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 (KD) 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 KD 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.

Cancer cell invasion is one of the crucial events in local spreading, growth, and metastasis of tumors. The present study investigated the antiinvasive and antimetastatic action of gambogic acid (GA) in MDA-MB-435 human breast carcinoma cells. GA caused a concentration-dependent suppression of cell invasion through Matrigel and significantly inhibited lung metastases of the cells transplanted in vivo. The potent effects of GA have been attributed to its ability to reduce the expression of matrix metalloproteinases (MMP) 2 and 9 in vitro and in vivo both at the protein and mRNA levels, which were associated with protein kinase C (PKC) signaling pathway as supported by the diminished antiinvasive effect of GA in the presence of specific activator of the pathway. Collectively, our data demonstrated that GA exhibited antiinvasion properties on highly invasive cancer cells via PKC mediated MMP-2/9 expression inhibition. This indicated that GA can be served as a potential novel therapeutic candidate for the treatment of cancer metastasis.

•Chloroperoxidase efficiently converts diclofenac and naproxen under mild conditions.•Complete conversion is reached in 9 min and 7 min respectively with 10−9 mol L−1 enzyme.•The enzymatic conversion pathways of the drugs was proposed according to MS and NMR assay.•Converted products are significantly less toxic than the parent drugs.•CPO-H2O2 conversion followed by activated sludge treatment greatly improved the drugs removal.Non-steroidal anti-inflammatory drugs diclofenac and naproxen are widely used for the treatment of arthritis, ankylosing spondylitis, and acute muscle pain. However, most of them are usually not metabolized and simply pass through human body. These drugs are difficult to be decomposed by general waste treatment strategies and have caused serious environmental concerns. We report a rapid and efficient conversion of diclofenac and naproxen by chloroperoxidase-catalyzed H2O2-oxidation, a heme protein isolated from Caldarimyces fumago. Complete conversion of diclofenac and naproxen was achieved in only 9 and 7 min respectively with 0.1 mmol L−1 H2O2 and nanomolar enzyme concentration at pH 3.0. The converted products were identified by HPLC–MS and NMR, suggesting involvement of multiple steps in CPO catalyzed conversion. Our work demonstrated that CPO treatment (with COD removal of 4.9%, 9.1% for diclofenac and naproxen, respectively) followed by existing bioremediation technologies (activated sludge) greatly improved the decontaminating these two drugs from waste water (COD removal was enhanced to 85% and 86%, respectively). The eco-toxicity evaluation according to the 72-h EC50 value using the green algae Chlorella Pyrenoidos as ecological indicators showed that the converted products of diclofenac and naproxen had lower toxicity than the original drugs.Chloroperoxidase efficiently degrades diclofenac and naproxen under mild conditions. Complete degradation is in 9 min and 7 min respectively using 10−9 mol L−1 enzyme.Download high-res image (100KB)Download full-size image