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.

Fig. 1. Acarbose biosynthesis in Actinoplanes sp. SE50/110.

a Acarbose biosynthetic gene cluster from Actinoplanes sp. SE50/110; b Proposed acarbose biosynthetic pathways. The thick purple arrows (shaded in blue) indicate steps that have been characterized biochemically prior to this study. The blue and red arrows indicate previously proposed pathways. The thick black arrows (shaded in yellow) show the pathway elucidated in this study. SH7P, sedoheptulose 7-phosphate; EEV, 2-epi-5-epi-valiolone; EEV7P, 2-epi-5-epi-valiolone 7-phosphate; EV7P, 5-epi-valiolone 7-phosphate; V1P, valienol 1-phosphate; V7P, valienol 7-phosphate; 1-epi-V7P, 1-epi-valienol 7-phosphate; V1,7PP, valienol 1,7-diphosphate; 1-epi-V1,7PP, 1-epi-valienol 1,7-diphosphate; NDP-V, nucleoside diphosphate-valienol; NDP-V7P, nucleoside diphosphate-valienol 7-phosphate; NDP-1-epi-V7P, nucleoside diphosphate-1-epi-valienol 7-phosphate; dTDP-4a6dGlc, dTDP-4-amino-4,6-dideoxyglucose; 4-aminoDGG, O-4-amino-(4,6-dideoxy-α-D-glucopyranosyl)-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose.

Fig. 2. Biochemical characterization of the kinase AcbU and the phosphatase AcbJ.

a ESI (–) EIC (m/z 334.9940) of AcbU reaction with V7P; b ESI (–) EIC (m/z 334.9940 and 255.0275) of AcbU reaction with V1P; c ESI (–) EIC (m/z 255.0275) of AcbU reaction with V; d PK/LDH coupled enzyme assay of AcbU reactions with V7P, V, or V1P as substrates, and no substrate (NS) in the presence of ATP. Reaction with boiled AcbU was used as a blank; e PK/LDH coupled enzyme assay of AcbU reactions with V7P, V, or V1P as substrates in the presence of ATP. Reactions of AcbU without substrate but with ATP were used as blanks; f MESG assay of AcbJ reaction with V1,7PP, 1-epi-V1,7PP, V7P, or Aca7P as substrates; g ESI (–) EIC (m/z 255.0275) of AcbJ reactions with V1,7PP. For d–f, error bars indicate standard deviation (SD) (n = 3 analytical replicates), and data are presented as mean values ± SD. All experiments were carried out at least three times with similar results. ESI, electrospray ionization mass spectrometry; EIC, extracted ion chromatogram; V, valienol; V1P, valienol 1-phosphate; V7P, valienol 7-phosphate; V1,7PP, valienol 1,7-diphosphate; 1-epi-V1,7PP, 1-epi-valienol 1,7-diphosphate; Aca7P, acarbose 7-phosphate.

Fig. 3. Biochemical characterizations of AcbR and AcbI.

a–d The activity of AcbR with V1P, 1-epi-V1P, V1,7PP, or 1-epi-V1,7PP as substrates incubated with various NTPs. The blank was a reaction mixture lacking AcbR. A: ATP, U: UTP, G: GTP, C: CTP, dT: dTTP; e Partial LC-QTOF/MS EIC chromatograms (negative ion mode) of AcbR reaction with V1P and GTP (in red) and the negative control lacking AcbR (in black). Both chromatograms are the extraction of corresponding calculated exact mass for GDP-valienol (m/z 600.0750 [M-H]^–). f Coupling between dTDP4a6dGlc and maltose catalyzed by AcbI; g MS/MS analysis of the product of the AcbI reaction; h ESI( + ) EIC for 4-aminoDGG (m/z 510.1793, [M + Na]⁺) from AcbI reactions with maltose, maltotriose, maltotetraose, and maltopentaose; and (i) ESI( + ) EIC for 4-aminoDGG (m/z 510.1793 [M + Na]⁺) from AcbQ, AcbS, and AcbI reactions with maltose. For a–d, error bars indicate standard deviation (SD) (n = 3 analytical replicates), and data are presented as mean values ± SD. All experiments were carried out at least three times with similar results. ESI, electrospray ionization mass spectrometry; EIC, extracted ion chromatogram; V1P, valienol 1-phosphate; V1,7PP, valienol 1,7-diphosphate; 1-epi-V1,7PP, 1-epi-valienol 1,7-diphosphate; A, ATP; U, UTP; G, GTP; C, CTP; dT, dTTP; dTDP-4a6dGlc, dTDP-4-amino-4,6-dideoxyglucose; 4-aminoDGG, O-4-amino-(4,6-dideoxy-α-D-glucopyranosyl)-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose.

Fig. 4. Characterization of the pseudoglycosyltransferase AcbS.

a ESI( + ) EIC for acarbose at m/z 646.2553 of reaction mixtures with AcbI, AcbQ, or with or without AcbS; b ESI-MS/MS of the AcbS product; c ESI-MS/MS of acarbose standard; d reaction scheme of AcbR and AcbS; e ESI( + ) EIC for acarbose at m/z 646.2553 of reaction mixtures with AcbI, AcbR, and AcbS added in different sequence and combination (see text and experimental section). All experiments were carried out at least three times with similar results. ESI, electrospray ionization mass spectrometry; EIC, extracted ion chromatogram; V1P, valienol 1-phosphate; GDP-V, GDP-valienol; dTDP-4a6dGlc, dTDP-4-amino-4,6-dideoxyglucose; 4-aminoDGG, O-4-amino-(4,6-dideoxy-α-D-glucopyranosyl)-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose.

Fig. 5. Homologues of AcbI and AcbS.

a Homology searches for AcbI and AcbS; b Phylogenetic tree of AcbI and AcbS. The ones shaded in blue are predicted to be AcbS homologues (PsGTs). The ones shaded in red are AcbI homologues (GTs). The ones shaded in dark blue are AcbS whose genes do not have an AcbI partner gene within a biosynthetic gene cluster (BGC). The ones shaded in purple do not pair with another AcbI/AcbS homologue in a BGC. The ones shaded in dark purple are AcbI/AcbS homologues whose genes are part of a BGC that lacks an aminotransferase gene. The ones shaded in yellow are AcbI/AcbS homologue whose genes are part of a BGC that does not contain cyclitol biosynthetic genes.