Biosynthetic Studies on the α-Glucosidase Inhibitor Acarbose in Actinoplanes sp.:  2-epi-5-epi-Valiolone Is the Direct Precursor of the Valienamine Moiety

Abstract

The biosynthetic pathway leading to the mC7N cyclitol (valienamine) moiety of acarbose (1) in Actinoplanes sp. strain SN 223/29 has been studied using 3H-, 2H-, and 13C-labeled cyclitols. These precursors were synthesized from d-glucose or d-mannose as starting materials. The feeding experiments demonstrated that cyclitols having the same stereochemistry at C-2 as the valienamine moiety of acarbose; i.e., valienone, valienamine, valiolone, valiolamine, and 1-epi-valienol, were not incorporated and thus are not plausible intermediates in 1 biosynthesis. 2-epi-Valiolone (10b), which has the same stereochemistry as the presumed open-chain precursor, sedoheptulose 7-phosphate, was also not incorporated. However, its C-5 epimer (10a) was incorporated efficiently and specifically into the valienamine moiety of 1. Surprisingly, the dehydrated form of 2-epi-5-epi-valiolone, 2-epi-valienone, was not incorporated. This suggests that 2-epi-5-epi-valiolone must be converted directly into the pseudodisaccharide moiety of acarbose without the intervention of other free cyclitol intermediates. This may occur by linkage to the amino group of TDP-4-amino-4,6-dideoxyglucose to form the imine, epimerization at C-2 to the correct stereochemistry, dehydration between C-5 and C-6 aided by enamine formation, and finally reduction to the amine. It is proposed that these reaction steps all take place on a single enzyme without free intermediates. Alternative mechanistic possibilities are also discussed.

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

“…This conversion step was unexpected because it seemed more likely that either dehydration at positions 5 and 6 or reduction of the keto group at position 1 of the cyclitol came first. The fact that 2-epi-5-epi-valiolone is first phosphorylated and only thereafter further modified by other biosynthetic enzymes such as AcbO is in good accordance with all feeding experiments with whole cells that have been performed so far (8). In these studies, 2-epi-5-epi-valiolone was the only extracellularly provided C 7 -cyclitol that was incorporated into acarbose.…”

“…Earlier work has demonstrated that 2-epi-5-epi-valiolone is the precursor of the C 7 -cyclitol unit of the acarviosyl moiety of acarbose (5,8). The expectation was that a series of enzymecatalyzed steps involving dehydration, reduction, and epimerization converted this precursor to valienol (or valienamine) before its incorporation into the pseudodisaccharide acarviosine.…”

“…The expectation was that a series of enzymecatalyzed steps involving dehydration, reduction, and epimerization converted this precursor to valienol (or valienamine) before its incorporation into the pseudodisaccharide acarviosine. Although a number of feeding experiments with possible intermediates were described, all other steps and intermediates of this biosynthetic route remained unclear so far (8).…”

“…In additional experiments, we have found evidence that 1-epi-valienol is phosphorylated by a crude extract from Actinoplanes sp. SE50/110 (data not shown); experiments with 1-epi-valienol 7-phosphate as the more likely substrate (because extracellularly applied 1-epi-valienol is not incorporated) (8) in similar assays are in progress. Phosphorylation at C-1 would be a prerequisite for a subsequent nucleotidylation step.…”

“…1) (6,7). In contrast, similar feeding experiments revealed 2-epi-5-epi-valiolone to be the only precursor that was incorporated into acarbose (8). Therefore, fundamental differences in * The costs of publication of this article were defrayed in part by the payment of page charges.…”