Allosteric inhibitor of SHP2 enhances macrophage endocytosis and bacteria elimination by increasing caveolae activation and protects against bacterial sepsis.

The uncontrolled bacterial infection-induced cytokine storm and sequential immunosuppression are commonly observed in septic patients, which indicates that the activation of phagocytic cells and the efficient and timely elimination of bacteria are crucial for combating bacterial infections. However, the role of dysregulated immune cells and their disrupted function in sepsis remains unclear. Here, we found that macrophages exhibited the impaired endocytosis capabilities in sepsis by Single-cell RNA sequencing and bulk RNA sequencing. Caveolae protein Caveolin-1 (Cav-1) of macrophages was inactivated by SHP2 rapidly during Escherichia coli (E.coli) infection. Allosteric inhibitor of SHP2 effectively maintains Cav-1 phosphorylation to enhance macrophage to endocytose and eliminate bacteria. Additionally, TLR4 endocytosis of macrophage was also enhanced upon E.coli infection by SHP099, inducing an increased and rapidly resolved inflammatory response. In vivo, pretreatment or posttreatment with inhibitor of SHP2 significantly reduced the bacterial burden in organs and mortality of mice subjected E.coli infection or CLP-induced sepsis. The cotreatment of inhibitor of SHP2 with an antibiotic conferred complete protection against mortality in mice. Our findings suggest that Cav-1-mediated endocytosis and bacterial elimination may play a critical role in the pathogenesis of sepsis, highlighting inhibitor of SHP2 as a potential therapeutic agent for sepsis.

A novel l-leucine 5-hydroxylase from Nostoc piscinale unravels unexpected sulfoxidation activity toward l-methionine.

Hydroxy amino acids are produced by Fe(II)/αKG-dependent dioxygenases and used widely as medicinal intermediates for chemical synthesis. A novel l-leucine 5-hydroxylase gene from Nostoc piscinale (NpLDO) was cloned into pET28a (+), pColdI and pQE-80 L plasmids. Using a two-step purification process (Ni-affinity chromatography and gel filtration), highly purified recombinant NpLDO was obtained. Recombinant NpLDO displayed unexpectedly high sulfoxidation activity toward l-methionine. The reaction products were analyzed by high-performance liquid chromatography. Sequence alignment analysis implied that residues of His150, His236 and Asp152 constitute the catalytic triad of NpLDO, which is completely conserved in the Fe(II)/αKG-dependent dioxygenase superfamily. Biochemical data showed that NpLDO catalyzed regio- and stereoselective hydroxylation of l-leucine and sulfoxidation of l-methionine with Fe(II) and l-ascorbic acid as cofactor, and αKG as cosubstrate, respectively.

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates.

BACKGROUND: Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor. RESULTS: Recombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively). CONCLUSIONS: The successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.

Biodegradation of vulcanized rubber by a gut bacterium from plastic-eating mealworms.

The disposal of vulcanized rubber waste is difficult due to the presence of three-dimensional crosslinking network structure. Here, we report that a bacterium Acinetobacter sp. BIT-H3, isolated from the gut of plastic-eating mealworm, can grow on and degrade vulcanized poly(cis-1,4-isoprene) rubber (vPR). Scanning electronic microscopy (SEM) shows that strain BIT-H3 can penetrate into the vPR and produce craters and cracks. The tensile strength and the crosslink density of vPR decreased by 53.2% and 29.3% after ten weeks' incubation, respectively. The results of Horikx analysis, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, and X-ray absorption near-edge structure (XANES) spectroscopy reveal that strain BIT-H3 can break down both sulfide bridges and double bonds of polymeric backbone within vPR. Sulfate and oligo(cis-1,4 isoprene) with terminal aldehyde and keto groups were identified as metabolic products released during vPR degradation. Through genomic and transcriptional analyses, five enzymes of dszA, dszC1, dszC2, Laccase2147, and Peroxidase1232 were found to be responsible for vPR degradation. Based on the chemical structure characterizations and molecular analyses, a vPR biodegradation pathway was proposed for strain BIT-H3. These findings pave a way for exploiting vulcanized rubber-degrading microorganisms from insect gut and contribute to establish a biodegradation method for vulcanized rubber waste disposal.

