The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism

The final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (<b>1</b>) is the highly unusual cytochrome P450-catalyzed, oxidative rearrangement of pentalenolactone F (<b>2</b>), involving the transient generation and rearrangement of a neopentyl cation. In <i>Streptomyces arenae</i> this reaction is catalyzed by CYP161C2 (PntM), with highly conserved orthologs being present in at least 10 other Actinomycetes. Crystal structures of substrate-free PntM, as well as PntM with bound substrate <b>2</b>, product <b>1</b>, and substrate analogue 6,7-dihydropentalenolactone F (<b>7</b>) revealed interactions of bound ligand with three residues, F232, M77, and M81 that are unique to PntM and its orthologs and absent from essentially all other P450s. Site-directed mutagenesis, ligand-binding measurements, steady-state kinetics, and reaction product profiles established there is no special stabilization of reactive cationic intermediates by these side chains. Reduced substrate analogue <b>7</b> did not undergo either oxidative rearrangement or simple hydroxylation, suggesting that the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of <b>2</b>. The crystal structures also revealed plausible proton relay networks likely involved in the generation of the key characteristic P450 oxidizing species, Compound I, and in mediating stereospecific deprotonation of H-3_<i>re</i> of the substrate. We conclude that the unusual carbocation intermediate results from outer shell electron transfer from the transiently generated C1 radical to the tightly paired heme-•Fe³⁺–OH radical species. The oxidative electron transfer is kinetically dominant as a result of the unusually strong steric barrier to oxygen rebound to the neopentyl center C-1_<i>si</i>, which is flanked on each neighboring carbon by <i>syn</i>-axial substituents

Beyond the Spitfire: Re-visioning Latinas in Sylvia Morales’ A Crushing Love

From Dolores del Río to Salma Hayek and from Lupe Vélez to Eva Longoria, the portrayal of Latinas in the United States has provoked debate, criticism and controversy. Since the era of silent movies, Hollywood’s depiction of the Latina has been rigidly prescribed and reductive, while Latinos and Latinas themselves have contested these problematic portrayals in more nuanced and multifaceted visions of their communities. Sylvia Morales’s documentary A Crushing Love: Chicanas, Motherhood and Activism (2009) presents a vision of activists and mothers that is radically at odds with the stereotypes of Latinas in Hollywood film and in the US media more generally.1 This chapter examines the film against the context of persistently problematic representation of Latinas in the media and the ways in which Morales’ film counters demeaning images of Chicanas through a multilayered examination of different experiences of motherhood by politically radical women

A long 15-bp motif increases CLAMP binding to DNA.

<p><b>A)</b> Motifs obtained from the custom gcPBMs are shown: I) The PBM+ChIP+ motif represents the sequences that CLAMP binds both <i>in vivo</i> and <i>in vitro</i>. II) The PBM-ChIP- motif represents the sequences that are on the array but not bound by CLAMP <i>in vivo</i> or <i>in vitro</i>. The most conserved 8-bp core element is indicated by vertical dashed lines. <b>B)</b> A representation of the methodology to define the minimal CLAMP-bound motif by scanning both 5’ and 3’ of the core motif. The table shows the percentage of PBM+ChIP+ (CLAMP binding both <i>in vitro</i> and <i>in vivo</i>) sequences that overlap with PBM-ChIP- sequences (CLAMP binding neither <i>in vitro</i> nor <i>in vivo</i>) at the specified motif size. The y-axis shows the nucleotides 5’ of the 8-bp core motif and the x-axis shows the nucleotides 3’. The scale ranges from green for the maximal values to red for the minimal values. Values show the percentage of PBM+ChIP+ sequences shared with PBM-ChIP- sequences for the length window selected. A value of zero overlapping sequences represents complete separation between PBM+ChIP+ and PBM-ChIP- sequences and is obtained at 4-bp 5’ and 3-bp 3’ of the core motif. <b>C)</b> CLAMP and MSL ChIP-seq enrichments are shown for sequences containing the 8-bp core with and without additional flanking sequence matching the motif. Motif hits were found using the FIMO tool (p < E-4). All 8-bp core hits were found first and the ones overlapping with the full 15-bp motif were separated as ‘8bp + matched endogenous flank’ and the rest were grouped as ‘8bp + unmatched endogenous flank’. Since the ‘8bp + unmatched endogenous flank’ group has ~10,000 sites, the top 10,000 enrichments are shown in the CLAMP enrichment plot. Since there are ~300 CES, the top 300 enrichments are shown in the MSL enrichment plot. <b>D)</b> Binding intensities are shown for the following classes of probes: 1) probes with the optimal motif (8-bp + matched endogenous flank, red); 2) probes that have matching 8-bp core regions but the endogenous flanks do not match the motif (8-bp + unmatched endogenous flanks, blue); 3) probes that have 8-bp cores with synthetic constant flanking sequences (8-bp + unmatched synthetic flank, cyan); 4) probes that do not have the 8-bp core motif (without 8-bp, brown); 5) Intensities for C-terminal 4 zinc finger GST fusion proteins are shown for probes containing the 15-bp CLAMP motif (15–bp, 4ZF, orange). <b>E)</b> CLAMP binds to DNA containing a high affinity, 8 bp + matched flank motif in an electrophoretic mobility shift assay. Biotin-labeled DNA alone (lane 1) and DNA with MBP (lane 2) do not shift, while MBP-CLAMP forms a complex with DNA to shift the signal. This was competed away with specific (high affinity) competitor but not a non-specific competitor that contains the 8-bp core but lacks endogenous flanking sequences (8-bp + unmatched synthetic flank).</p

CLAMP binds to similar <i>in vivo</i> X-enriched binding sites in <i>D</i>. <i>miranda</i> and <i>D</i>. <i>melanogaster</i>.

