Effects of elevated levels of intracellular nitric oxide on Pseudomonas aeruginosa biofilm in chronic skin wound and slow-killing infection models.

Nitric oxide (NO), produced through the denitrification pathway, regulates biofilm dynamics through the quorum sensing system in Pseudomonas aeruginosa. NO stimulates P. aeruginosa biofilm dispersal by enhancing phosphodiesterase activity to decrease cyclic di-GMP levels. In a chronic skin wound model containing a mature biofilm, the gene expression of nirS, encoding nitrite reductase to produce NO, was low, leading to reduced intracellular NO levels. Although low-dose NO induces biofilm dispersion, it is unknown whether it influences the formation of P. aeruginosa biofilms in chronic skin wounds. In this study, a P. aeruginosa PAO1 strain with overexpressed nirS was established to investigate NO effects on P. aeruginosa biofilm formation in an ex vivo chronic skin wound model and unravel the underlying molecular mechanisms. Elevated intracellular NO levels altered the biofilm structure in the wound model by inhibiting the expression of quorum sensing-related genes, which was different from an in vitro model. In Caenorhabditis elegans as a slow-killing infection model, elevated intracellular NO levels increased worms' lifespan by 18%. Worms that fed on the nirS-overexpressed PAO1 strain for 4 h had complete tissue, whereas worms that fed on empty plasmid-containing PAO1 had biofilms on their body, causing severe damage to the head and tail. Thus, elevated intracellular NO levels can inhibit P. aeruginosa biofilm growth in chronic skin wounds and reduce pathogenicity to the host. Targeting NO is a potential approach to control biofilm growth in chronic skin wounds wherein P. aeruginosa biofilms are a persistent problem.

A single amino acid residue tunes the stability of the fully reduced flavin cofactor and photorepair activity in photolyases.

The UV-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 photoproducts), can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. The fully reduced flavin (hydroquinone, HQ) cofactor is required for the catalysis of both types of these photolyases. On the other hand, flavin cofactor in the semireduced state, semiquinone, can be utilized by photolyase homologs, the cryptochromes. However, the evolutionary process of the transition of the functional states of flavin cofactors in photolyases and cryptochromes remains mysterious. In this work, we investigated three representative photolyases (Escherichia coli CPD photolyase, Microcystis aeruginosa DASH, and Phaeodactylum tricornutum 6-4 photolyase). We show that the residue at a single site adjacent to the flavin cofactor (corresponding to Ala377 in E. coli CPD photolyase, hereafter referred to as site 377) can fine-tune the stability of the HQ cofactor. We found that, in the presence of a polar residue (such as Ser or Asn) at site 377, HQ was stabilized against oxidation. Furthermore, this polar residue enhanced the photorepair activity of these photolyases both in vitro and in vivo. In contrast, substitution of hydrophobic residues, such as Ile, at site 377 in these photolyases adversely affected the stability of HQ. We speculate that these differential residue preferences at site 377 in photolyase proteins might reflect an important evolutionary event that altered the stability of HQ on the timeline from expression of photolyases to that of cryptochromes.

Antioxidant, anti-inflammatory, and anti-apoptotic effects of genkwanin against aflatoxin B1-induced testicular toxicity.

