Phylogenetic tree of ABBA prenyltransferases of the phenol / phenazine family.

<p>Data include previously biochemically characterised ABBA prenyltransferases and those investigated in this study. The tree was constructed with MEGA6 using default parameter for multiple sequence alignment (CLUSTALW) and neighbour-joining method. Bootstrap values (in percent) calculated from 1000 replications are shown at the respective nodes. The fungal indole prenyltransferase DMATS (shares PT barrel) serves as a root.</p

Genome-Based Discovery of a Novel Membrane-Bound 1,6-Dihydroxyphenazine Prenyltransferase from a Marine Actinomycete

<div><p>Recently, novel prenylated derivatives of 1,6-dihydroxyphenazine have been isolated from the marine sponge-associated <i>Streptomyces</i> sp. SpC080624SC-11. Genome sequencing of this strain now revealed a gene cluster containing all genes necessary for the synthesis of the phenazine and the isoprenoid moieties. Unexpectedly, however, the cluster did not contain a gene with similarity to previously investigated phenazine prenyltransferases, but instead a gene with modest similarity to the membrane-bound prenyltransferases of ubiquinone and menaquinone biosynthesis. Expression of this gene in <i>E. coli</i> and isolation of the membrane fraction proved that the encoded enzyme, Mpz10, catalyzes two successive prenylations of 1,6-dihydroxyphenazine. Mpz10 is the first example of a membrane-bound enzyme catalyzing the prenylation of a phenazine substrate, and one of few examples of membrane-bound enzymes involved in the prenylation of aromatic secondary metabolites in microorganisms.</p></div

Mixed-terpenoid secondary metabolites of <i>Streptomyces</i> sp. CNQ-509.

<p>Mixed-terpenoid secondary metabolites of <i>Streptomyces</i> sp. CNQ-509.</p

HPLC and LC-MS analysis of the reaction products of Mpz10.

<p>(A) and (B): Incubations containing 1,6-dihydroxyphenazine, dimethylallyl diphosphate (DMAPP), and Mg²⁺ with the membrane fractions of <i>E. coli</i> harboring either the empty vector pET-28a(+) (A) or the Mpz10 expression vector pPH23 (B). (C) Authentic DHDMP (structure see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#pone-0099122-g002" target="_blank">Figure 2B</a>). (D) and (E): Authentic JBIR-47 and JBIR-46 (structures see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#pone-0099122-g001" target="_blank">Figure 1B</a>). (F) Incubation containing 1-hydroxyphenazine, DMAPP, and Mg²⁺ with the membrane fraction of <i>E. coli</i> harboring the Mpz10 expression vector pPH23 (for the structure of <b>3</b> see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#pone-0099122-g005" target="_blank">Figure 5</a>). Mass spectra of the three enzymatic products are shown on the right. Detection: A-E, UV 275 nm; F, UV 368 nm.</p

Localization of the Mpz10 activity in the membrane fraction.

<p>Comparison of the prenyltransferase activity of different cell fractions. Activity was tested with 1,6-dihydroxyphenazine, DMAPP, and Mg²⁺ as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#s4" target="_blank">Experimental Procedures</a>. The membrane fraction was obtained by centrifugation at 100,000×<i>g</i>.</p

Phenazine biosynthesis in <i>Streptomyces</i> sp. SpC080624SC-11.

<p>(A) Putative biosynthetic gene cluster of JBIR-46, -47, and -48. (B) Proposed pathway for the biosynthesis of JBIR-46, -47, and -48. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#pone-0099122-t001" target="_blank">Table 1</a> for additional information on the genes contained in the depicted gene cluster.</p

Prenylation in the biosynthesis of phenazines.

<p>(A) Reaction catalyzed by the previously discovered prenyltransferases PpzP and EpzP. (B) Structures of the phenazines JBIR-46, -47, and -48 from <i>Streptomyces</i> sp. SpC080624SC-11.</p

Genes in the putative biosynthetic gene cluster for JBIR-46, -47, and -48.

<p>Genes in the putative biosynthetic gene cluster for JBIR-46, -47, and -48.</p

Biochemical investigation of the Mpz10 reaction.

<p>(A) HPLC analysis of the time-dependent formation of products <b>1</b> and <b>2</b>. Detection: UV, 275 nm. (B) and (C): Product formation at different concentrations of 1,6-dihydroxyphenazine and dimethylallyl diphosphate (DMAPP). In the experiments shown in (B), DMAPP was kept constant at 0.5 mM. In the experiments shown in (C), 1,6-dihydroxyphenazine was kept constant at 0.2 mM. <i>K</i>_m values were determined by nonlinear regression, using GraphPad Prism software. Data represent mean ± SD of triplicate determinations.</p

Structure of the enzymatic product 1-hydroxy-4-dimethylallyl-phenazine (3).

<p>Bold lines indicate key ¹H-¹H COSY and arrows key ¹H-¹³C HMBC correlations.</p