achieved via several mechanisms, some of which rely on sensor histidine kinases (HKs). The best studied nitrate- and nitrite-sensing HKs (NSHKs) are NarQ and NarX from Escherichia coli. Here, we review the function of NSHKs, analyze their natural diversity, and describe the available structural information. In particular, we show that around 6000 different NSHK sequences forming several distinct clusters may now be found in genomic databases, comprising mostly the genes from Beta- and Gammaproteobacteria as well as from Bacteroidetes and Chloroflexi, including those from anaerobic ammonia oxidation (annamox) communities. We show that the architecture of NSHKs is mostly conserved, although proteins from Bacteroidetes lack the HAMP and GAF-like domains yet sometimes have PAS. We reconcile the variation of NSHK sequences with atomistic models and pinpoint the structural elements important for signal transduction from the sensor domain to the catalytic module over the transmembrane and cytoplasmic regions spanning more than 200 Å.

Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

Post-translational modification of histone H4 by the small ubiquitin-like modifier (SUMO) protein was associated with gene repression. However, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using H4 chemically modified at Lys12 by SUMO-3 (H4K12su) that was incorporated into mononucleosomes and chromatinized plasmids. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed H4K12su inhibits RNA polymerase II-mediated transcription and H4 tail acetylation. Thus, we uncovered negative crosstalk with acetylation/methylation and the direct inhibition of RNAPII-mediated transcription by H4K12su.

Expression, Characterization and Structure Analysis of a New GH26 Endo-β-1, 4-Mannanase (Man26E) from Enterobacter aerogenes B19

β-mannanase is one of the key enzymes to hydrolyze hemicellulose. At present, most β-mannanases are not widely applied because of their low enzyme activity and unsuitable enzymatic properties. In this work, a new β-mannanase from Enterobacter aerogenes was studied, which laid the foundation for its further application. Additionally, we will further perform directed evolution of the enzyme to increase its activity, improve its temperature and pH properties to allow it more applications in industry. A new β-mannanase (Man26E) from Enterobacter aerogenes was successfully expressed in Escherichia coli. Man26E showed about 40 kDa on SDS-PAGE gel. The SWISS-MODEL program was used to model the tertiary structure of Man26E, which presented a core (α/β)8-barrel catalytic module. Based on the binding pattern of CjMan26 C, Man26E docking Gal1Man4 was investigated. The catalytic region consisted of a surface containing four solvent-exposed aromatic rings, many hydrophilic and charged residues. Man26E displayed the highest activity at pH 6.0 and 55 °C, and high acid and alkali stability in a wide pH range (pH 4–10) and thermostability from 40 to 50 °C. The enzyme showed the highest activity on locust bean gum, and the Km and Vmax were 7.16 mg mL−1 and 508 U mg−1, respectively. This is the second β-mannanase reported from Enterobacter aerogenes B19. The β-mannanase displayed high enzyme activity, a relatively high catalytic temperature and a broad range of catalytic pH values. The enzyme catalyzed both polysaccharides and manno-oligosaccharides.

Gαs directly drives PDZ-RhoGEF signaling to Cdc42

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13. Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227L's ability to guide PRG's interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

A systematic analysis of the parameters of disk-type membrane-catalytic devices for producing high-purity hydrogen from methane and diesel fuel

Mathematical simulation is used to analyze systematically the results of testing an individual disk-type membrane-catalytic module for producing high-purity hydrogen from methane, with a capacity of about 0.3 m3H2/h, and the design data of a membrane-catalytic reactor based on 32 individual disk-type modules for producing high-purity hydrogen from diesel fuel, with a capacity of 7.45 m3H2/h. The used mathematical model adequately and on a good quantitative level describes the experimental and design data known from the literature. In terms of the used model representations, possible ways of increasing both the capacity of disk-type membrane-catalytic devices and the efficiency of extracting high-purity hydrogen from the original hydrocarbon material are considered.

A novel β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation activity and its use in generating long-chain N-acetyl chitooligosaccharides

Background: N-acetyl glucosamine (GlcNAc) and N-acetyl chitooligosaccharides (N-acetyl COSs) exhibit antitumor and antimicrobial activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Thus, it is crucial to discover a NAGase that can both synthesize GlcNAc and N-acetyl COSs. Results: The gene encoding the novel β-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity with the corresponding domain of the well-characterized NAGases. CmNAGase gene was highly expressed with soluble form in Escherichia coli BL21 (DE3) cells, whereupon it had a specific activity of 4,878.6 U/mg of protein toward p-nitrophenyl-N-acetyl glucosaminide. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The Vmax, Km, and Kcat of CmNAGase were 833.33 μmol·L-1 ·min-1, 10.9 mmol, and 6.37 ´ 108 mM·mg-1, respectively. Analysis of the hydrolysis products of N-acetyl chitooligosaccharides and colloidal chitin revealed that CmNAGase exhibits exo-acting activities. Particularly, it possesses transglycosylation activity, which can synthesize (GlcNAc)n+1 from (GlcNAc)n (n=1−6), respectively. In addition, CmNAGase also can catalyze GlcNAc to its dimers with various linked forms. Conclusions: The observations recorded in this study that CmNAGase is an exo NAGase with unique transglycosylation activity, suggests a possible application in the production of long-chain N-acetyl CHOs. This is first report of a bacterial NAGase, which can produce long-chain N-acetyl COSs via transglycosylation activity.

The guide sRNA sequence determines the activity level of box C/D RNPs

2’-O-rRNA methylation, which is essential in eukaryotes and archaea, is catalysed by the Box C/D RNP complex in an RNA-guided manner. Despite the conservation of the methylation sites, the abundance of site-specific modifications shows variability across species and tissues, suggesting that rRNA methylation may provide a means of controlling gene expression. As all Box C/D RNPs are thought to adopt a similar structure, it remains unclear how the methylation efficiency is regulated. Here, we provide the first structural evidence that, in the context of the Box C/D RNP, the affinity of the catalytic module fibrillarin for the substrate–guide helix is dependent on the RNA sequence outside the methylation site, thus providing a mechanism by which both the substrate and guide RNA sequences determine the degree of methylation. To reach this result, we develop an iterative structure-calculation protocol that exploits the power of integrative structural biology to characterize conformational ensembles.