Aflatoxin B1 (AFB1) is the most hazardous aflatoxin that causes significant damage to the male reproductive system. Genkwanin (GNK) is a bioactive flavonoid that shows antioxidant and anti-inflammatory potential. Therefore, the current study was planned to evaluate the effects of GNK against AFB1-induced testicular toxicity. Forty-eight male rats were distributed into four groups (n = 12 rats). AFB1 (50 µg/kg) and GNK (20 mg/kg) were administered to the rats for eight weeks. Results of the current study revealed that AFB1 exposure induced adverse effects on the Nrf2/Keap1 pathway and reduced the expressions and activities of antioxidant enzymes. Additionally, it increased the levels of oxidative stress markers. Furthermore, expressions of steroidogenic enzymes were down-regulated by AFB1 intoxication. Besides, AFB1 exposure reduced the levels of gonadotropins and plasma testosterone, which subsequently reduced the epididymal sperm count, motility, and hypo-osmotic swelled (HOS) sperms, while increasing the number of dead sperms and causing morphological anomalies of the head, midpiece, and tail of the sperms. In addition, AFB1 decreased the activities of testicular function marker enzymes and the levels of inflammatory markers. Moreover, it severely affected the apoptotic profile by up-regulating the expressions of Bax and Casp3, while down-regulating the Bcl2 expression. Besides, AFB1 significantly damaged the histoarchitecture of testicular tissues. However, GNK treatment reversed all the AFB1-induced damages in the rats. Taken together, the current study reports the potential use of GNK as a therapeutic agent to prevent AFB1-induced testicular toxicity due to its antioxidant, anti-inflammatory, and anti-apoptotic properties.

Recent advances in taste transduction mechanism, analysis methods and strategies employed to improve the taste of taste peptides.

Taste peptides are oligopeptides that enhance both aroma and taste of food, and they are classified into five categories based on their taste characteristics: salty, sour, umami, sweet, bitter, and kokumi peptide. Recently, taste peptides have attracted the attention of several fields of research in food science and commercial applications. However, research on taste receptors of taste peptides and their taste transduction mechanisms are not clearly understood and we present a comprehensive review about these topics here. This review covers the aspects of taste peptides perceived by their receptors in taste cells, the proposed transduction pathway, as well as structural features of taste peptides. Apart from traditional methods, molecular docking, peptidomic analysis, cell and animal models and taste bud biosensors can be used to explore the taste mechanism of taste peptides. Furthermore, synergistic effect, Maillard reaction, structural modifications and changing external environment are employed to improve the taste of taste peptides. Consequently, we discussed the current challenges and future trends in taste peptide research. Based on the summarized developments, taste peptides derived from food proteins potentially appear to be important taste substances. Their applications meet the principles of "safe, nutritious and sustainable" in food development.

The impact of RNA binding proteins and the associated long non-coding RNAs in the TCA cycle on cancer pathogenesis.

The tricarboxylic acid (TCA) cycle is a central route for generating cellular energy and precursors for biosynthetic pathways. Emerging evidences have shown that the aberrations of metabolic enzymes which affect the integrity of TCA cycle are implicated in various tumour pathological processes. Interestingly, several TCA enzymes exhibit the characteristics of RNA binding properties, and their long non-coding RNA (lncRNA) partners play critical regulatory roles in regulating the function of TCA cycle and tumour progression. In this review, we will discuss the functional roles of RNA binding proteins and their lncRNA partners in TCA cycle, with emphasis placed on the cancer progression. A further understanding of RNA binding proteins and their lncRNA partners in TCA cycle, as well as their molecular mechanisms in oncogenesis, will aid in developing novel layers of metabolic targets for cancer therapy in the near future.Abbreviations: CS: citrate synthase. AH: aconitase, including ACO1, and ACO2. IDH: isocitrate dehydrogenase, including IDH1, IDH2, and IDH3. KGDHC: α-ketoglutarate dehydrogenase complex, including OGDH, DLD, and DLST. SCS: succinyl-CoA synthase, including SUCLG1, SUCLG2, and SUCLA2. SDH: succinate dehydrogenase, including SDHA, SDHB, SDHC, and SDHD. FH: fumarate hydratase. MDH: malate dehydrogenase, including MDH1 and MDH2. PC: pyruvate carboxylase. ACLY: ATP Citrate Lyase. NIT: nitrilase. GAD: glutamate decarboxylase. ABAT: 4-aminobutyrate aminotransferase. ALDH5A1: aldehyde dehydrogenase 5 family member A1. ASS: argininosuccinate synthase. ASL: adenylosuccinate synthase. DDO: D-aspartate oxidase. GOT: glutamic-oxaloacetic transaminase. GLUD: glutamate dehydrogenase. HK: hexokinase. PK: pyruvate kinase. LDH: lactate dehydrogenase. PDK: pyruvate dehydrogenase kinase. PDH: pyruvate dehydrogenase complex. PHD: prolyl hydroxylase domain protein.