Engineering a thermostable version of D-allulose 3-epimerase from Rhodopirellula baltica via site-directed mutagenesis based on B-factors analysis.

D-allulose has received increasing attention due to its excellent physiological properties and commercial potential. The D-allulose 3-epimerase from Rhodopirellula baltica (RbDAEase) catalyzes the conversion of D-fructose to D-allulose. However, its poor thermostability has hampered its industrial application. Site-directed mutagenesis based on homologous structures in which the residuals on high flexible regions were substituted according to B-factors analysis, is an effective way to improve the thermostability and robustness of an enzyme. RbDAEase showed substrate specificity toward D-allulose with a Km of 58.57 mM and kcat of 1849.43 min-1. It showed a melting temperature (Tm) of 45.7 °C and half-life (t1/2) of 52.3 min at pH 8.0, 60 °C with 1 mM Mn2+. The Site-directed mutation L144 F strengthened the thermostability to a Δt1/2 of 50.4 min, ΔTm of 12.6 °C, and ΔT5060 of 22 °C. It also improved the conversion rate to 28.6%. Structural analysis reveals that a new hydrophobic interaction was formed by the mutation. Thus, site-directed mutagenesis based on B-factors analysis would be an efficient strategy to enhance the thermostability of designed ketose 3-epimerases.

Soluble expression, purification and biochemical characterization of a C-7 cholesterol dehydrogenase from Drosophila melanogaster.

The C-7 cholesterol dehydrogenase (NVD), which converts cholesterol to 7-dehydrocholesterol (7-DHC) by 7,8-dehydrogenation, plays a pivotal role in the metabolism of cholesterol and steroid intermediates. The NVD protein was successfully expressed in insect Sf9 cells. To reduce the production cost for industrial application, the NVD gene was cloned into E. coli BL21(DE3). However, the His-tagged NVD protein showed poor binding to Ni-NTA resin, mainly due to the formation of inclusion bodies. Consequently, the expression and solubility of NVD were optimized by respectively fusing it with maltose-binding protein (MBP), glutathione S-transferase (GST), and the nonspecific DNA binding protein from Sulfolobus solfataricus (Sso7d) as solubility tags. The NVD fusion with MBP at the N-terminus and His-tag at the C-terminus achieved a high yield of the soluble enzyme. It was further purified by ion-exchange chromatography with 95.4% purity and with a 10.4% yield. The product 7-DHC, which is produced in a reaction catalyzed by NVD and ferredoxin reductase KshB, was initially identified by GC-MS. An analysis of the amino acid sequence alignment revealed a distinct Rieske-type iron-sulfur cluster and non-heme Fe2+-binding domain, which are evolutionarily conserved among NVD family enzymes.

Efficient Biosynthesis of High-Value Succinic Acid and 5-Hydroxyleucine Using a Multienzyme Cascade and Whole-Cell Catalysis.

Succinic acid (SA) is applied in the food, chemical, and pharmaceutical industries. 5-Hydroxyleucine (5-HLeu) is a promising precursor for the biosynthesis of antituberculosis drugs. Here, we designed a promising synthetic route for the simultaneous production of SA and 5-HLeu by combining l-leucine dioxygenase (NpLDO), l-glutamate oxidase (LGOX), and catalase (CAT). Two bioconversion systems: "a multienzyme cascade catalysis in vitro" (MECCS) and "whole-cell catalysis system" (WCCS) were constructed. A high-activity NpLDO mutant was screened by error-prone polymerase chain reaction (PCR) and showed 6.1-fold improvement of catalytic activity. After optimization of reaction conditions, MECSS yielded 3.15 g/L SA and 3.92 g/L 5-HLeu, while the production of SA and 5-HLeu by the most effective WCSS reached 15.12 and 18.83 g/L, respectively. This is the first attempt to use ferrous iron/α-ketoglutarate-dependent dioxygenases for the simultaneous production of SA and hydroxy-amino-acid. This research provides a tool for industrial production of food of high-value products from low-cost raw materials.

Biochemical characterization of a novel ulvan lyase from Pseudoalteromonas sp. strain PLSV.

Ulvans, complex polysaccharides found in the ulvales (green seaweed) cell wall, contain predominantly 3-sulfated rhamnose (Rha3S) linked to either d-glucuronic acid, l-iduronic acid or d-xylose. The ulvan lyase endolytically cleaves the glycoside bond between Rha3S and uronic acid via a ß-elimination mechanism. Ulvan lyase has been identified as belonging to the polysaccharide lyase family PL24 or PL25 in the carbohydrate active enzymes database, in which fewer members have been characterized. We present the cloning and characterization of a novel ulvan lyase from Pseudoalteromonas sp. strain PLSV (PsPL). The enzymes were heterologously expressed in Escherichia coli BL21 (DE3) and purified as the His-tag fusion protein using affinity chromatography, ion-exchange chromatography and size-exclusion chromatography. The degradation products were determined by thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS) to be mainly disaccharides and tetrasaccharides. Ulvan lyase provides an example of degrading ulvales into oligosaccharides. Arg265, His152 and Tyr249 were considered to serve as catalytic residues based on PsPL structural model analysis.

[Advances in hydroxylation of hydrophobic amino acid].

Hydroxy amino acids, constituents of chiral pharmaceutical intermediates or precursors, have a variety of unique functions in the research fields of biotechnology and molecular biology, i.e. antifungal, antibacterial, antiviral and anticancer properties. Biosynthesis of hydroxy amino acids is preferred because of its high specificity and selectivity. The hydroxylation of hydrophobic amino acids is catalyzed by hydroxylase, which belongs to the mononuclear non-heme Fe(Ⅱ)/α-ketoglutarate-dependent dioxygenases (Fe/αKGDs). Fe/αKGDs utilize an (Fe(Ⅳ)=O) intermediate to activate diverse oxidative transformations with key biological roles in the process of catalytic reaction. Here, we review the physiological properties and synthesis of hydroxy amino acids, especially for the 4-HIL and hydroxyproline. The catalytic mechanism of Fe/αKGDs is elucidated, and the applications of hydroxy amino acids in industrial engineering are also discussed.

A Framework for Flood Risk Analysis and Benefit Assessment of Flood Control Measures in Urban Areas.

Flood risk analysis is more complex in urban areas than that in rural areas because of their closely packed buildings, different kinds of land uses, and large number of flood control works and drainage systems. The purpose of this paper is to propose a practical framework for flood risk analysis and benefit assessment of flood control measures in urban areas. Based on the concept of disaster risk triangle (hazard, vulnerability and exposure), a comprehensive analysis method and a general procedure were proposed for urban flood risk analysis. Urban Flood Simulation Model (UFSM) and Urban Flood Damage Assessment Model (UFDAM) were integrated to estimate the flood risk in the Pudong flood protection area (Shanghai, China). S-shaped functions were adopted to represent flood return period and damage (R-D) curves. The study results show that flood control works could significantly reduce the flood risk within the 66-year flood return period and the flood risk was reduced by 15.59%. However, the flood risk was only reduced by 7.06% when the flood return period exceeded 66-years. Hence, it is difficult to meet the increasing demands for flood control solely relying on structural measures. The R-D function is suitable to describe the changes of flood control capacity. This frame work can assess the flood risk reduction due to flood control measures, and provide crucial information for strategy development and planning adaptation.