Synthesis of a natural core substrate with lignin-xylan cross-linkage for unveiling the productive kinetic parameters of glucuronoyl esterase.

Lignin-carbohydrate complexes (LCCs) present a considerable hurdle to the economic utilization of lignocellulosic biomass. Glucuronoyl esterase (GE) is an LCC-degrading enzyme that catalyzes the cleavage of the cross-linkages between lignin and xylan in LCCs. Benzyl-d-glucuronate (Bn-GlcA), a commercially available substrate, is widely used to evaluate GE activity assays. However, since Bn-GlcA lacks the structural backbone of naturally occurring LCCs, the mechanisms underlying the activity of GEs and their diversity in the structure-activity relationship are not fully understood. Herein, we provided a synthesis scheme for designing 1,2 ³ -α-d-(6-benzyl-4-O-methyl-glucuronyl)-1,4-β-d-xylotriose (Bn-MeGlcA ³ Xyl ₃ ) as a natural core substrate bearing cross-linkage between lignin and glucuronoxylan. A well-defined and yet more realistic synthetic substrate was successfully synthesized via a key step of the benzyl esterification of 4-O-methyl-glucuronyl-1,4-β-d-xylotriose (MeGlcA ³ Xyl ₃ ), a minimized fragment of glucuronoxylan enzymatically digested by β-1,4-xylanase. To the best of our knowledge, this is the first report of the productive GE kinetic analysis using this substrate. Kinetic parameters of the GE from the fungal Pestalotiopsis sp. AN-7 (PesGE), i.e., the K _m , V _max , and k _cat of Bn-MeGlcA ³ Xyl ₃ , were 0.43 mM, 55.5 μmol min ^-1 ·mg ^-1 , and 35.8 s ^-1 , respectively. On the other hand, as reported to date, the productive kinetic parameters for Bn-GlcA were not obtained because of its excessively high K _m value (>16 mM). The substantial variance in the enzymatic activity of PesGE regarding substrate-binding affinity between Bn-MeGlcA ³ Xyl ₃ and Bn-GlcA was also demonstrated using in silico docking simulation. These results suggested that the extended xylan fragment is a key structural determinant affecting PesGE's substrate recognition. Furthermore, the presence of a natural xylan backbone allows for evaluating the enzyme activity of xylan-degrading enzymes. Accordingly, the synthesized substrate with the natural core structure of LCC allowed us to unveil the productive kinetic parameters of GEs, serving as a versatile substrate for further elucidating the cascade reaction of GE and xylan-degrading enzymes.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sangho Koh reports financial support was provided by Japan Society for the Promotion of Science. Sangho Koh reports financial support was provided by Shinshu UniversityJapan Society for the Promotion of Science. Masahiro Mizuno reports financial support was provided by Sugiyama Sangyou Kagaku Research Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)