Transesterification with CE15 glucuronoyl esterase from Cerrena unicolor reveals substrate preferences

Purpose Glucuronoyl esterases (GE, family CE15) catalyse the cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), and this study demonstrate how transesterification reactions with a fungal GE from Cerrena unicolor (CuGE) can reveal the enzyme’s preference for the alcohol-part of the ester-bond. Methods This alcohol-preference relates to where the ester-LCCs are located on the lignin molecule, and has consequences for how the enzymes potentially interact with lignin. It is unknown exactly what the enzymes prefer; either the α-benzyl or the γ-benzyl position. By providing the enzyme with a donor substrate (the methyl ester of either glucuronate or 4-O-methyl-glucuronate) and either one of two acceptor molecules (benzyl alcohol or 3-phenyl-1-propanol) we demonstrate that the enzyme can perform transesterification and it serves as a method for assessing the enzyme’s alcohol preferences. Conclusion CuGE preferentially forms the γ-ester from the methyl ester of 4-O-methyl-glucuronate and 3-phenyl-1-propanol and the enzyme’s substrate preferences are primarily dictated by the presence of the 4-O-methylation on the glucuronoyl donor, and secondly on the type of alcohol. Supplementary Information The online version contains supplementary material available at 10.1007/s10529-023-03456-x.


Figure S1 .
Figure S1.Reaction progression of the transesterification of BnzOH with Me-GlcA by 260 µg/mL (4.3 µM) CuGE over 100 min.Extracted Ion Chromatogram (EIC) of Me-GlcA (black) at a retention time (RT) 2.9 min and m/z 230.91 decreased intensity over reaction time.EIC of BnzGlcA (blue) at a RT 8.0 min and m/z 307.02 increased intensity over reaction time.All ions are shown as [M+Na] + .MS2 spectra of m/z 230.91 and m/z 307.02 are also shown.

Figure S2 .
Figure S2.Reaction progression of the transesterification of PhPrOH with Me-GlcA by 52 µg/mL (0.87 µM) CuGE over 100 min.EIC of Me-GlcA (black) at a retention time (RT) 3.4 min and m/z 230.91 decreased intensity over reaction time.EIC of PhPr-GlcA (red) at a RT 7.7 min and m/z 335.12 increased intensity over reaction time.All ions are shown as [M+Na] + .MS2 spectra of m/z 230.91and m/z 335.12 are also shown.m/z 335.12 splits in two peaks (a minor shoulder in front of the main peak), which could be indicative for chirality, however not investigated further.

Figure S3 .
Figure S3.Reaction progression of the transesterification of BnzOH with Me-4-O-MeGlcA by 6.5 µg/mL (0.11 µM) CuGE over 100 min.EIC of Me-4-O-MeGlcA (black) at a retention time (RT) 6.5 min and m/z 244.96 decreased intensity over reaction time.Bnz-4-O-MeGlcA (green) at a RT 9.8 min and m/z 321.08 increased intensity over reaction time.All ions are shown as [M+Na] + .MS2 spectra of m/z 244.96 and m/z 321.08 are also shown.

Figure S4 .
Figure S4.Reaction progression of the transesterification of PhPrOH with Me-4-O-MeGlcA by 1.3 µg/mL (0.02 µM) CuGE over 100 min.EIC of Me-4-O-MeGlcA (black) at a retention time (RT) 6.4 min and m/z 244.97 decreased intensity over reaction time.PhPr-4-O-MeGlcA (blue) at a RT 9.3 min and m/z 349.14 increased intensity over reaction time.All ions are shown as [M+Na] + .MS2 spectra of m/z 244.97 and m/z 349.14 are also shown.

Figure S5 .
Figure S5.Control reaction with BnzOH and Me-GlcA where CuGE loading was substituted with 10 mM Na acetate buffer pH 6 over 100 min.Extracted Ion Chromatogram (EIC) of Me-GlcA (violet) at a retention time (RT) 3.0 min and m/z 230.91 remained constant in intensity over reaction time.Ion is shown as [M+Na] + .MS2 spectrum of m/z 230.91 is also shown.

Figure S6 .
Figure S6.Control reaction with PhPrOH and Me-GlcA where CuGE loading was substituted with 10 mM Na acetate buffer pH 6 over 100 min.Extracted Ion Chromatogram (EIC) of Me-GlcA (violet) at a retention time (RT) 3.4 min and m/z 230.90 remained constant in intensity over reaction time.Ion is shown as [M+Na] + .MS2 spectrum of m/z 230.90 is also shown.

Figure S7 .
Figure S7.Control reaction with BnzOH and Me-4-O-MeGlcA where CuGE loading was substituted with 10 mM Na acetate buffer pH 6 over 100 min.Extracted Ion Chromatogram (EIC) of Me-4-O-MeGlcA (blue) at a retention time (RT) 6.5 min and m/z 244.95 remained constant in intensity over reaction time.Ion is shown as [M+Na] + .MS2 spectrum of m/z 244.95 is also shown.

Figure S8 .
Figure S8.Control reaction with PhPrOH and Me-4-O-MeGlcA where CuGE loading was substituted with 10 mM Na acetate buffer pH 6 over 100 min.Extracted Ion Chromatogram (EIC) of Me-4-O-MeGlcA (blue) at a retention time (RT) 6.4 min and m/z 244.94 remained constant in intensity over reaction time.Ion is shown as [M+Na] + .MS2 spectrum of m/z 244.95 is also shown.

Figure S9 .
Figure S9.Calibration curves.a) Calibration curve used for Me-GlcA hydrolysis by CuGE determined as the area below the EIC of m/z 230.92 and m/z 439.07 at different MeGlcA concentrations.b) Calibration curve used for Me-4-O-MeGlcA hydrolysis by CuGE determined as the area below the EIC of m/z 244.95 and m/z 467.03 at different Me-4-O-MeGlcA concentrations.c) Calibration curve used for transesterification products of BnzOH with Me-4-O-MeGlcA and Me-GlcA.Quantification is performed relative to the concentration of BnzGlcA (dissolved in BnzOH, as the area below the EIC of m/z 307.04 and m/z 591.01).d) Calibration curve used for transesterification of PhPrOH with Me-4-O-MeGlcA and Me-GlcA.Quantification is performed relative to the concentration of Bnz-GlcA (dissolved in PhPrOH, area below the EIC of m/z 307.04 and m/z 591.01).All calibrations are performed in triplicate.