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.

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.

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.

Biochemical Characterization and Crystal Structure of a Novel NAD+-Dependent Isocitrate Dehydrogenase from Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum originated from a series of secondary symbiotic events and has been used as a model organism for studying diatom biology. A novel type II homodimeric isocitrate dehydrogenase from P. tricornutum (PtIDH1) was expressed, purified, and identified in detail through enzymatic characterization. Kinetic analysis showed that PtIDH1 is NAD+-dependent and has no detectable activity with NADP+. The catalytic efficiency of PtIDH1 for NAD+ is 0.16 µM-1·s-1 and 0.09 µM-1·s-1 in the presence of Mn2+ and Mg2+, respectively. Unlike other bacterial homodimeric NAD-IDHs, PtIDH1 activity was allosterically regulated by the isocitrate. Furthermore, the dimeric structure of PtIDH1 was determined at 2.8 Å resolution, and each subunit was resolved into four domains, similar to the eukaryotic homodimeric NADP-IDH in the type II subfamily. Interestingly, a unique and novel C-terminal EF-hand domain was first defined in PtIDH1. Deletion of this domain disrupted the intact dimeric structure and activity. Mutation of the four Ca2+-binding sites in the EF-hand significantly reduced the calcium tolerance of PtIDH1. Thus, we suggest that the EF-hand domain could be involved in the dimerization and Ca2+-coordination of PtIDH1. The current report, on the first structure of type II eukaryotic NAD-IDH, provides new information for further investigation of the evolution of the IDH family.

Phenolic compounds and antioxidants from Eucalyptus camaldulensis as affected by some extraction conditions, a preparative optimization for GC-MS analysis.

Four organic solvents along with water were applied for the conventional extraction of Eucalyptus camaldulensis (Myrtaceae), phenolic contents and antioxidant activities were investigated through variable protocols and correlation coefficients were considered, the phenolic composition was also characterized by Gas chromatography-mass spectrometry (GC-MS). Using solvents with dissimilar polarities affected the phenolic yields extracted from E. camaldulensis and their related antioxidant activities varied significantly among the four investigated plant organs. The leaf extract of acetone 70% contained the highest amount of phenolic compounds (46.56 mg/g dry weight); while the bud-water boiled extract maintained the maximum value of tannins (45.68 mg/g dry weight). Correlation coefficients indicated that phenolic compounds were mostly accountable for the phosphomolybednum antioxidant potentials (0.520), followed by tannins (0.460). Also, both the reducing power activities and hydrogen peroxide scavenging of E. camaldulensis extracts positively correlated with tannins, but at different significance degrees. However, the GC-MS analysis revealed that most of the detected phenolic constituents were more abundant in the plant seed. So, the existence of some other compounds such as organic acids, along with phenolics, may have increased the antioxidant potentials of leaf and bud. Undeniably, the optimization of extraction conditions could stimulate the antioxidant capabilities of the plant extracts of E. camaldulensis.

[DNA barcoding identification of Dendrobium huoshanense and its adulterants].

This research preliminarily discusses the relations of Dendrobium system growth through chloroplast gene rbcL, matK and the nuclear genome ITS2. The DNA barcoding universal sequence for authentication of the Dendrobium medical plants was slected and the possibility concerning utilizing the DNA barcoding to distinguish the D. huoshanenseand its adulterants was analyzed. Using the universal primer pair of ITS2, rbcL and matK, series of extended sequencing in the Dendrobium were conducted. Meanwhile, considering the different index about amplification and sequencing success rate of each sequence, the intraspecific and interspecific aberrance, the employment of BioEdit and MEGA 5.0 software were applied to establish the systematic tree of the NJ molecular and evaluate the diversified authentication capability of various sequences. The consequence demonstrates that the sequence of ITS2 is not only the largest one both in the intraspecific and interspecific aberrance of the Dendrobium but also has obvious barcoding gap. Considering the few overlap between the intraspecific and interspecific aberrance and the highest percentage regarding the formation of unilateral branch in diverse Dendrobium which have different ITS2 sequences, it can differentiate the species of Dendrobium. Furthermore, due to the inferior success rate of the rbcL and thematK and the lower reliability of NJ systematic tree, the percentage of the unilateral species which are generated by the systematic tree of rbcL and matK sequences is deficient. Therefore, the sequence of ITS2 can serves as DNA barcoding to distinguish the D. huoshanense, the D. moniliform and the D. officinale.

