Functions of Genes and Enzymes Involved in Phenalinolactone Biosynthesis

Abstract

Phenalinolactones are novel terpene glycoside antibiotics produced by Streptomyces sp. Tü6071. Inactivation of three oxygenase genes (plaO2, plaO3 and plaO5), two dehydrogenase genes (plaU, plaZ) and one putative acetyltransferase gene (plaV) led to the production of novel phenalinolactone derivatives (PL HS6, PL HS7, PL HS2 and PL X1). Furthermore, the exact biosynthetic functions of two enzymes were determined, and their in vitro activities were demonstrated. PlaO1, an Fe(II)/alpha-ketoglutarate-dependent dioxygenase, is responsible for the key step in gamma-butyrolactone formation, whereas PlaO5, a cytochrome P450-dependent monooxygenase, catalyses the 1-C-hydroxylation of phenalinolactone D. In addition, stable isotope feeding experiments with biosynthetic precursors shed light on the origin of the carbons in the gamma-butyrolactone moiety.

Metabolic pathway monitoring of phenalinolactone biosynthesis from Streptomyces sp. Tü6071 by liquid chromatography/mass spectrometry coupling

“…The structures of all analysed phenalinolactone intermediates of the biosynthetic pathway, as well as PL A and D, were identified in the precursor ion scan. The search with the precursor ion m/z 124.2 revealed the intermediate structures which were described in previous studies . Aside from the already known structures, two new phenalinolactone intermediate (PL IM) structures were found with m/z 672.6 (PL IM1) by the precursor ion m/z 124 and 433.4 (PL IM2) by the precursor ion m/z 109 (Fig.…”

“…Phenalinolactones have a structurally unique architecture, which results from different biosynthetic pathways . Besides the terpenoid core structure, PL A and D consist of different moieties, such as γ‐butyrolactone, 5‐methylpyrrole, an acetate unit, and the O ‐glycosidically linked 4‐ O ‐methyl‐ l ‐amicetose (Fig.…”

Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function

“…3). 82,83 PlaO2 was determined to be nonessential in phenalinolactone formation, 83 although it is not clear whether it is nonfunctional, can be complemented by another P450, or has another biological role in the organism. An alternative strategy to functionalize one scaffold multiple times is the utilization of a multifunctional P450, with TamI being the quintessential example.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…Simple hydroxylation is seen in the formation of the diterpene glycoside antibiotic phenalinolactones where at least three different P450s oxidise a methylene (P450 PlaO5) or diastereotopic methyl (PlaO4 and PlaO3) groups. [52][53][54] In the case of the methyl groups, these pendant hydroxyls provide points of attachment for acyl or glycosyl moieties (cf. polyketides vide infra) and result in the P450s acting on structurally quite distinct substrates.…”

“…Interestingly, as with many biosynthetic monooxygenases, the operon encodes no obvious redox partners for these P450s but does contain another P450 of so far unknown function. [52][53][54] Similarly, CYP1051A1 is encoded in a diterpene biosynthetic operon in the marine organism Salinispora arenicola without any redox partner and is responsible for diastereoselective methyl hydroxylation. 54 The cyclooctatin gene cluster in Streptomyces melanosporofaciens is responsible for the biosynthesis of the unusual eponymous diterpene, the skeleton of which is formed via an unusually complex and cryptic rearrangement pathway.…”