Extended Data Fig. 5: Sugar-donor specificity of SoGH1.
a. Purified recombinant SoGH1. SoGH1 was expressed with C-terminal His-tag in N. benthamiana by Agrobacterium-mediated transient expression. Lane 1, cleared lysate before purification. Lane 2, purified fraction by TALON metal affinity purification. RbcL, Rubisco large subunit which is highly abundant in plant leaf soluble extracts. The unprocessed gel image is available as Source Data. b. Structure of surrogate saponin acceptor, 3-O-{α-l-rhamnopyranosyl-(1 → 2)-[β-d-galactopyranosyl-(1 → 2)]-β-d-glucopyranosiduronic acid}-28-O-{β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-β-d-fucopyranosyl ester}-quillaic acid (QA-TriR-FRX) used. A table with relevant calculated adducts and modifications is also shown. c. Enzyme assay of purified SoGH1 incubated with QA-TriR-FRX and various sugar donors analyzed using HPLC-MS. Extracted ion chromatograms (EIC) and MS/MS spectrum are shown. EIC displayed are for m/z 1555.6810, the calculated mass of [M-H]- adduct of QA-TriR-FRX plus hexose. SoGH1 incubated only with QA-TriR-FRX without any sugar donors is used as a negative control (-no donor). A noticeable product peak is observed when benzoyl-glc is given as the sugar donor, but less prominent product peaks are also observed with coumaroyl-glc, feruloyl-glc and naringenin-7glc as sugar donors. MS/MS fragmentation pattern of the product peaks suggests an addition of hexose (d-glucose) to the C-28 sugar chain, which then fragments to m/z 969.4703, corresponding to the expected [M-H]- of QA-TriR-FRX without the C-28 sugar chain. This experiment was repeated independently three times with similar results. UDP-glc, UDP-β-d-glucose; 4NP-glc, 4-nitrophenyl-β-d-glucoside; Phenyl-glc, phenyl-β-d-glucoside; arbutin, hydroquinone-β-d-glucoside; benzoyl-glc, benzoyl-β-d-glucoside; galloyl-glc, 1-O-galloyl-β-d-glucoside; coumaroyl-glc, 1-O-coumaroyl-β-d-glucoside; feruloyl-glc, 1-O-feruloyl-β-d-glucoside; naringenin-7glc, naringenin-7-O-β-d-glucoside; quercetin-3glc, quercetin-3-O-β-d-glucoside.
