Miyoun Yoo

Trisindoline synthesis and anticancer activity

Expression of a Rhodococcus-derived oxygenase gene in Escherichia coli yielded indigo metabolites with cytotoxic activity against cancer cells. Bioactivity-guided fractionation of these indigo metabolites led to the isolation of trisindoline as the agent responsible for the observed in vitro cytotoxic activity against cancer cells. While the cytotoxicity of etoposide, a common anticancer drug, was dramatically decreased in multidrug-resistant (MDR) cancer cells compared with treatment of parental cells, trisindoline was found to have similar cytotoxicity effects on both parental and MDR cell lines. In addition, the cytotoxic effects of trisindoline were resistant to P-glycoprotein overexpression, one of the most common mechanisms of drug resistance in cancer cells, supporting its use to kill MDR cancer cells.

Draft genome sequence and comparative analysis of the superb aromatic-hydrocarbon degrader Rhodococcus sp. strain DK17.

Rhodococcus sp. strain DK17 is capable of utilizing various derivatives of benzene and bicyclics containing both aromatic and alicyclic moieties as sole carbon and energy sources. Here, we present the 9,107,362-bp draft genome sequence of DK17 and its genomic analysis in comparison with other members of the genus Rhodococcus.

Differential degradation of bicyclics with aromatic and alicyclic rings by Rhodococcus sp. strain DK17

ABSTRACT The metabolically versatile Rhodococcus sp. strain DK17 is able to grow on tetralin and indan but cannot use their respective desaturated counterparts, 1,2-dihydronaphthalene and indene, as sole carbon and energy sources. Metabolite analyses by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry clearly show that (i) the meta-cleavage dioxygenase mutant strain DK180 accumulates 5,6,7,8-tetrahydro-1,2-naphthalene diol, 1,2-indene diol, and 3,4-dihydro-naphthalene-1,2-diol from tetralin, indene, and 1,2-dihydronaphthalene, respectively, and (ii) when expressed in Escherichia coli, the DK17 o-xylene dioxygenase transforms tetralin, indene, and 1,2-dihydronaphthalene into tetralin cis-dihydrodiol, indan-1,2-diol, and cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, respectively. Tetralin, which is activated by aromatic hydroxylation, is degraded successfully via the ring cleavage pathway to support growth of DK17. Indene and 1,2-dihydronaphthalene do not serve as growth substrates because DK17 hydroxylates them on the alicyclic ring and further metabolism results in a dead-end metabolite. This study reveals that aromatic hydroxylation is a prerequisite for proper degradation of bicyclics with aromatic and alicyclic rings by DK17 and confirms the unique ability of the DK17 o-xylene dioxygenase to perform distinct regioselective hydroxylations.

Benzylic and aryl hydroxylations of m-xylene by o-xylene dioxygenase from Rhodococcus sp. strain DK17

Escherichia coli cells expressing Rhodococcus DK17 o-xylene dioxygenase genes were used for bioconversion of m-xylene. Gas chromatography–mass spectrometry analysis of the oxidation products detected 3-methylbenzylalcohol and 2,4-dimethylphenol in the ratio 9:1. Molecular modeling suggests that o-xylene dioxygenase can hold xylene isomers at a kink region between α6 and α7 helices of the active site and α9 helix covers the substrates. m-Xylene is unlikely to locate at the active site with a methyl group facing the kink region because this configuration would not fit within the substrate-binding pocket. The m-xylene molecule can flip horizontally to expose the meta-position methyl group to the catalytic motif. In this configuration, 3-methylbenzylalcohol could be formed, presumably due to the meta effect. Alternatively, the m-xylene molecule can rotate counterclockwise, allowing the catalytic motif to hydroxylate at C-4 yielding 2,4-dimethylphenol. Site-directed mutagenesis combined with structural and functional analyses suggests that the alanine-218 and the aspartic acid-262 in the α7 and the α9 helices play an important role in positioning m-xylene, respectively.

Anthranilate degradation by a cold-adapted Pseudomonas sp.

An alpine soil bacterium Pseudomonas sp. strain PAMC 25931 was characterized as eurypsychrophilic (both psychrophilic and mesotolerant) with a broad temperature range of 5–30 °C both for anthranilate (2‐aminobenzoate) degradation and concomitant cell growth. Two degradative gene clusters (antABC and catBCA) were detected from a fosmid clone in the PAMC 25931 genomic library; each cluster was confirmed to be specifically induced by anthranilate. When expressed in Escherichia coli, the recombinant AntABC (anthranilate 1,2‐dioxygenase, AntDO) converted anthranilate into catechol, exhibiting strict specificity toward anthranilate. Recombinant CatA (catechol 1,2‐dioxygenase, C12O) from the organism was active over a broad temperature range (5–37 °C). However, CatA rapidly lost the enzyme activity when incubated at above 25 °C. For example, 1 h‐preincubation at 37 °C resulted in 100% loss of enzyme activity, while a counterpart from mesophilic Pseudomonas putida mt‐2 did not show any negative effect on the initial enzyme activity. These results suggest that CatA is a new cold‐adapted thermolabile enzyme, which might be a product through the adaptation process of PAMC 25931 to naturally cold environments and contribute to its ability to grow on anthranilate there.

Characterization and engineering of an o-xylene dioxygenase for biocatalytic applications.

Depending on the size and position of the substituent groups on the aromatic ring, the o-xylene dioxygenase from Rhodococcus sp. strain DK17 possesses the unique ability to perform distinct regioselective hydroxylations via differential positioning of substrates within the active site. The substrate-binding pocket of the DK17 o-xylene dioxygenase is large enough to accommodate bicyclics and can be divided into three regions (distal, central, and proximal), and hydrophobic interactions in the distal position are important for substrate binding. Current molecular and functional knowledge contribute insights into how to engineer this enzyme to create tailor-made properties for chemoenzymatic syntheses.

Abstract C200: Effects of indirubin derivatives on the FLT3 activity and growth of acute myeloid leukemia cell lines

Indirubin is an active constituent of traditional Chinese recipe used for the treatment of chronic myelocytic leukemia. In this study, inhibitory activity of indirubin and its derivatives toward Fms‐like tyrosine kinase 3 (FLT3) was examined. Indirubin‐3′‐oxime(IO) and 6‐bromoindirubin‐3′‐oxime(BIO) showed potent inhibitory activity against FLT3 with 50% inhibitory concentration (IC50) of 79 nM and 254 nM, respectively. Meanwhile, indirubin and 6‐bromoindirubin exhibited negligible effect on FLT3 inhibitory activity up to 10 µM. We also tested the cytotoxicity of those compounds against acute myeloid leukemia cell lines; MV4;11 cells harboring constitutively activated form of FLT3 and RS4;11 cells with wild type FLT3. IO and BIO potently inhibited the growth of MV4;11 cells with IC50 of 30 nM and 61 nM, respectively. On the other hand, RS4;11 cells were far less sensitive to those compounds with IC50 values of 3600 nM and 820 nM, respectively. In the cell cycle assay, IO arrested the cell cycle of MV4;11 cells at G1 phase, and increased dead cell population at sub‐G1 phase about 25 and 50% at 0.1 and 1.0 µM after 48 hours, respectively. These results strongly suggest that the derivatives of IO have potentials to be developed for novel anti‐leukemic agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C200.