A unique homodimeric NAD⁺-linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri.

In eukaryotes, NAD(+)-dependent isocitrate dehydrogenase (IDH) is strictly mitochondrial and is a key enzyme in the Krebs cycle. To date, all known NAD(+)-specific IDHs (NAD-IDHs) in the mitochondria are believed to be heteromeric in solution. Here, a unique homodimeric NAD-IDH from Ostreococcus tauri (OtIDH), the smallest autotrophic picoeukaryote, was unveiled. Active OtIDH has a molecular weight of ∼93 kDa with each subunit of 46.7 kDa. In the presence of Mn(2+) and Mg(2+), OtIDH displayed 42-fold and 51-fold preference for NAD(+) over NADP(+), respectively. Interestingly, OtIDH exhibited a sigmoidal kinetic behavior in response to isocitrate unlike other homodimeric homologs, and a remarkably high affinity for isocitrate (S0.5 < 10 µM) unlike other hetero-oligomeric homologs. Furthermore, its coenzyme specificity can be completely converted from NAD(+) (ancient trait) to NADP(+) (adaptive trait) by rational mutagenesis based on the evolutionary trace. Mutants D344R and D344R/M345H displayed a 15-fold and 72-fold preference for NADP(+) over NAD(+), respectively, indicating that D344 and M345 are the determinants of NAD(+) specificity. These findings also suggest that OtIDH may be an ancestral form of type II IDHs (all reported members are NADP(+)-linked enzymes) and may have evolved into NADP(+)-dependent IDH for adaptation to the increased demand of NADPH under carbon starvation.

Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum.

Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP⁺-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP⁺ over NAD⁺ in the presence of Mg(2+) and Mn(2+), respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn(2+)) and 65 °C (with Mg(2+)), and pH 7.5 (with Mn(2+)) and pH 8.0 (with Mg(2+)). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn(2+) or Mg(2+). Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP⁺ to NAD⁺ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP⁺ use by the IDH family.

Identification of a novel fumarase C from Streptomyces lividans TK54 as a good candidate for L-malate production.

Fumarase is a key enzyme that catalyzes the reversible hydration of fumarate to L-malate in the tricarboxylic acid cycle. This reaction has been extensively utilized for industrial applications in producing L-malate. In this study, a fumarase C gene from Streptomyces lividans TK54 (slFumC) was cloned and expressed as a fused protein (SlFumC) in Escherichia coli. The molecular mass of SlFumC was about 49 kDa determined by SDS-PAGE. Kinetic studies showed that the K m value of SlFumC for L-malate increased by approximately 8.5-fold at pH 6.5 (6.7 ± 0.81 mM) to 8.0 (57.0 ± 1.12 mM), which was higher than some known fumarases. The catalytic efficiency (k cat) and the specific activity increased by about 9.5-fold at pH 6.5 (65 s(-1)) to 8.0 (620 s(-1)) and from 79 U/mg at pH 6.5 to 752 U/mg at pH 8.0, respectively. Therefore, SlFumC may acquire strong catalytic ability by increasing pH to partially compensate for the loss of substrate affinity. The enzyme also showed substrate inhibition phenomenon, which is pH-dependent. Specific activity of SlFumC was gradually enhanced with increasing phosphate concentrations. However, no inhibition was observed at high concentration of phosphate ion, which was distinctly different in case of other Class II fumarases. In industrial process, the reaction temperatures for L-malate production are usually set between 40 and 60 °C. The recombinant SlFumC displayed maximal activity at 45 °C and remained over 85 % of original activity after 48 h incubation at 40 °C, which was more thermostable than other fumarases from Streptomyces and make it an efficient enzyme for use in the industrial production of L-malate.