Hypoxia is fine-tuned by Hif-1α and regulates mesendoderm differentiation through the Wnt/ß-Catenin pathway.

BACKGROUND: Hypoxia naturally happens in embryogenesis and thus serves as an important environmental factor affecting embryo development. Hif-1α, an essential hypoxia response factor, was mostly considered to mediate or synergistically regulate the effect of hypoxia on stem cells. However, the function and relationship of hypoxia and Hif-1α in regulating mesendoderm differentiation remains controversial. RESULTS: We here discovered that hypoxia dramatically suppressed the mesendoderm differentiation and promoted the ectoderm differentiation of mouse embryonic stem cells (mESCs). However, hypoxia treatment after mesendoderm was established promoted the downstream differentiation of mesendoderm-derived lineages. These effects of hypoxia were mediated by the repression of the Wnt/ß-Catenin pathway and the Wnt/ß-Catenin pathway was at least partially regulated by the Akt/Gsk3ß axis. Blocking the Wnt/ß-Catenin pathway under normoxia using IWP2 mimicked the effects of hypoxia while activating the Wnt/ß-Catenin pathway with CHIR99021 fully rescued the mesendoderm differentiation suppression caused by hypoxia. Unexpectedly, Hif-1α overexpression, in contrast to hypoxia, promoted mesendoderm differentiation and suppressed ectoderm differentiation. Knockdown of Hif-1α under normoxia and hypoxia both inhibited the mesendoderm differentiation. Moreover, hypoxia even suppressed the mesendoderm differentiation of Hif-1α knockdown mESCs, further implying that the effects of hypoxia on the mesendoderm differentiation were Hif-1α independent. Consistently, the Wnt/ß-Catenin pathway was enhanced by Hif-1α overexpression and inhibited by Hif-1α knockdown. As shown by RNA-seq, unlike hypoxia, the effect of Hif-1α was relatively mild and selectively regulated part of hypoxia response genes, which fine-tuned the effect of hypoxia on mESC differentiation. CONCLUSIONS: This study revealed that hypoxia is fine-tuned by Hif-1α and regulates the mesendoderm and ectoderm differentiation by manipulating the Wnt/ß-Catenin pathway, which contributed to the understanding of hypoxia-mediated regulation of development.

Fatty acid composition and energy allocation in muscle and gonad tissues indicate that the female mackerel icefish Champsocephalus gunnari is an income breeder.

The energy density and fatty acid composition profiles of the muscle and gonad tissues of female mackerel icefish Champsocephalus gunnari from the South Orkney Islands in Antarctica were investigated throughout ovarian development to better understand the reproductive allocation strategy and the role of specific fatty acids in the reproductive process. Energy density in gonads increased from resting to spawning stages as the ovaries developed (19.60-25.10 kJ g-1 dry mass [DM]). In contrast, energy density in muscles remained constant throughout ovarian development (20.13-22.87 kJ g-1 DM), suggesting that the spawning events of the C. gunnari rely on energy income from feeding rather than on the energy stored in body. In addition, the variation in fatty acid composition between muscle and gonad tissues may reflect the role of main FAs as energy source. These results suggest that C. gunnari may utilize an income breeding strategy.

Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications.

