Evolution of a new class of prenyl transferases of secondary metabolism

LUTZ HEIDE
Pharmazeutisches Institut der Universität Tübingen
Auf der Morgenstelle 8
D-72076 Tübingen
heide@uni-tuebingen.de
http://www.uni-tuebingen.de/pharmazie/abteilungen/biologie/

References
Keller S, Pojer F, Heide L, Lawson DM (2006) Crystallization and preliminary X-ray analysis of the aromatic prenyltransferase CloQ from the clorobiocin biosynthetic cluster of Streptomyces roseochromogenes. Acta Cryst. F 62, 1153-1155
Crystals of recombinant CloQ (subunit MW = 35 626 Da; 324 amino acids), an aromatic prenyltransferase from Streptomyces roseochromogenes, were grown by vapour diffusion. The protein crystallizes in space group I4122, with unit-cell parameters a = b = 135.19, c = 98.13 A ° . Native data from a single crystal were recorded to a resolution of 2.2 A ° in-house. Preliminary analysis of these data indicated that the asymmetric unit corresponds to a monomer, giving an estimated solvent content of 60.6%. CloQ is involved in the biosynthesis of the aminocoumarin antibiotic clorobiocin, which targets the essential bacterial enzyme DNA gyrase.

Haagen Y, Glück K, Fay K, Kammerer B, Gust B, Heide L (2006) A gene cluster for prenylated naphthoquinone and prenylated phenazine biosynthesis in Streptomyces cinnamonensis DSM 1042. Chembiochem 7: 2016-2027
Streptomyces cinnamonensis DSM 1042 produces two classes of secondary metabolites of mixed isoprenoid/non-isoprenoid origin: the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and several prenylated phenazines, predominantly endophenazine A. We now report the cloning and sequence analysis of a 55 kb gene cluster for the biosynthesis of these compounds. Several inactivation experiments confirmed the involvement of this gene cluster in the biosynthesis of FNQ I and endophenazine A. The six identified genes for endophenazine biosynthesis showed close similarity to phenazine biosynthetic genes from Pseudomonas. Of the 28 open reading frames identified in the adjacent FNQ I cluster, 13 showed close similarity to genes contained in the cluster for furaquinocin, a structurally similar metabolite from another Streptomyces strain. These genes included a type III polyketide synthase gene, a momA-like monooxygenase gene, and two cloQ-like prenyltransferase genes designated as fnq26 and fnq28, respectively. Inactivation experiments confirmed the involvement of fnq26 into FNQ I biosynthesis, whereas no change of secondary metabolite formation was observed after fnq28 inactivation. The FNQ I cluster contains a contigous group of five genes which together encode all enzymatic functions required for the recycling of S-adenosylhomocysteine (SAH) to S-adenosylmethionine (SAM). Two SAM-dependent methyltransferases are encoded within the cluster. Inactivation experiments showed that fnq9 is responsible for the 7-O-methylation and fnq27 for the 6-C-methylation reaction in FNQ I biosynthesis.

Haagen Y, Unsöld I, Westrich L, Gust B, Heide L (2007) A soluble, magnesium-independent prenyltransferase catalyzes reverse and regular C-prenylations and O-prenylations of aromatic substrates. FEBS Letters 581: 2889-2893
Fnq26 from Streptomyces cinnamonensis DSM 1042 is a new member of the recently identified CloQ/Orf2 class of prenyltransferases. The enzyme was overexpressed in E. coli and purified to apparent homogeneity, resulting in a soluble, monomeric protein of 33.2 kDa. The catalytic activity of Fnq26 is independent of the presence of Mg²⁺ or other divalent metal ions. With flaviolin (2,5,7-trihydroxy-1,4-naphthoquinone) as substrate, Fnq26 catalyzes the formation of a carbon-carbonbond between C-3 (rather than C-1) of geranyl diphosphate and C-3 of flaviolin, i.e. an unusual ''reverse'' prenylation. With 1,3- dihydroxynaphthalene and 4-hydroxybenzoate as substrates Fnq26 catalyzes O-prenylations.

Bringmann G, Haagen Y, Gulder TAM, Gulder T, Heide L (2007) Biosynthesis of the isoprenoid moieties of furanonaphthoquinone i and endophenazine A in Streptomyces cinnamonensis DSM 1042. J Org Chem 72: 4198-4204.

Streptomyces cinnamonensis DSM 1042 produces the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and isoprenylated phenazines, predominantly endophenazine A. However, the recently identified biosynthetic gene cluster for these compounds only contains a single gene for a mevalonate pathway enzyme, i.e., a putative mevalonate kinase gene. This is in strong contrast to all Streptomyces strains examined so far, where all six genes encoding the mevalonate pathway enzymes are clustered in a single operon of 6.8kb and thus raised the question about the biosynthetic origin of the isoprenoid moieties of FNQ I and endophenazine A. In this study, we investigated the incorporation of [¹³C₂]acetate and [2-¹³C]glycerol into FNQ I and endophenazine A. The results unequivocally prove that the isoprenoid building blocks of both compounds are predominantly formed via the mevalonate pathway (approximately 80%), but that the MEP pathway (approximately 20%) contributes to the biosynthesis of these molecules, too. In Actinomycetes, this is the first experimentally proven example of the utilization of both biosynthetic routes for the formation of one single secondary metabolite. The incorporation pattern of [2-¹³C]glycerol was consistent with a "reverse" prenyl transfer, i.e., with the formation of a C-C bond from C-3 of GPP to the polyketide nucleus of FNQ I.

[back]