Xiaotang Wang

The chemical identification of the modified heme (the green heme) during chloroperoxidase catalyzed epoxidation of allylbenzene remains unestablished due to its high instability within the protein matrix, the absence of paramagnetically shifted signals, and the difficulty in obtaining crystals of the modified enzyme. We have established the unambiguous structure of the modified prosthetic heme group, which was extracted from the protein matrix using ²D NMR spectroscopy and LC-MS spectrometry. The modified heme was isolated as a µ-oxo dimer that can be quantitatively converted to the corresponding monomer. The depolymerized green heme displayed characteristic NMR signatures of iron porphyrin complexes, but no Nuclear Overhauser Effect was observable to assist in signal assignment. An alternative strategy was applied by removing the iron center of the green heme, resulting in a stable demetallated green porphyrin species. Complete assignment of all the NMR resonances in the demetallated green heme allowed us to establish the molecular architecture of the modified species as a novel N-alkylated heme. Decisive space correlations between the propyl protons of allylbenzene and the γ meso proton coupled with clear dipolar connectivities between the propyl-2H of the substrate and the β proton in the side chain of the propionic acid at carbon-6 of the porphyrin ring, clearly indicate that allylbenzene was covalently attached to the nitrogen atom of the pyrrole ring III of the prosthetic heme. In this study, the mechanism of green CPO formation and its relation to CPO catalyzed chiral transformations are also discussed. It is concluded that the double-phenyl clamp formed by two phenylalanine residues at the distal heme pocket plays a critical role in fine-tuning substrate orientation that determines the outcome of CPO catalyzed epoxidation of substituted styrenes.

Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.

Squamous cell carcinoma (SCC) is a usually responds poorly to treatment suffers from poor therapeutic benefits while oroxylin A (OA) is a promising flavonoid with high anticancer efficacy against various cancer types. Here in our study, in order to reveal the potential of OA based drug delivery systems (DDSs) in the treatment of SCC, we firstly revealed that OA had a certain pharmacodynamic effect on skin SCC (A431 cells). Afterwards, OA was loaded into a newly synthesized aggregation-induced emission (AIE)-active polymer to construct OA-loaded PDots for the first time. Our results revealed that OA-loaded PDots showed preferable drug loading and enhanced stability. Moreover, the DDS was also capable of self-illumination in the aggregate state to reveal the uptake profile. Most importantly, the DDS showed much more elevated anticancer benefits than free OA in vitro and advanced tumor targetability in vivo, suggesting that it might be a promising system against SCC.

Non-small-cell lung carcinoma (NSCLC) continues to be a vital disease worldwide for its high incidence and consequent mortality rate. In this study, we investigated the anti-cancer effect of LZ-101, a new derivative of danofloxacin, against non-small-cell lung cancer and the underlying mechanisms. In vitro, LZ-101 inhibited the viability of human non-small cell lung cancer cell lines. We demonstrated that LZ-101 induced mitochondrial-mediated apoptosis by increasing Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (ΔΨm), release of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) in A549 cells. Further research illuminated that LZ-101 induced apoptosis was related to the activation of FOXO3a/Bim pathway. Moreover, we found that LZ-101 increased the stability of FOXO3a by blocking autophagy-dependent FOXO3a degradation. However, inhibition of autophagosome formation abolished FOXO3a stabilization and apoptosis induced by LZ-101. In vivo, LZ-101 exerted a remarkable anti-tumor activity with high safety in xenograft model inoculated A549 tumor through the same mechanism as in our in vitro study. In conclusion, our findings indicated that LZ-101 induces mitochondrial apoptosis and stabilizes FOXO3a by blocking autophagy flux.

Starch is an inexpensive and renewable raw material for numerous industrial applications. However, most starch-based products are not cost-efficient due to high-energy input needed in traditional enzymatic starch conversion processes. Therefore, α-amylase with high efficiency to directly hydrolyze high concentration raw starches at a relatively lower temperature will have a profound impact on the efficient application of starch. A novel raw starch digesting α-amylase (named AmyZ1) was screened and cloned from a deep-sea bacterium Pontibacillus sp. ZY. Phylogenetic analysis showed that AmyZ1 was a member of subfamily 5 of glycoside hydrolase family 13. When expressed in Escherichia coli, the recombinant AmyZ1 showed high activity at pH 6.0–7.5 and 25–50 °C. Its optimal pH and temperature were 7.0 and 35 °C, respectively. Similar to most α-amylases, AmyZ1 activity was enhanced (2.4-fold) by 1.0 mM Ca2+. Its half-life time at 35 °C was also extended from about 10 min to 100 min. In comparison, AmyZ1 showed a broad substrate specificity toward raw starches, including those derived from rice, corn, and wheat. The specific activity of AmyZ1 towards raw rice starch was 12,621 ± 196 U/mg, much higher than other reported raw starch hydrolases. When used in raw starch hydrolyzing process, AmyZ1 hydrolyzed 52%, 47% and 38% of 30% (w/v) rice, corn, and wheat starch after 4 h incubation. It can also hydrolyze marine raw starch derived from Chlorella pyrenoidosa, resulting in 50.9 mg/g DW (dry weight of the biomass) of reducing sugars after 4 h incubation at 35 °C. Furthermore, when hydrolyzing raw corn starch using the combination of AmyZ1 and commercial glucoamylase, the hydrolysis rate reached 75% after 4.5 h reaction, notably higher than that obtained in existing starch-processing industries. As a novel raw starch-digesting α-amylase with high specific activity, AmyZ1 efficiently hydrolyzed raw starches derived from both terrestrial and marine environments at near ambient temperature, suggesting its application potential in starch-based industrial processes.