Pseudomonas aeruginosa PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP+-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient counterpart NAD-IDHs. For a better understanding of PaIDH1 and PaIDH2, we cloned the genes, overexpressed them in Escherichia coli and purified them to homogeneity. PaIDH1 displayed higher affinity to NADP+ and isocitrate, with lower Km values when compared to PaIDH2. Moreover, PaIDH1 possessed higher temperature tolerance (50 °C) and wider pH range tolerance (7.2-8.5) and could be phosphorylated. After treatment with the bifunctional PaIDH kinase/phosphatase (PaIDH K/P), PaIDH1 lost 80% of its enzymatic activity in one hour due to the phosphorylation of Ser115. Small-molecule compounds like glyoxylic acid and oxaloacetate can effectively inhibit the activity of PaIDHs. The mutant PaIDH1-D346I347A353K393 exhibited enhanced affinity for NAD+ while it lost activity towards NADP+, and the Km value (7770.67 µM) of the mutant PaIDH2-L589 I600 for NADP+ was higher than that observed for NAD+ (5824.33 µM), indicating a shift in coenzyme specificity from NADP+ to NAD+ for both PaIDHs. The experiments demonstrated that the mutation did not alter the oligomeric state of either protein. This study provides a foundation for the elucidation of the evolution and function of two NADP-IDHs in the pathogenic bacterium P. aeruginosa.

Enzymatic Characterization of the Isocitrate Dehydrogenase with Dual Coenzyme Specificity from the Marine Bacterium Umbonibacter marinipuiceus.

Isocitrate dehydrogenase (IDH) can be divided into NAD+-dependent and NADP+-dependent types based on the coenzyme specificity. It is worth noting that some IDHs exhibit dual coenzyme specificity characteristics. Herein, a dual coenzyme-dependent IDH from Umbonibacter Marinipuiceus (UmIDH) was expressed, purified, and identified in detail for the first time. SDS-PAGE and Gel filtration chromatography analyses showed that UmIDH is an 84.7 kDa homodimer in solution. The Km values for NAD+ and NADP+ are 1800.0 ± 64.4 µM and 1167.7 ± 113.0 µM in the presence of Mn2+, respectively. Meanwhile, the catalytic efficiency (kcat/Km) of UmIDH is only 2.3-fold greater for NADP+ than NAD+. The maximal activity for UmIDH occurred at pH 8.5 (with Mn2+) or pH 8.7 (with Mg2+) and at 35 °C (with Mn2+ or Mg2+). Heat inactivation assay revealed that UmIDH sustained 50% of maximal activity after incubation at 57 °C for 20 min with either Mn2+ or Mg2+. Moreover, three putative core coenzyme binding residues (R345, L346, and V352) of UmIDH were evaluated by site-directed mutagenesis. This recent work identified a unique dual coenzyme-dependent IDH and achieved the groundbreaking bidirectional modification of this specific IDH's coenzyme dependence for the first time. This provides not only a reference for the study of dual coenzyme-dependent IDH, but also a basis for the investigation of the coenzyme-specific evolutionary mechanisms of IDH.

Identification of a Novel Class of Photolyases as Possible Ancestors of Their Family.

UV irradiation induces the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts in DNA. These two types of lesions can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. Recently, a new class of 6-4 photolyases named iron-sulfur bacterial cryptochromes and photolyases (FeS-BCPs) were found, which were considered as the ancestors of all photolyases and their homologs-cryptochromes. However, a controversy exists regarding 6-4 photoproducts only constituting ∼10-30% of the total UV-induced lesions that primordial organisms would hardly survive without a CPD repair enzyme. By extensive phylogenetic analyses, we identified a novel class of proteins, all from eubacteria. They have relatively high similarity to class I/III CPD photolyases, especially in the putative substrate-binding and FAD-binding regions. However, these proteins are shorter, and they lack the "N-terminal α/ß domain" of normal photolyases. Therefore, we named them short photolyase-like. Nevertheless, similar to FeS-BCPs, some of short photolyase-likes also contain four conserved cysteines, which may also coordinate an iron-sulfur cluster as FeS-BCPs. A member from Rhodococcus fascians was cloned and expressed. It was demonstrated that the protein contains a FAD cofactor and an iron-sulfur cluster, and has CPD repair activity. It was speculated that this novel class of photolyases may be the real ancestors of the cryptochrome/photolyase family.