Bioactive Compounds from Vicia sativa L. and Vicia monantha Retz. with Unveiling Antiviral Potentials in Newly Green Synthesized CdO Nanoparticles.

BACKGROUND: in the current study, a comparative phytochemical analysis was carried out to explore the phenolic and flavonoid contents in the aerial parts of Vicia sativa L and Vicia monantha Retz growing in cultivated, reclaimed, and desert habitats. METHODS: High-performance liquid chromatography (HPLC) was used to detect Vicia methanolic extracts' individual phenolic and flavonoid constituents. The first-time synthesis of cadmium oxide nanoparticles (CdO NPs) using the aqueous extract of V. monantha has been developed using a green approach. Also, the cytotoxicity of V. monantha extract and CdO NPs was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for unveiling them as anti-HAV and anti-AdV. RESULTS: Our results indicated that in the case of desert habitat, the contents of total phenolics (76.37 mg/g) and total flavonoids (65.23 mg/g) of V. monantha were higher than those of V. sativa (67.35 mg/g and 47.34 mg/g, respectively) and the contents of these secondary metabolites were even increased in V. monantha collected from reclaimed land (phenolics: 119.77 mg/g, flavonoids: 88.61 mg/g). Also, V. monantha surpassed V. sativa in the contents of some individual HPLC constituents, and hence, V. monantha was used to synthesize the green CdO NPs and subsequent antiviral tests. The average size of CdO NPs was determined to be 24.28 nm, and the transmission electron microscopy (TEM) images of CdO NPs clearly showed their spherical form and varying particle sizes, with different diameters in the range of 19-29 nm. MTT assay was positive to the exposure of CdO NPs in the normal cell line, proposing that CdO NPs can reduce cell viability. V. monantha extract showed promising antiviral activity against Hepatitis A virus (HAV) and Adenovirus (AdV) with SI of 16.40 and 10.54. On the other hand, CdONPs had poor antiviral activity against HAV with an SI of 4.74 and moderate antiviral activity against AdV with an SI of 10.54. CONCLUSION: V. monantha is now considered a new, valuable natural resource for phenolics and flavonoids, especially when grown in reclaimed soil. The green CdO NPs based on V. monantha extract showed a promising antiviral effect against HAV and AdV.

Expression and characterization of a novel isocitrate dehydrogenase from Streptomyces diastaticus No. 7 strain M1033.

Isocitrate dehydrogenase (IDH) is one of the key enzymes in tricarboxylic acid cycle, widely distributed in Archaea, Bacteria and Eukarya. Here, we report for the first time the cloning, expression and characterization of a monomeric NADP(+)-dependent IDH from Streptomyces diastaticus No. 7 strain M1033 (SdIDH). Molecular mass of SdIDH was about 80 kDa and showed high amino acid sequence identity with known monomeric IDHs. Maximal activity of SdIDH was observed at pH 8.0 (Mn(2+)) and 9.0 (Mg(2+)), and the optimal temperature was 40 °C (Mn(2+)) and 37 °C (Mg(2+)). Heat-inactivation studies showed that SdIDH remained about 50 % activity after 20 min of incubation at 47 °C. SdIDH displayed a 19,000 and 32,000-fold (k (cat)/K (m)) preference for NADP(+) over NAD(+) with Mn(2+) and Mg(2+), respectively. Our work implicate that SdIDH is a divalent metal ion-dependent monomeric IDH with remarkably high coenzyme preference for NADP(+). This work may provide fundamental information for further investigation on the catalytic mechanism of monomeric IDH and give a clue to disclose the real cause of IDH monomerization.

Biochemical characterization of NADP⁺-dependent isocitrate dehydrogenase from Microcystis aeruginosa PCC7806.