P53 plays important roles in regulating the metabolic reprogramming of cancer, such as aerobic glycolysis. Oroxylin A is a natural active flavonoid with strong anticancer effects both in vitro and in vivo. wt-p53 (MCF-7 and HCT116 cells) cancer cells and p53-null H1299 cancer cells were used. The glucose uptake and lactate production were analyzed using Lactic Acid production Detection kit and the Amplex Red Glucose Assay Kit. Then, the protein levels and RNA levels of p53, mouse double minute 2 (MDM2), and p53-targeted glycolytic enzymes were quantified using Western blotting and quantitative polymerase chain reaction (PCR), respectively. Immunoprecipitation were performed to assess the binding between p53, MDM2, and sirtuin-3 (SIRT3), and the deacetylation of phosphatase and tensin homolog (PTEN). Reporter assays were performed to assess the transcriptional activity of PTEN. In vivo, effects of oroxylin A was investigated in nude mice xenograft tumor-inoculated MCF-7 or HCT116 cells. Here, we analyzed the underlying mechanisms that oroxylin A regulated p53 level and glycolytic metabolism in wt-p53 cancer cells, and found that oroxylin A inhibited glycolysis through upregulating p53 level. Oroxylin A did not directly affect the transcription of wt-p53, but suppressed the MDM2-mediated degradation of p53 via downregulating MDM2 transcription in wt-p53 cancer cells. In further studies, we found that oroxylin A induced a reduction in MDM2 transcription by promoting the lipid phosphatase activity of phosphatase and tensin homolog, which was upregulated via sirtuin3-mediated deacetylation. In vivo, oroxylin A inhibited the tumor growth of nude mice-inoculated MCF-7 or HCT116 cells. The expression of MDM2 protein in tumor tissue was downregulated by oroxylin A as well. These results provide a p53-independent mechanism of MDM2 transcription and reveal the potential of oroxylin A on glycolytic regulation in both wt-p53 and mut-p53 cancer cells. The studies have important implications for the investigation on anticancer effects of oroxylin A, and provide the academic basis for the clinical trial of oroxylin A in cancer patients.

Laccases have potential applications in detoxification of lignocellulosic biomass after thermochemical pretreatment and production of value-added products or biofuels from renewable biomass. However, their application in large-scale industrial and environmental processes has been severely thwarted by the high cost of commercial laccases. Therefore, it is necessary to identify new laccases with lower cost but higher activity to detoxify lignocellulosic hydrolysates and better efficiency to produce biofuels such as bioethanol. Laccases from Ganoderma lucidum represent proper candidates in processing of lignocellulosic biomass. G. lucidum 77002 produces three laccase isoenzymes with a total laccase activity of 141.1 U/mL within 6 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A new isoenzyme named Glac15 was identified, purified, and characterized. Glac15 possesses an optimum pH of 4.5 to 5.0 and a temperature range of 45°C to 55°C for the substrates tested. It was stable at pH values ranging from 5.0 to 7.0 and temperatures lower than 55°C, with more than 80% activity retained after incubation for 2 h. When used in bioethanol production process, 0.05 U/mL Glac15 removed 84% of the phenolic compounds in prehydrolysate, and the yeast biomass reached 11.81 (optimal density at 600 nm (OD600)), compared to no growth in the untreated one. Addition of Glac15 before cellulase hydrolysis had no significant effect on glucose recovery. However, ethanol yield were improved in samples treated with laccases compared to that in control samples. The final ethanol concentration of 9.74, 10.05, 10.11, and 10.81 g/L were obtained from samples containing only solid content, solid content treated with Glac15, solid content containing 50% prehydrolysate, and solid content containing 50% prehydrolysate treated with Glac15, respectively. The G. lucidum laccase Glac15 has potentials in bioethanol production industry.

To unravel the mechanism of chloroperoxidase (CPO)-catalyzed regioselective oxidation of indole, we studied the structure of the CPO–indole complex using nuclear magnetic resonance (NMR) relaxation measurements and computational techniques. The dissociation constant (K_D) of the CPO–indole complex was calculated to be approximately 21 mM. The distances (r) between protons of indole and the heme iron calculated via NMR relaxation measurements and molecular docking revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. Both K_D and r values are independent of pH in the range of 3.0–6.5. The stability and structure of the CPO–indole complex are also independent of the concentration of chloride or iodide ion. Molecular docking suggests the formation of a hydrogen bond between the NH group of indole and the carboxyl O of Glu 183 in the binding of indole to CPO. Simulated annealing of the CPO–indole complex using r values from NMR experiments as distance restraints reveals that the van der Waals interactions were much stronger than the Coulomb interactions in the binding of indole to CPO, indicating that the association of indole with CPO is primarily governed by hydrophobic rather than electrostatic interactions. This work provides the first experimental and theoretical evidence of the long-sought mechanism that leads to the “unexpected” regioselectivity of the CPO-catalyzed oxidation of indole. The structure of the CPO–indole complex will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications.