Characterization and structure-based protein engineering of a regiospecific saponin acetyltransferase from Astragalus membranaceus

Abstract

Acetylation contributes to the bioactivity of numerous medicinally important natural products. However, little is known about the acetylation on sugar moieties. Here we report a saponin acetyltransferase from Astragalus membranaceus. AmAT7-3 is discovered through a stepwise gene mining approach and characterized as the xylose C3'/C4'-O-acetyltransferse of astragaloside IV (1). To elucidate its catalytic mechanism, complex crystal structures of AmAT7-3/1 and AmAT7-3_A310G/1 are obtained, which reveal a large active pocket decided by a specific sequence AADAG. Combining with QM/MM computation, the regiospecificity of AmAT7-3 is determined by sugar positioning modulated by surrounding amino acids including #A310 and #L290. Furthermore, a small mutant library is built using semi-rational design, where variants A310G and A310W are found to catalyze specific C3'-O and C4'-O acetylation, respectively. AmAT7-3 and its variants are also employed to acetylate other bioactive saponins. This work expands the understanding of saponin acetyltransferases, and provide efficient catalytic tools for saponin acetylation.

Fig. 1. Functional characterization of AmAT7-3.

a Maximum likelihood tree of acetyltransferases with diverse functions (Supplementary Data 1). b Maximum likelihood tree of AmAT7-3 with representative acyltransferases. Evolutionary analyses were conducted in MEGA7 with 1000 bootstrap replicates^,. Yellow and green arrows indicate the first- and second-round gene screening, respectively. HCT/HQT, shikimic acid or quinic acid acyltransferase. c Acetylation of astragaloside IV (1) catalyzed by AmAT7-3. d UPLC/MS total ion current chromatograms of the reaction, control, and the standard samples. e HR-MS and HR-MS/MS analysis of products 1a, 1b, 1c in positive ion mode. f Time-course of the enzymatic reaction of AmAT7-3 using 1 as the substrate. Data are presented in mean ± SD (n = 2 or 3 independent experiments). Source data are provided as a Source Data file. g Peak area ratio of 1a-1c in the reaction mixture of AmAT7-3 using 1a as the substrate. Glc, glucosyl group; Xyl, xylosyl group; Ac-CoA, acetyl-coenzyme A; Reaction: acetylated reaction catalyzed by AmAT7-3; Control: boiled AmAT7-3 was used in the reaction mixture.

Fig. 2. Contribution of AmAT7-3 to C2′-OH acetylated saponins in A. membranaceus.

a UHPLC/CAD chromatogram of Astragalus Root. b Subcellular localization of AmAT7-3. Three independent experiments showed the similar results. c Proportions of degraded products of 1a at different pH. Data are presented as the average of triplicate independent experiments. d–f Time-course of the acetyl migration for 1a, 1b, and 1d, respectively. Data are presented in mean ± SD (n = 2 or 3 independent experiments). Source data are provided as a Source Data file for (c–f). g Transient expression of AmAT7-3 in N. benthamiana. UHPLC/MS traces for extracts from agroinfiltrated leaves are presented. EV: fluorescent signal of empty pSuper 1300-GFP vector; Reaction: transient expression AmAT7-3 in N. benthamiana; Control: transient expression empty pEAQ-HT vector in N. benthamiana.

Fig. 3. Crystal structure and catalytic mechanism of AmAT7-3.

a The complex crystal structure of AmAT7-3/astragaloside IV. b The active pocket surface of AmAT7-3. c The active pocket surface of SbHCT. d Comparison of the molecular sizes of astragaloside IV and shikimic acid, substrates for AmAT7-3 and SbHCT, respectively. e The distance population between acyl C of Ac-CoA and sugar O sites of astragaloside IV. f The distance between acyl C and various sugar O sites of astragaloside IV. g A reactive snapshot of conformation-1 and its interaction with surrounding amino acids from MD simulations. h A reactive snapshot of conformation-2 and its interaction with surrounding amino acids from MD simulations. i QM/MM-optimized reaction states of the O3′ acetylation reaction catalyzed by AmAT7-3. j QM/MM-calculated energy profile for AmAT7-3/ astragaloside IV/Ac-CoA at a reactive snapshot of conformation-1. k QM/MM-calculated energy profile for AmAT7-3/astragaloside IV/Ac-CoA at a reactive snapshot of conformation-2. AsIV astragaloside IV, PS p-coumaroyl shikimic acid, RC reactant complex, TS1 transition state 1, IM1 intermediate 1, TS2 transition state2, PC product complex. Energies are given in kcal/mol, while the distances are given in angstrom.

Fig. 4. Engineering the regioselectivity of AmAT7-3 and the regioselective mechanism of its mutants.

a Amino acids within 5 Å of 1 in the active pocket. b Mutant library of AmAT7-3 and the ratio of 1a/1b/1c produced different mutants. c UHPLC/MS chromatograms of the reaction samples catalyzed by AmAT7-3 and its mutants. d Mutants with specific C3′-OH or C4′-OH acetylation activities. e Superposition between the QM/MM-optimized reactant complex of AmAT7-3_A310G (cyan) and that of the wild-type (from conformation-1, gray). f Superposition between the QM/MM-optimized reactant complex of AmAT7-3_A310W (cyan) and that of the wild-type (from conformation-1, gray). g QM/MM-calculated energy profile (in kcal/mol) for AmAT7-3_A310G/W/1/Ac-CoA. AsIV, astragaloside IV (1). h Sequence alignment for AmAT7-3 and other flavonoid or saponin acetyltransferases. i Location of YFGN and AADAG motifs in the β10 barrel of AmAT7-3. RC reactant complex, TS1 transition state 1, IM1 intermediate 1.

Fig. 5. Acetylation of medicinally important saponins using AmAT7-3 and its mutants.

a Structures and bioactivities of compounds 1-8. b Acetylated reaction of 6 catalyzed by AmAT7-3 or by chemical reagents. PPT protopanaxatriol, a/b/c indicated mono-/di-/try-acetylated products. c Conversion rates of acetylated products catalyzed by AmAT7-3. O: identified by comparing with the standards; #: prepared and identified by NMR; *: compounds which were not reported previously. d Conversion rates of acetylated products catalyzed by AmAT7-3_A310G and AmAT7-3_A310W.