Microcystis aeruginosa is the key symptom of water eutrophication and produces persistent microcystins. Our special attention was paid to the isocitrate dehydrogenase (IDH) of M. aeruginosa (MaIDH) because it plays important roles in energy and biosynthesis metabolisms and its catalytic product 2-oxoglutarate provides the carbon skeleton for ammonium assimilation and also constitutes a signaling molecule of nitrogen starvation in cyanobacteria. Sequence alignment showed that MaIDH shared significant sequence identity with IDHs from other cyanobacteria (>80 %) and other bacteria (>45 %). The subunit molecular weight of MaIDH was determined to be 52.6 kDa by filtration chromatography, suggesting MaIDH is a typical homodimer. The purified recombinant MaIDH was completely NADP(+)-dependent and no NAD(+)-linked activity was detectable. The K m values for NADP(+) were 32.24 and 71.71 µM with Mg(2+) and Mn(2+) as a sole divalent cation, and DL-isocitrate linked K m values were 32.56 µM (Mg(2+)) and 124.3 µM (Mn(2+)), respectively. As compared with Mn(2+), MaIDH showed about 2.5-times and 4-times higher affinities (1/K m) to NADP(+) and DL-isocitrate with Mg(2+). The optimum activity of MaIDH was found at pH 7.5, and its optimum temperature was 45 °C (Mn(2+)) and 50 °C (Mg(2+)). Heat-inactivation studies showed that heat treatment for 20 min at 45 °C caused a 50 % loss of enzyme activity. MaIDH was completely divalent cation dependent as other typical dimeric IDHs and Mn(2+) was its best activator. Our study is expected to give a better understanding of primary metabolic enzymes in M. aeruginosa. This would provide useful basic information for the research of controlling the blue-green algae blooms through biological techniques.

Isolation and characterization of eleven polymorphic microsatellite loci for the valuable medicinal plant Dendrobium huoshanense and cross-species amplification.

Dendrobium huoshanense (Orchidaceae) is a perennial herb and a widely used medicinal plant in Traditional Chinese medicine (TCM) endemic to Huoshan County town in Anhui province in Southeast China. A microsatellite-enriched genomic DNA library of D. huoshanense was developed and screened to identify marker loci. Eleven polymorphic loci were isolated and analyzed by screening 25 individuals collected from a natural population. The number of alleles per locus ranged from 2 to 5. The observed and expected heterozygosities ranged from 0.227 to 0.818 and from 0.317 to 0.757, respectively. Two loci showed significant deviations from Hardy-Weinberg equilibrium and four of the pairwise comparisons of loci revealed linkage disequilibrium (p < 0.05). These microsatellite loci were cross-amplified for five congeneric species and seven loci can be amplified in all species. These simple sequence repeats (SSR) markers are useful in genetic studies of D. huoshanense and other related species and in conservation decision-making.

[Using shape analysis to inform variation in otolith morphology with life stages of Dissostichus mawsoni]. / 基于形态分析探究鳞头犬牙南极鱼各生活史阶段耳石形态变化.

As the most important domestic fish in the Antarctic Ocean, Antarctic toothfish (Dissostichus mawsoni) has an important ecological role and high commercial value. The otolith morphology of fish species differs across stages of life history. Therefore, otolith shape analysis can be used to infer life history of D. mawsoni. In this study, otoliths from 120 D. mawsoni individuals with four life stages randomly collected from the Ross Sea, Amundsen Sea, Weddell Sea and Lazarev Sea were used to analyze the otolith morphological differences of D. mawsoni at life stages by conventional measurement and elliptical Fourier analysis. The results showed that variation in otolith morphology occurred across life stages. Generally, the morphology of otolith was changed from smooth and low comple-xity to zigzagging and high complexity. The growth rate of otolith along the longitudinal direction was lower than that along the transverse direction. The characteristic parts of otolith, such as antirostrum, changed significantly across life stages. Compared to the linear discriminant analysis (71.9%), the elliptical Fourier analysis had the higher discrimination rate (85.4%), indicating that the elliptical Fourier analysis was more suitable to analyze the otolith morphology of D. mawsoni.

Genomic and phenotypic biology of a novel Dickeya zeae WH1 isolated from rice in China: Insights into pathogenicity and virulence factors.

