Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase

Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen <i>Phomopsis amygdali</i> that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While <i>in vitro</i> incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly

Construction of the <i>orf13</i> disruptant and LC/MS analyses of the product accumulated in a culture broth of the <i>orf13</i> disruptants.

<p>(A) A strategy for disruption of the <i>orf13</i> by a double crossover is schematically shown. Arrows indicate the primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042090#pone.0042090.s006" target="_blank">Table S1</a>) used in PCR analysis, which correspond to the upstream and downstream of the <i>orf13</i>. (B) Disruption was confirmed by agarose gel electrophoresis of the PCR-amplified fragment. (C) The product accumulated in a culture broth of the <i>orf10</i> disruptant (peak B) was confirmed to be <b>13</b> by LC/MS analysis.</p

Fusicoccin A biosynthetic gene clusters cloned in our previous (A) and this study (B), respectively.

<p>Fusicoccin A biosynthetic gene clusters cloned in our previous (A) and this study (B), respectively.</p

Effects of abscisic acid (ABA) treatment on the expression levels of gibberellin (GA) metabolism genes in imbibed lettuce seeds

(A) The expression levels of the genes were analysed by QRT-PCR. The results were normalized to the expression of 18S rRNA (internal control), and the expression levels of all genes examined are given relative to the reference value of the transcript level of at 0 h, set to 1. Three independent experiments were performed, and means with standard errors are shown. (B) Expression analysis using seeds that had been cut in half. The results were normalized to the expression of 18S rRNA (internal control), and the highest value was set to 100. Two independent experiments were performed, and means with standard errors are shown.<p><b>Copyright information:</b></p><p>Taken from "Germination of photoblastic lettuce seeds is regulated via the control of endogenous physiologically active gibberellin content, rather than of gibberellin responsiveness"</p><p></p><p>Journal of Experimental Botany 2008;59(12):3383-3393.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2529229.</p><p></p

HPLC traces of the broths of the orf13 disruptant (upper), the orf10 disruptant (middle), and the parental strain (lower).

<p>HPLC traces of the broths of the orf13 disruptant (upper), the orf10 disruptant (middle), and the parental strain (lower).</p

Changes in the transcript levels of gibberellin (GA) metabolism genes in the cotyledon end and hypocotyl end of lettuce seeds

(A) Frozen seeds were divided into two parts: cotyledon end, including the cotyledons (Cot), fruit wall (FW), seed coat (SC), and endosperm (ES); and the hypocotyl end, including the hypocotyl (Hyp), root apical meristem (RAM), shoot apical meristem (SAM), and part of the Cot, FW, SC, and ES. (B) Expression levels of GA metabolism genes after light treatment, determined using QRT-PCR. See for light treatments. The results were normalized to the expression of 18S rRNA (internal control), and the highest value was set to 100. Two independent experiments were performed, and means with standard errors are shown.<p><b>Copyright information:</b></p><p>Taken from "Germination of photoblastic lettuce seeds is regulated via the control of endogenous physiologically active gibberellin content, rather than of gibberellin responsiveness"</p><p></p><p>Journal of Experimental Botany 2008;59(12):3383-3393.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2529229.</p><p></p

Changes in the transcript levels of gibberellin (GA) metabolism genes in imbibed lettuce seeds after various light treatments

(A) Time-course of lettuce seed germination after light treatment. Zero (0) h indicates seeds imbibed for 3 h in the dark that received no light treatment. FR, FR/R, and FR/R/FR indicate seeds treated with far-red light, far-red followed by red light, and far-red followed by red and then far-red light, respectively. FR/R+ABA indicates seeds treated with far-red followed by red light and 0.1 mM ABA. Triplicate experiments were performed, and means with standard errors are shown. (B) Expression levels of GA metabolism genes after light treatment. The expression levels of these genes were analysed by QRT-PCR. The results were normalized to the expression of 18S rRNA (internal control); the expression levels of all genes examined are given relative to the reference value of the transcript level of at 0 h, set to 1. Three independent experiments were performed, and means with standard errors are shown.<p><b>Copyright information:</b></p><p>Taken from "Germination of photoblastic lettuce seeds is regulated via the control of endogenous physiologically active gibberellin content, rather than of gibberellin responsiveness"</p><p></p><p>Journal of Experimental Botany 2008;59(12):3383-3393.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2529229.</p><p></p

Expression of and during germination, and gibberellin (GA) responsiveness in lettuce seeds

(A) Expression levels of and after light treatment. The expression analysis was carried out by QRT-PCR. See for light treatments. The results were normalized to the expression of 18S rRNA (internal control), and the expression levels of all genes examined are given relative to the reference value of the transcript level of at 0 h, set to 1. Three independent experiments were performed, and means with standard errors are shown. (B) Expression levels of and in the cotyledon end and the hypocotyl end of lettuce seeds after light treatment. The results were normalized to the expression of 18S rRNA (internal control), and the highest value was set to 100. Two independent experiments were performed, and means with standard errors are shown. (C) Germination frequency of lettuce seeds in the presence of a GA biosynthesis inhibitor and various concentrations of GA. Five sets of 20 decoated lettuce seeds were incubated in the dark at 25 °C in medium containing 50 μM uniconazol-P and various concentrations of GA. After red light treatment, seeds were incubated at 25 °C in the dark for 24 h and the germination frequency was recorded. Means with standard errors are shown.<p><b>Copyright information:</b></p><p>Taken from "Germination of photoblastic lettuce seeds is regulated via the control of endogenous physiologically active gibberellin content, rather than of gibberellin responsiveness"</p><p></p><p>Journal of Experimental Botany 2008;59(12):3383-3393.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2529229.</p><p></p

SDS-PAGE analysis of the purified enzymes.

<p>(A) molecular mass markers (lane 1) and purified methyltransferase (lane 2). (B) molecular mass markers (lane 1) and purified acetyltransferase (lane 2). (C) molecular mass markers (lane 1) and purified glycosyltransferase (lane 2).</p

Construction of the <i>orf10</i> disruptant and LC/MS analyses of the product accumulated in a culture broth of the <i>orf10</i> disruptants.

<p>(A) A strategy for disruption of the <i>orf10</i> by a double crossover is schematically shown. Arrows indicate the primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042090#pone.0042090.s006" target="_blank">Table S1</a>) used in PCR analysis, which correspond to the upstream and downstream of the <i>orf10</i>. (B) Disruption was confirmed by agarose gel electrophoresis of the PCR-amplified fragment. (C) The product accumulated in a culture broth of the <i>orf10</i> disruptant (peak A) was confirmed to be <b>10</b> by LC/MS analysis.</p