Evolutionary Relationships of Microbial Aromatic Prenyltransferases

The linkage of isoprenoid and aromatic moieties, catalyzed by aromatic prenyltransferases (PTases), leads to an impressive diversity of primary and secondary metabolites, including important pharmaceuticals and toxins. A few years ago, a hydroxynaphthalene PTase, NphB, featuring a novel ten-stranded β-barrel fold was identified in Streptomyces sp. strain CL190. This fold, termed the PT-barrel, is formed of five tandem ααββ structural repeats and remained exclusive to the NphB family until its recent discovery in the DMATS family of indole PTases. Members of these two families exist only in fungi and bacteria, and all of them appear to catalyze the prenylation of aromatic substrates involved in secondary metabolism. Sequence comparisons using PSI-BLAST do not yield matches between these two families, suggesting that they may have converged upon the same fold independently. However, we now provide evidence for a common ancestry for the NphB and DMATS families of PTases. We also identify sequence repeats that coincide with the structural repeats in proteins belonging to these two families. Therefore we propose that the PT-barrel arose by amplification of an ancestral ααββ module. In view of their homology and their similarities in structure and function, we propose to group the NphB and DMATS families together into a single superfamily, the PT-barrel superfamily

Structure-based engineering increased the catalytic turnover rate of a novel phenazine prenyltransferase.

Prenyltransferases (PTs) catalyze the regioselective transfer of prenyl moieties onto aromatic substrates in biosynthetic pathways of microbial secondary metabolites. Therefore, these enzymes contribute to the chemical diversity of natural products. Prenylation is frequently essential for the pharmacological properties of these metabolites, including their antibiotic and antitumor activities. Recently, the first phenazine PTs, termed EpzP and PpzP, were isolated and biochemically characterized. The two enzymes play a central role in the biosynthesis of endophenazines by catalyzing the regiospecific prenylation of 5,10-dihydrophenazine-1-carboxylic acid (dhPCA) in the secondary metabolism of two different Streptomyces strains. Here we report crystal structures of EpzP in its unliganded state as well as bound to S-thiolodiphosphate (SPP), thus defining the first three-dimensional structures for any phenazine PT. A model of a ternary complex resulted from in silico modeling of dhPCA and site-directed mutagenesis. The structural analysis provides detailed insight into the likely mechanism of phenazine prenylation. The catalytic mechanism suggested by the structure identifies amino acids that are required for catalysis. Inspection of the structures and the model of the ternary complex furthermore allowed us to rationally engineer EpzP variants with up to 14-fold higher catalytic reaction rate compared to the wild-type enzyme. This study therefore provides a solid foundation for additional enzyme modifications that should result in efficient, tailor-made biocatalysts for phenazines production

Mutational analysis of a phenazine biosynthetic gene cluster in Streptomyces anulatus 9663

The biosynthetic gene cluster for endophenazines, i.e., prenylated phenazines from Streptomyces anulatus 9663, was heterologously expressed in several engineered host strains derived from Streptomyces coelicolor M145. The highest production levels were obtained in strain M512. Mutations in the rpoB and rpsL genes of the host, which result in increased production of other secondary metabolites, had no beneficial effect on the production of phenazines. The heterologous expression strains produced, besides the known phenazine compounds, a new prenylated phenazine, termed endophenazine E. The structure of endophenazine E was determined by high-resolution mass spectrometry and by one- and two-dimensional NMR spectroscopy. It represented a conjugate of endophenazine A (9-dimethylallylphenazine-1-carboxylic acid) and L-glutamine (L-Gln), with the carboxyl group of endophenazine A forming an amide bond to the α-amino group of L-Gln. Gene inactivation experiments in the gene cluster proved that ppzM codes for a phenazine N-methyltransferase. The gene ppzV apparently represents a new type of TetR-family regulator, specifically controlling the prenylation in endophenazine biosynthesis. The gene ppzY codes for a LysR-type regulator and most likely controls the biosynthesis of the phenazine core. A further putative transcriptional regulator is located in the vicinity of the cluster, but was found not to be required for phenazine or endophenazine formation. This is the first investigation of the regulatory genes of phenazine biosynthesis in Streptomyces

Structures of EpzP.