Soft rot caused by Dickeya zeae is an important bacterial disease affecting rice and other plants worldwide. In this study, Nanopore and Illumina sequencing platforms were used to sequence the high-quality complete genome of a novel D. zeae strain WH1 (size: 4.68 Mb; depth: 322.37x for Nanopore, 243.51x for Illumina; GC content: 53.59%), which was isolated from healthy rice root surface together with Paenibacillus polymyxa, a potential biocontrol bacterium against D. zeae strain WH1. However, the pure WH1 culture presented severe pathogenicity. Multilocus sequence analysis (MLSA) indicated that strains WH1, EC1, and EC2 isolated from rice were grouped into a clade differentiated from other D. zeae strains. The average nucleotide identity (ANI) and DNA-DNA hybridization (DDH) analyses demonstrated that WH1 was phylogenetically closest to EC2. Furthermore, the pathogenicity determinants and virulence factors of WH1 were mainly analyzed through genomic comparison with complete genomes of other D. zeae strains with high virulence (EC1, EC2, MS1, and MS2). The results revealed that plant cell wall-degrading extracellular enzymes (PCWDEs), flagellar and chemotaxis, and quorum sensing were highly conserved in all analyzed genomes, which were confirmed through phenotypic assays. Besides, WH1 harbored type I, II, III, and VI secretion systems (T1SS, T2SS, T3SS, and T6SS), but lost T4SS and T5SS. Like strains MS1 and MS2 isolated from bananas, WH1 harbored genes encoding both capsule polysaccharide (CPS) and exopolysaccharide (EPS) biosynthesis. The results of pathogenicity assays demonstrated that WH1 produced severe soft rot symptoms on potato tubers, carrots, radishes, and Chinese cabbage. Meanwhile, WH1 also produced phytotoxin(s) to inhibit rice seed germination with an 87% inhibitory rate in laboratory conditions. More importantly, we confirmed that phytotoxin(s) produced by WH1 are different from zeamines produced by EC1. Comparative genomics analyses and phenotypic and pathogenicity assays suggested that WH1 likely evolved through a pathway different from the other D. zeae strains from rice, producing a new type of rice foot rot pathogen. These findings highlight the emergence of a new type of D. zeae strain with high virulence, causing soft rot in rice and other plants.

Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680.

A malate dehydrogenase (MDH) from Streptomyces avermitilis MA-4680 (SaMDH) has been expressed and purified as a fusion protein. The molecular mass of SaMDH is about 35 kDa determined by SDS-PAGE. The recombinant SaMDH has a maximum activity at pH 8.0. The enzyme shows the optimal temperature around 42 °C and displays a half-life (t(1/2)) of 160 min at 50°C which is more thermostable than reported MDHs from most bacteria and fungi. The k(cat) value of SaMDH is about 240-fold of that for malate oxidation. In addition, the k(cat)/K(m) ratio shows that SaMDH has about 1,246-fold preference for oxaloacetate (OAA) reduction over L-malate oxidation. The recombinant SaMDH may also use NADPH as a cofactor although it is a highly NAD(H)-specific enzyme. There was no activity detected when malate and NADP(+) were used as substrates. Substrate inhibition studies show that SaMDH activity is strongly inhibited by excess OAA with NADH, but is not sensitive to excess L-malate. Enzymatic activity is enhanced by the addition of Na(+), NH(4)(+), Ca(2+), Cu(2+) and Mg(2+) and inhibited by addition of Hg(2+) and Zn(2+). MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors. The enzymatic analysis could provide the important basic knowledge for its utilizations.

Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680.

A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45°C, respectively. Heat-inactivation studies showed that heating for 20 min at 50°C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP+ as electron acceptor. Apparent Km values were 4.98 µM for NADP+ and 6,620 µM for NAD+, respectively, using Mn2+ as divalent cation. The enzyme performed a 33,000-fold greater specificity (kcat/Km) for NADP+ than NAD+. Moreover, SaIDH activity was entirely dependent on the presence of Mn2+ or Mg2+, but was strongly inhibited by Ca2+ and Zn2+. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP+.

Persistent organic pollution in Antarctic marine biota: Level, transport and risk assessment.