Electrochemical Ammonia Synthesis via NO Reduction on 2D-MOF.

Developing new catalysts that effectively promote electrocatalytic NO reduction (ENOR) is a very important industrial field. A two-dimensional (2D) metal-organic framework (MOF) with hexaaminobenzene (HAB) ligands (TM-HAB MOF, TM=Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Mo, Ru, Rh and Pd) as an electrocatalyst for ENOR was systematically explored in this work by means of well-defined density functional theory (DFT) calculations. We predicted the impact of the coordination structure of different MOFs on its catalytic performance and found that the suitable candidates are Co- and Rh-HAB MOFs due to moderate binding strength between NO and substrates. Further calculations indicated that Co-HAB MOF has the best ENOR catalytic activity with a limiting potential of - 0.26 V toward NH3 production at low NO coverage, yet NO reduction to N2 O at high NO coverage was limited due to high limiting potential. The scaling relationship with a good correlation coefficient between several electronic properties and the adsorption Gibbs free energy change of *NO (ΔG*NO ) were found, which implies that ΔG*NO can be used as a simple descriptor for screening out suitable electrocatalysts. This work offers a new paradigm for ENOR toward NH3 production under ambient conditions.

Purification and Identification of Peptides from Oyster (Crassostrea hongkongensis) Protein Enzymatic Hydrolysates and Their Anti-Skin Photoaging Effects on UVB-Irradiated HaCaT Cells.

This study aimed to purify and identify antiphotoaging peptides from oyster (Crassostrea hongkongensis) protein enzymatic hydrolysates (OPEH) and to investigate the possible mechanism underlying its antiphotoaging effect. Multiple methods (Ultrafiltration, G25 Chromatography, RP-HPLC, and LC/MS/MS) had been used for this purpose, and eventually, two peptides, including WNLNP and RKNEVLGK, were identified. Particularly, WNLNP exerted remarkable antiphotoaging effect on the UVB-irradiated HaCaT photoaged cell model in a dose-dependent manner. WNLNP exerted its protective effect mainly through inhibiting ROS production, decreasing MMP-1 expression, but increasing extracellular pro-collagen I content. Furthermore, WNLNP downregulated p38, JNK, ERK, and p65 phosphorylation in the MAPK/NF-κB signaling pathway and attenuated bax over-expressions but reversed bcl-2 reduction in UVB- irradiated HaCaT cells. The molecular docking analysis showed that WNLNP forms five and seven hydrogen bonds with NF-κB (p65) and MMP-1, respectively. This study suggested that a pentapeptide WNLNP isolated from OPEH had great potential to prevent and regulate skin photoaging.

Biological-physical processes regulate autumn prey availability of spiny icefish Chaenodraco wilsoni in the Bransfield Strait, Antarctic.

This study examines the adaptability of a Southern Ocean predator, which is dependent on Antarctic krill (Euphausia superba), to potential changes in food availability. Muscle fatty acids (FAs) of the spiny icefish Chaenodraco wilsoni collected from three areas in the Bransfield Strait (BS), northern Antarctic Peninsula during February-April 2016 give a good representation of their feeding variability. The compositions of 22:6n3 (DHA) and 20:5n3 (EPA) were both higher in the Transitional Zonal Water with Bellingshausen influence (TBW)-controlled C. wilsoni than in the Transitional Zonal Water with Weddell Sea influence (TWW)-controlled fish. This was positively correlated with photoadaptation and carbon sequestration in TBW-controlled phytoplankton. Results for the FAs 16:1n7, 16:0, DHA and EPA indicate the presence of dinoflagellates in all three areas, suggesting that during late summer and early fall, there is a seasonal phytoplankton succession, where small phytoplankton become dominant, in the BS. In addition, the compositions of some long-chain FAs (>20, such as 20:0, 20:1, 22:0 and 22:1n9) and ∑18 indicated that the food chain based on flagellates and copepods was more apparent in TWW-controlled C. wilsoni, especially the effect of El Niño-Southern Oscillation (ENSO) on the variation of prey communities in TWW-controlled areas. FA markers such as SFA/(PUFA+MUFA), ∑15 + ∑17 and ARA were more pronounced in TWW-controlled C. wilsoni, indicating a more strongly carnivorous and benthic food source. In the TBW-TWW confluence, the complex hydrological structure, including the presence of a large number of mesoscale eddies, allows rich nutrients and krill larvae to remain in it, providing a rich food source for the C. wilsoni. Overall, the FA data of this study show that the diet of C. wilsoni varies in different marine environments, aiding their survivability at the face of climate change.

Mapping leprosy-associated coding variants of interleukin genes by targeted sequencing.

Previous genotyping-based assays have identified non-coding variants of several interleukins (ILs) being associated with genetic susceptibility to leprosy. However, understanding of the involvement of coding variants within all IL family genes in leprosy was still limited. To obtain the full mutation spectrum of all ILs in leprosy, we performed a targeted deep sequencing of coding regions of 58 ILs genes in 798 leprosy patients (age 56.2 ± 14.4; female 31.5%) and 990 healthy controls (age 38.1 ± 14.0; female 44.3%) from Yunnan, Southwest China. mRNA expression alterations of ILs in leprosy skin lesions or in response to M. leprae treatment were estimated by using publicly available expression datasets. Two coding variants in IL27 (rs17855750, p.S59A, p = 4.02 × 10-8 , odds ratio [OR] = 1.748) and IL1RN (rs45507693, p.A106T, p = 1.45 × 10-5 , OR = 3.629) were significantly associated with leprosy risk. mRNA levels of IL27 and IL1RN were upregulated in whole blood cells after M. leprae stimulation. These data showed that IL27 and IL1RN are leprosy risk genes. Further functional study is required for characterizing the exact role of ILs in leprosy.

Crystal structures of NAD+-linked isocitrate dehydrogenase from the green alga Ostreococcus tauri and its evolutionary relationship with eukaryotic NADP+-linked homologs.

NAD+-linked isocitrate dehydrogenases (NAD-IDHs) catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate. Previously, we identified a novel phylogenetic clade including NAD-IDHs from several algae in the type II subfamily, represented by homodimeric NAD-IDH from Ostreococcus tauri (OtIDH). However, due to its lack of a crystalline structure, the molecular mechanisms of the ligand binding and catalysis of OtIDH are little known. Here, we elucidate four high-resolution crystal structures of OtIDH in a ligand-free and various ligand-bound forms that capture at least three states in the catalytic cycle: open, semi-closed, and fully closed. Our results indicate that OtIDH shows several novel interactions with NAD+, unlike type I NAD-IDHs, as well as a strictly conserved substrate binding mode that is similar to other homologs. The central roles of Lys283' in dual coenzyme recognition and Lys234 in catalysis were also revealed. In addition, the crystal structures obtained here also allow us to understand the catalytic mechanism. As expected, structural comparisons reveal that OtIDH has a very high structural similarity to eukaryotic NADP+-linked IDHs (NADP-IDHs) within the type II subfamily rather than with the previously reported NAD-IDHs within the type I subfamily. It has also been demonstrated that OtIDH exhibits substantial conformation changes upon ligand binding, similar to eukaryotic NADP-IDHs. These results unambiguously support our hypothesis that OtIDH and OtIDH-like homologs are possible evolutionary ancestors of eukaryotic NADP-IDHs in type II subfamily.

A bifunctional enzyme belonging to cytochrome P450 family involved in the O-dealkylation and N-dealkoxymethylation toward chloroacetanilide herbicides in Rhodococcus sp. B2.