<p>a) Overall structure of EpzPwt reveals an ABBA PT-fold. Secondary structural elements are colored according to their r.m.s. deviations of C_α-atom positions compared to the most homologous structure of NphB (PDB-Code: 1ZB6) from blue (zero r.m.s.d.) to orange (r.m.s.d. above 3 Å). The substrates dhPCA and DMAPP are modeled and shown in stick representation. <b>b</b>) View into the active site of EpzPwt. Side chains which showed a different orientation in EpzPm-nat are depicted in transparent green color. R267 points towards the cavity in EpzPwt and away from it in the EpzPm-nat and EpzPm-SPP structures. Water molecule W1, which is proposed to deprotonate the Wheland complex, is shown in magenta. Two sulfate ions occupy the diphosphate binding site of EpzPwt. The (F_obs-F_calc)-omit map (cyan) is shown at σ-level of 3.2 for both sulfate ions and inside the barrel. The remaining electron density in the proposed substrate binding site of EpzPwt could not be explained; it does not fit with any molecule used in downstream purification and crystallization experiments and may represent a molecule inserted into the enzyme during protein production. <b>c</b>) The active site of EpzPm-SPP viewed along the same axis. The (F_obs-F_calc)-omit map (cyan) is shown at σ-level of 3.0 and clearly reveals the presence of the <i>S</i>-thiolodiphosphate moiety (cyan) and a PEG molecule (purple). Hydrogen bonds and hydrophobic interactions are represented with red and green lines, respectively.</p

Data collection and refinement statistics^a.

a<p>Values in parentheses are for the highest resolution shell. All data sets were recorded at wavelength λ = 1.0 Å.</p

Model of catalysis.

<p><b>a)</b> Model of phenazine prenylation based on crystal structures, <i>in silico</i> docking and site-directed mutagenesis. The reactions centers of DMAPP (blue) and dhPCA (purple) are emphasized with spheres. Water molecule W1 (magenta) deprotonates the intermediate Wheland complex. EpzP mutants that confirm the model of catalysis or have been engineered to modify turnover-rates are colored orange and green, respectively. 1,6-dihydroxy naphthalene (green), the substrate of NphB, occupies essentially the same binding pocket as dhPCA in EpzP. <b>b)</b> Schematic view of substrate binding in EpzP. <b>c)</b> and <b>d</b>) Relative enzymatic activities of EpzP variants compared to the wild-type EpzP.</p

Comparison of EpzP structure and sequence with other PTs of the NphB/CloQ family. a)

<p>Superposition of NphB (black lines) and EpzP. <b>b)</b> Superposition of CloQ (black lines) with EpzP. The superpositions reveal variability in structure and sequence in the aromatic binding site at the C-terminal part of the enzymes. Side chain residues of EpzP that differ from NphB or CloQ are shown orange, whereas conserved residues are shown grey. The catalytic water molecules W1 and W2 are shown as spheres in magenta and red, respectively. The substrates of NphB and CloQ are shown in stick representation (green). <b>c)</b> Structure-based sequence comparison of EpzP, CloQ and NphB and sequence comparison of PpzP, NphB and Fnq28. NphB and Fnq28 are magnesium-dependent enzymes, whereas the others are not. Amino acids of the diphosphate binding site (cyan), mutations that confirm the <i>in silico</i> docking model (orange), and mutations that modify the enzymatic turnover (green) are emphasized.</p

EpzP and PpzP catalyze the regioselective C-prenylation of 5,10-dihydrophenazine-1-carboxylate (dhPCA) to yield 5,10-dihydroendophenazine A.

<p>EpzP and PpzP catalyze the regioselective C-prenylation of 5,10-dihydrophenazine-1-carboxylate (dhPCA) to yield 5,10-dihydroendophenazine A.</p