Discovery and Characterization of BlsE, a Radical <i>S</i>-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

<div><p>BlsE, a predicted radical <i>S</i>-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted <i>in vitro</i> to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in <i>blsE</i> mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe–4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe–4S]²⁺ cluster. Mutagenesis studies demonstrated that Cys₃₁, Cys_35, Cys₃₈ in the C×××C×MC motif and Gly₇₃, Gly₇₄, Glu₇₅, Pro₇₆ in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met₃₇ had little effect on its function. Our results indicate that BlsE represents a typical [4Fe–4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.</p></div

<i>In vitro</i> assays of the mutants.

<p>The BlsEC31A, C35A and C38A (abbreviated as BC31A, BC35A and BC38A in figure) represent respective mutant proteins in which cysteine in the C×××C×MC motif of BlsE was replaced by alanine; BM37F, BM37Y and BM37W (similarly abbreviated) represent mutant proteins in which methionine in the same motif was replaced by phenylalanine, tyrosine or tryptophan, respectively. BG73A, BG74A, BE75A and BP76A represent mutant versions of BlsE in which each amino acid in the GGEP motif was changed into an alanine.</p

Kinetic parameters for BlsE and select mutant versions of BlsE.

*<p>Enzyme kinetic parameters were tested using CGA as the substrate with the concentration ranged from 0.025–800 <i>μ</i>M, as indicated. All the reactions were incubated at 25°C.</p

Silencing of APE1 Enhances Sensitivity of Human Hepatocellular Carcinoma Cells to Radiotherapy <em>In Vitro</em> and in a Xenograft Model

<div><p>Resistance to radiotherapy is a key limitation for the treatment of human hepatocellular carcinoma (HCC). To overcome this problem, we investigated the correlation between radioresistance and the human apurinic/apyrimidinic endonuclease (APE1), a bifunctional protein, which plays an important role in DNA repair and redox regulation activity of transcription factors. In the present study, we examined the radiosensitivity profiles of three human HCC cell lines, HepG2, Hep3B, and MHCC97L, using the adenoviral vector Ad5/F35-mediated APE1 siRNA (Ad5/F35-siAPE1). The p53 mutant cell lines MHCC97L showed radioresistance, compared with HepG2 and Hep3B cells. APE1 was strongly expressed in MHCC97L cells and was induced by irradiation in a dose-dependent manner, and Ad5/F35-siAPE1 effectively inhibited irradiation-induced APE1 and p53 expression. Moreover, silencing of APE1 significantly potentiated the growth inhibition and apoptosis induction by irradiation in all tested human HCC cell lines. In addition, Ad5/F35-siAPE1 significantly enhanced inhibition of tumor growth and potentiated cell apoptosis by irradiation both in HepG2 and MHCC97L xenografts. In conclusion, down regulation of APE1 could enhance sensitivity of human HCC cells to radiotherapy <em>in vitro</em> and <em>in vivo</em>.</p> </div

Proposed mechanism of BlsE-catalyzed reaction.

<p>BlsE catalyzes the decarboxylation at C5 position of <b>8</b>, leading to the formation of <b>9</b>. SAM (<b>10</b>) serves as the source of methionine (<b>12</b>) and 5′-Ado radical (<b>13</b>), the latter could abstract the hydrogen from <b>8</b> and generate 5′-AdoH (<b>11</b>).</p

Activity of iron–sulfur cluster-reconstituted purified BlsE.

<p>(A) HPLC traces of BlsE catalytic reactions with <b>8</b>: (a) standard <b>8</b>; (b) standard <b>10</b>; (c) standard <b>11</b>; (d) <b>8</b>+<b>10</b>+BlsE. (B) Composite HPLC traces of various treatment. Sample 1 represents full reaction (CGA, SAM, dithiothreitol, sodium dithionite, and BlsE). Samples 2 and 3 represent <i>in vitro</i> BlsE reaction incubated aerobically and BlsE purified under aerobic conditions, respectively. Samples 4–7 represent full reactions without dithiothreitol, <b>8</b>, <b>10</b>, or sodium dithionite, respectively. All the experiments used sodium dithionite as the reductant.</p

UV-vis spectrum of BlsE.

<p>The purified BlsE has a size of 40.2 KD as estimated by SDS-PAGE analysis; the red trace represents purified and reconstituted BlsE (50 <i>μ</i>M), whereas the blue trace represents BlsE reduced by 1 mM sodium dithionite.</p

EPR spectra of BlsE under various conditions.

<p>(A) As-isolated sample of BlsE (120 <i>μ</i>M). (B) Reconstituted BlsE reduced by dithionite before loading into an EPR tube anaerobically. (C) B with 1 mM SAM. (D) C with 1 mM CGA. EPR conditions used were as follows: microwave frequency, 9.390 GHz; microwave power, 10 <i>μ</i>W; modulation amplitude, 2 Gauss; modulation frequency, 100 kHz; temperature, 13K. Each spectrum is the average of 80 scans.</p

Time dependence of CAP and 5′-AdoH production.

<p>Time dependence of CAP (◊) and 5′-AdoH (Δ) formation in the reaction with CGA. Error bars show SD, each data point represents the average of three replicates.</p

Activity assay of BlsE-catalyzed reaction by Fld, Fpr and NADPH.

<p>(A) Gel electrophoresis analysis (SDS-PAGE) of Fpr and Fld on the left side. Fpr and Fld came out as golden and light brown in 50 mM HEPES (pH 8.0) on the right side, respectively. (B) Activity of the natural reduction system. Sample 1 represents the reaction mixture containing SAM, CGA, sodium dithionite, and BlsE. Sample 2 has same reaction mixture as sample 1 but with sodium dithionite substituted by NADPH, Fpr and Fld. Samples 3–5 represent reaction 2 without NADPH, Fpr and Fld, respectively. Error bars show SD, each data point represents the average of three replicates.</p