Biosynthesis of the C7-cyclitol Moiety of Acarbose inActinoplanes Species SE50/110

Abstract

We have previously demonstrated that the biosynthesis of the C 7 -cyclitol, called valienol (or valienamine), of the ␣-glucosidase inhibitor acarbose starts from the cyclization of sedo-heptulose 7-phosphate to 2-epi-5-epivaliolone (Stratmann, A., Mahmud, T., Lee, S., Distler, J., Thus, a phosphotransferase activity was identified modifying 2-epi-5-epi-valiolone by ATP-dependent phosphorylation. This activity could be attributed to the AcbM protein by verifying this activity in S. lividans strain TK64/pCW4123M, expressing His-tagged AcbM. The Histagged AcbM protein was purified and subsequently characterized as a 2-epi-5-epi-valiolone 7-kinase, presumably catalyzing the first enzyme reaction in the biosynthetic route, leading to an activated form of the intermediate 1-epi-valienol. The AcbK protein could not catalyze the same reaction nor convert any of the other C 7 -cyclitol monomers tested. The 2-epi-5-epi-valiolone 7-phosphate was further converted by the AcbO protein to another isomeric and phosphorylated intermediate, which was likely to be the 2-epimer 5-epi-valiolone 7-phosphate. The products of both enzyme reactions were characterized by mass spectrometric methods. The product of the AcbM-catalyzed reaction, 2-epi-5-epi-valiolone 7-phosphate, was purified on a preparative scale and identified by NMR spectroscopy. A biosynthetic pathway for the pseudodisaccharidic acarviosyl moiety of acarbose is proposed on the basis of these data.

Genetic localization and heterologous expression of validamycin biosynthetic gene cluster isolated from Streptomyces hygroscopicus var. limoneus KCCM 11405 (IFO 12704)

“…The recombinant VldA protein, obtained from E. coli, efficiently consumed sedo-heptulose 7-phosphate to generate a new product that showed a TLC-analytical pattern consistent with that of 2-epi-5-epi-valiolone, as in the literature (Stratmann et al, 1999;Zhang et al, 2002;Yu et al, 2005) (Fig. 3d, e).…”

“…The biochemical characterization of AcbM in the acarbose pathway as 2-epi-5-epi-valiolone 7-kinase reveals that the majority of the intermediates to the valienamine moiety of acarbose are 7-phosphorylated (Zhang et al, 2002). It also leads to the prediction that an AcbM-like kinase is not engaged in the validamycin biosynthesis.…”

“…After cell-lysis by sonication, the cell debris was removed by centrifugation at 12,000 rpm for 15 min to give the cell-free enzyme extract. The enzyme assay was carried out using a coupled reaction with transketolase, as previously described (Zhang et al, 2002).…”

Glucoamylase gene, vldI, is linked to validamycin biosynthesis in Streptomyces hygroscopicus var. limoneus, and vldADEFG confers validamycin production in Streptomyces lividans, revealing the role of VldE in glucose attachment

“…Precursor-feeding experiments firmly established the intermediacies of 2-epi-5-epivaliolone, 5-epi-valiolone, valienone, and validone in validamycin biosynthetic pathways and substantiated that no phosphorylated intermediate is involved (Dong et al, 2001). However, the product of vldC is homologous to 2-epi-5-epi-valiolone 7-kinase in the acarbose pathway, implying there is a phosphorylated intermediate in the validamycin m-C 7 N cyclitol pathway (Singh et al, 2006;Zhang et al, 2002). Later, VldC was characterized as glucokinase, contradicting the involvement of a phosphorylated intermediate in the validamycin m-C 7 N cyclitol biosynthesis (Seo et al, 2006).…”

Isotope tracer investigations of natural products biosynthesis: the discovery of novel metabolic pathways

“…44 The C 7 N-aminocyclitol-containing natural products, 45 e.g. acarbose (50), validamycin A (51), cetoniacytone (52), and pyralomicin (53), were initially considered to be the aliphatic version of the aromatic C 7 N-units found in many antibiotic natural products, e.g. rifamycin (54), mitomycin (55), and geldanamycin (56) (Scheme 9), which are derived from the aminoshikimate pathway -a metabolic pathway parallel to the shikimate pathway.…”