Although the Antarctic is considered as a pristine region away from human pollution, traditional and emerging persistent organic pollutants (POPs), such as organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and per-/polyfluoroalkyl substances (PFASs), have been constantly detected in the Antarctic, which received global concerns. POPs are persistent and toxic, prone to accumulate in organisms and further pose environmental risks. In order to understand their biogeochemical processes as well as impacts on organisms in the Antarctic, we summarized the research status of POPs in different trophic levels in the Antarctic marine, combining their living habits. We also proposed the future research directions of POPs in the vulnerable Antarctic ecosystem. Researches showed that the Antar-ctic was the region under the lowest pollution level all over the world. However, the types of POPs in Antarctic marine organisms were increasing in the past decades, meaning the area was affected increasingly by the activities inside and/or outside of this area. Scattered research data and different techniques hamper red the elucidation about the mechanism of POPs transport along the food chain. It is urgent to establish long-term monitoring and assessment program on POPs dynamics of Antarctic marine ecosystem.

Isocitrate dehydrogenase 1 from Acinetobacter baummanii (AbIDH1) enzymatic characterization and its regulation by phosphorylation.

Acinetobacter baumannii encodes all enzymes required in the tricarboxylic acid (TCA) cycle and glyoxylate bypass except for isocitrate dehydrogenase kinase/phosphatase (IDHKP), which can phosphorylate isocitrate dehydrogenase (IDH) at a substrate-binding Ser site and control the carbon flux in enterobacteria, such as Escherichia coli. The potential kinase was not successfully pulled down from A. baumannii cell lyase; therefore, whether the IDH 1 from A. baumannii (AbIDH1) can be phosphorylated to regulate intracellular carbon flux has not been clarified. Herein, the AbIDH1 gene was cloned, the encoded protein was expressed and purified to homogeneity, and phosphorylation and enzyme kinetics were evaluated in vitro. Gel filtration and SDS-PAGE analyses showed that AbIDH1 is an 83.5 kDa homodimer in solution. The kinetics showed that AbIDH1 is a fully active NADP-dependent enzyme. The Michaelis constant Km is 46.6 (Mn2+) and 18.1 µM (Mg2+) for NADP+ and 50.5 (Mn2+) and 65.4 µM (Mg2+) for the substrate isocitrate. Phosphorylation experiments in vitro indicated that AbIDH1 is a substrate for E. coli IDHKP. The activity of AbIDH1 treated with E. coli IDHKP immediately decreased by 80% within 9 min. Mass spectrometry indicated that the conserved Ser113 of AbIDH1 is phosphorylated. Continuous phosphorylation-mimicking mutants (Ser113Glu and Ser113Asp) lack almost all enzymatic activity. Side-chain mutations at Ser113 (Ser113Thr, Ser113Ala, Ser113Gly and Ser113Tyr) remarkably reduce the enzymatic activity. Understanding the potential of AbIDH1 phosphorylation enables further investigations of the AbIDH1 physiological functions in A. baumannii.

Identification and biochemical characterization of a thermostable malate dehydrogenase from the mesophile Streptomyces coelicolor A3(2).

We identified and characterized a malate dehydrogenase from Streptomyces coelicolor A3(2) (ScMDH). The molecular mass of ScMDH was 73,353.5 Da with two 36,675.0 Da subunits as analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The detailed kinetic parameters of recombinant ScMDH are reported here. Heat inactivation studies showed that ScMDH was more thermostable than most MDHs from other organisms, except for a few extremely thermophile bacteria. Recombinant ScMDH was highly NAD(+)-specific and displayed about 400-fold (k(cat)) and 1,050-fold (k(cat)/K(m)) preferences for oxaloacetate reduction over malate oxidation. Substrate inhibition studies showed that ScMDH activity was inhibited by excess oxaloacetate (K(i)=5.8 mM) and excess L-malate (K(i)=12.8 mM). Moreover, ScMDH activity was not affected by most metal ions, but was strongly inhibited by Fe(2+) and Zn(2+). Taken together, our findings indicate that ScMDH is significantly thermostable and presents a remarkably high catalytic efficiency for malate synthesis.