BACKGROUND: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown. RESULTS: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1 °C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, harboring gene cluster (EthRABCDB2), was absent from the mutant strain TB2 which could not degrade pretilachlor. The Eth gene cluster, encodes an AraC/XylS family transcriptional regulator (EthRB2), a ferredoxin reductase (EthAB2), a cytochrome P450 monooxygenase (EthBB2), a ferredoxin (EthCB2) and a 10-kDa protein of unknown function (EthDB2). Complementation with EthABCDB2 and EthABDB2, but not EthABCB2 in strain TB2 restored its ability to degrade chloroacetamide herbicides. Subsequently, codon optimization of EthABCDB2 was performed, after which the optimized components were separately expressed in Escherichia coli, and purified using Ni-affinity chromatography. A mixture of EthABCDB2 or EthABDB2 but not EthABCB2 catalyzed the N-dealkoxymethylation of alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation of pretilachlor, revealing that EthDB2 acted as a ferredoxin in strain B2. EthABDB2 displayed maximal activity at 30 °C and pH 7.5. CONCLUSIONS: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation and N-dealkoxymethylation of pretilachlor and propisochlor, respectively. And the results of the present study provide a microbial resource for the remediation of chloroacetamide herbicides-contaminated sites.

Identification and Characterization of a Novel Soluble Pyridine Nucleotide Transhydrogenase from Streptomyces avermitilis.

Soluble pyridine nucleotide transhydrogenase (STH) transfers hydride between NADH and NADPH to maintain redox balance. In the present study, the sth gene from Gram-positive bacterium Streptomyces avermitilis (SaSTH) was expressed in Escherichia coli, and the recombinant STH protein was purified to homogeneity. Activity assays indicated that SaSTH was able to catalyze transhydrogenase reactions by using NADH or NADPH as reductants and thio-NAD+ as an oxidant. The apparent Km value for NADPH (74.5 µM) was lower than that for NADH (104.0 µM) and the apparent kcat/Km for NADPH (2704.7 mM-1 s-1) was higher than that for NADH (1129.8 mM-1 s-1). SaSTH showed optimal activity at 25 °C and at a pH of 6.2. Heat-inactivation studies revealed that SaSTH remained stable below 55 °C and that approximately 50% activity was preserved at 57 °C for 20 min. Analyses also showed that SaSTH activity was inhibited by divalent ions, particularly Co2+, Ni2+, and Zn2+. In addition, the transhydrogenase activity of SaSTH was inhibited by ATP and strongly stimulated by ADP and AMP. In summary, we characterized a recombinant enzyme exhibiting STH activity from Gram-positive bacteria for the first time. Our findings provide new options for cofactor engineering and industrial biocatalytic processes.

Biochemical and phylogenetic characterization of a monomeric isocitrate dehydrogenase from a marine methanogenic archaeon Methanococcoides methylutens.

Monomeric isocitrate dehydrogenase (IDH) stands for a separated subgroup among IDH protein family. Up to now, all reported monomeric IDHs are from prokaryotes. Here, a monomeric IDH from a marine methanogenic archaeon Methanococcoides methylutens (MmIDH) was reported for the first time. BLAST search demonstrated that only a few marine archaea encode the monomeric IDH and all these organisms are methylotrophic. MmIDH shows the highest homology (~ 70%) to the monomeric IDHs from some marine bacteria, suggesting a lateral gene transfer event between marine bacteria and archaea. The monomeric state of MmIDH was determined by size exclusion chromatography. MmIDH is divalent cation-dependent and Mn2+ is the most favored. Kinetic analysis showed that MmIDH is highly specific to NADP+ and cannot utilize the NAD+. The optimal temperature for MmIDH activity is 50 °C and the optimal pH is 8.2. Heat inactivation assay revealed that MmIDH is a mesophilic enzyme. It sustained 50% activity after incubation at 39 °C for 20 min. Moreover, the putative coenzyme binding residues (His590, Arg601, and Arg650) of MmIDH were explored by mutagenesis. The triple mutant H590L/R601D/R650S displayed a 5.93-fold preference for NAD+ over NADP+, indicating that the coenzyme specificity of MmIDH was significantly switched from NADP+ to NAD+ by three key mutations.

