Complete biosynthetic pathway to the antidiabetic drug acarbose

Abstract

Acarbose is a bacterial-derived α-glucosidase inhibitor clinically used to treat patients with type 2 diabetes. As type 2 diabetes is on the rise worldwide, the market demand for acarbose has also increased. Despite its significant therapeutic importance, how it is made in nature is not completely understood. Here, we report the complete biosynthetic pathway to acarbose and its structural components, GDP-valienol and O-4-amino-(4,6-dideoxy-α-D-glucopyranosyl)-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose. GDP-valienol is derived from valienol 7-phosphate, catalyzed by three cyclitol modifying enzymes, whereas O-4-amino-(4,6-dideoxy-α-D-glucopyranosyl)-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose is produced from dTDP-4-amino-4,6-dideoxy-D-glucose and maltose by the glycosyltransferase AcbI. The final assembly process is catalyzed by a pseudoglycosyltransferase enzyme, AcbS, which is a homologue of AcbI but catalyzes the formation of a non-glycosidic C-N bond. This study clarifies all previously unknown steps in acarbose biosynthesis and establishes a complete pathway to this high value pharmaceutical.

Biochemical Characterization of GacI, a Bifunctional Glycosyltransferase–Phosphatase Enzyme Involved in Acarbose Biosynthesis in Streptomyces glaucescens GLA.O

“…Although our previous studies showed that acarbose or acarbose-related BGCs are widely distributed in many actinomycetes, the degree to which the gacI -like bifunctional GT-PP gene is distributed among those BGCs was unclear. Therefore, a follow-up study of those BGCs was performed, and the results showed that from 139 strains that harbor acarbose or acarbose-related BGCs, 129 strains have a gacI -type gene, instead of a pair of acbI and acbJ genes, and all of them are strains of Streptomyces .…”

“…The results suggest that GacI can effectively dephosphorylate both V7P and V1,7PP but not 1-epi-V1,7PP or Aca7P, which are consistent with the substrate preference of AcbJ from the acarbose pathway in Actinoplanes sp. SE50/110 . Although both V7P and V1,7PP can be effectively dephosphorylated by GacI, the natural substrate for GacI is believed to be V1,7PP, as its dephosphorylation product valienol 1-phosphate (V1P, 9 ) has recently been identified as an intermediate in acarbose biosynthesis (Figure A) .…”

“…For LC-Q-TOF-MS analysis, a reaction mixture (10 μL) containing Tris–HCl buffer (20 mM, pH 7.5), GacI or GacI-PP (2 μM), and substrate (1 mM) was incubated at 30 °C for 3 h. All substrates, V1,7PP, 1- epi -V1,7PP, V7P, and Aca7P, have been prepared and reported in our previous publications. , The reaction mixtures were analyzed by LC-Q-TOF-MS using the below-described method.…”

“…Subsequently, EEV ( 3 ) undergoes phosphorylation by the kinase AcbM, epimerization by AcbO, dehydration by AcbL, and reduction by AcbN to give valienol 7-phosphate (V7P, 7 ) . V7P ( 7 ) is then phosphorylated to valienol 1,7-diphosphate (V1,7PP, 8 ) by the kinase AcbU, dephosphorylated to valienol 1-phosphate (V1P, 9 ) by the phosphatase AcbJ, and converted to GDP-valienol (GDP-V, 10 ) by the nucleotidyltransferase AcbR . The 4-aminodeoxysugar moiety of acarbose is derived from dTDP-4-amino-4,6-dideoxyglucose (dTDP4a6dGlc, 11 ), which is synthesized from glucose 1-phosphate catalyzed successively by the glucose 1-phosphate thymidylyltransferase AcbA, the dTDP-glucose 4,6-dehydratase AcbB, and the dTDP-4-amino-4,6-dideoxyglucose transaminase AcbV.…”

“…The 4-aminodeoxysugar moiety of acarbose is derived from dTDP-4-amino-4,6-dideoxyglucose (dTDP4a6dGlc, 11 ), which is synthesized from glucose 1-phosphate catalyzed successively by the glucose 1-phosphate thymidylyltransferase AcbA, the dTDP-glucose 4,6-dehydratase AcbB, and the dTDP-4-amino-4,6-dideoxyglucose transaminase AcbV. Subsequently, dTDP4a6dGlc ( 11 ) is coupled with maltose by AcbI to give 4-aminoDGG ( 12 ) . The final assembly of acarbose ( 1 ) is accomplished by coupling between GDP-valienol and 4-aminoDGG by AcbS, which is a new member of a growing family of pseudoglycosyltransferases (PsGTs) …”