<p><b>A)</b> The ratio of <i>in vitro</i> CLAMP binding site density (number of CLAMP binding sequence hits per Mb) for X versus autosomes is plotted for <i>D</i>. <i>miranda</i>, <i>D</i>. <i>melanogaster</i> and <i>A</i>. <i>gambiae</i>. Autosomes of <i>D</i>. <i>melanogaster</i> are chromosomes 2L (Muller-B), 2R (Muller-C), 3L (Muller-D), 3R (Muller-E) and 4 (Muller-F); autosomes of <i>D</i>.<i>miranda</i> are chromosomes 2 (Muller-E), 4 (Muller-B) and 5 (Muller-F); and autosomes of <i>A</i>. <i>gambiae</i> are chromosomes 2L (Muller-D), 2R (Muller-E), 3L (Muller-C), and 3R (Muller-B). <b>B)</b> The density ratios of GA-repeats on individual X-chromosome(s) vs. autosomes for different repeat lengths in the <i>D</i>. <i>melanogaster</i>, <i>D</i>. <i>miranda</i> and <i>A</i>. <i>gambiae</i> genomes are plotted. Any value above 1 indicates a higher repeat density on the X-chromosome compared to autosomes. <b>C)</b> CLAMP ChIP-seq motifs are shown for <i>D</i>. <i>melanogaster</i> and <i>D</i>. <i>Miranda</i> larval ChIP-seq data. Motifs are found using MEME-ChIP for the peak regions (peak centers +/-100bp).</p

Increasing the number of GA-dinucleotide repeats increases CLAMP occupancy.

<p><b>A)</b> A multiple linear regression to test contribution of sequence length (k-mer) and shape to overall binding. Adding dinucleotide (2mer) features to the sequence-only (1mer) model increases the performance more than adding DNA shape features, indicating the importance of dinucleotides in CLAMP-DNA recognition. Adding trinucleotide (3mer) features further increases the performance marginally. <b>B)</b> CLAMP PBM binding for GA-dinucleotide repeats of different lengths is plotted as box plot distributions. The y-axis is the PBM intensity score for each number of GA-repeats, which are plotted along the x-axis, e.g. 1 = GA, 2 = GAGA. <b>C)</b> An electrophoretic mobility shift assay to test MBP-CLAMP binding to increasing numbers of GA-repeats. The labeled probes contain GA-repeats of 4 (8-bp), 8 (16-bp), 10 (20-bp) and 15 (30-bp) centered within a 60-bp sequence. The first four lanes are reactions containing MBP control protein with DNA, and the next four are MBP-CLAMP with DNA. <b>D)</b> Input-normalized CLAMP ChIP-seq signal enrichments at GA-repeats of different lengths are given for the X-chromosome (red) and autosomes (blue) from male S2 (top) and female Kc cells (bottom). The x-axis shows the number of GA-repeats e.g. 1 = GA, 2 = GAGA.</p

EMSA probe sequences.

<p>EMSA probe sequences.</p

CLAMP directly binds to a long GA-rich motif that is enriched at <i>in vivo</i> targets.

<p><b>A)</b> The following motifs are shown: I) MRE motif [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006120#pgen.1006120.ref013" target="_blank">13</a>]. II) CLAMP <i>in vivo</i> motif derived from ChIP-seq data from S2 cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006120#pgen.1006120.ref020" target="_blank">20</a>]; III) CLAMP <i>in vitro</i> motif derived from the uPBM [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006120#pgen.1006120.ref020" target="_blank">20</a>] and IV) CLAMP <i>in vitro</i> motif that we derived from the custom gcPBM. The most conserved part of the motifs, their 8-bp core, is highlighted between two dashed lines. <b>B)</b> gcPBM intensities (intensity from genomic context PBM) (top) and CLAMP ChIP signal (input normalized RPKM: Reads per Kb per Million) for S2 (middle) and Kc (bottom) cells are plotted for 800 bp windows centered at each of two MSL complex CES (<i>roX1</i>, left, and <i>roX2</i>, <i>right</i>). MRE sequences are shown as blue dashes on each profile. <b>C)</b> A histogram of intensities from the gcPBM experiment. The intensity of 6500, between the two peaks of the bimodal distribution, is indicated with a dashed line. Probes with intensities higher than 6500 are designated PBM+ and those that are lower than 6500 are considered PBM-. <b>D)</b> Box plots are used to compare the intensities for probes sorted by whether they contain sequences that are bound or unbound from <i>in vivo</i> ChIP-seq data (ChIP +/-) and whether or not they conform to the previously characterized MRE motif (MRE+/-). In the right half of the panel, probes were resorted to add the category of bound or unbound on the PBM (PBM+/-). p-values for comparisons between categories are displayed above the relevant boxes, and the number of probes for each category are listed for each group. For each box plot used throughout this study, the collared box indicates that 95% percent confidence interval. If the notches at the center of the box are not overlapping, it indicates that two samples are statistically different from each other.</p