Ameliorative Effects of Peptides from the Oyster (Crassostrea hongkongensis) Protein Hydrolysates against UVB-Induced Skin Photodamage in Mice.

Chronic exposure to ultraviolet B (UVB) irradiation is a major cause for skin photoaging. UVB induces damage to skin mainly by oxidative stress, inflammation, and collagen degradation. This paper investigated the photo-protective effects of peptides from oyster (Crassostrea hongkongensis) protein hydrolysates (OPs) by topical application on the skin of UVB-irradiated mice. Results from mass spectrometry showed that OPs consisted of peptides with a molecular weight range of 302.17-2936.43 Da. In vivo study demonstrated that topical application of OPs on the skin significantly alleviated moisture loss, epidermal hyperplasia, as well as degradation of collagen and elastin fibers caused by chronic UVB irradiation. In this study, OPs treatment promoted antioxidant enzymes (SOD and GPH-Px) activities, while decreased malondialdehyde (MDA) level in the skin. In addition, OPs treatment significantly decreased inflammatory cytokines (IL-1ß, IL-6, TNF-α) content, and inhibited inflammation related (iNOS, COX-2) protein expression in the skin. Via inhibiting metalloproteinase 1(MMP1) expression, OPs treatment markedly decreased the degradation of collagen and elastin fibers as well as recovered the altered arrangement of extracellular matrix network in the dermis of skin. Our study demonstrated for the first time that OPs protected against UVB induced skin photodamage by virtue of its antioxidative and anti-inflammatory properties, as well as regulating the abnormal expression of MMP-1. The possible molecular mechanism underlying OPs anti-photoaging is possibly related to downregulating of the MAPK/NF-κB signaling pathway, while promoting TGF-ß production in the skin.

Characterization of a novel hyper-thermostable and chlorpyrifos-hydrolyzing carboxylesterase EstC: A representative of the new esterase family XIX.

Carboxylesterases have widely been used in a series of industrial applications, especially, the detoxification of pesticide residues. In the present study, EstC, a novel carboxylesterase from Streptomyces lividans TK24, was successfully heterogeneously expressed, purified and characterized. Phylogenetic analysis showed that EstC can be assigned as the first member of a novel family XIX. Multiple sequence alignment indicated that EstC has highly conserved structural features, including a catalytic triad formed by Ser155, Asp248 and His278, as well as a canonical Gly-His-Ser-Ala-Gly pentapeptide. Biochemical characterization indicated that EstC exhibited maximal activity at pH 9.0 (Tris-HCl buffer) and 55 °C. It also showed higher activity towards short-chain substrates, with the highest activity for p-nitrophenyl acetate (pNPA2) (Km = 0.31 ± 0.02 mM, kcat/Km = 1923.35 ± 9.62 s-1 mM-1) compared to other pNP esters used in this experiment. Notably, EstC showed hyper-thermostability and good alkali stability. The activity of EstC had no significant changes when it was incubated under 55 °C for 100 h and reached half-life after incubation at 100 °C for 8 h. Beyond that, EstC also showed stability at pH ranging from 6.0 to 11.0 and about 90% residual activity still reserved after treatment at pH 8.0 or 9.0 for 26 h, especially. Furthermore, EstC had outstanding potential for bioremediation of chlorpyrifos-contaminated environment. The recombinant enzyme (0.5 U mL-1) could hydrolyze 79.89% chlorpyrifos (5 mg L-1) at 37 °C within 80 min. These properties will make EstC have a potential application value in various industrial productions and detoxification of chlorpyrifos residues.