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)-alpha-d-(6-benzyl-4-O-methyl-glucuronyl)-1,4-beta-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-beta-d-xylotriose (MeGlcA(3)Xyl(3)), a minimized fragment of glucuronoxylan enzymatically digested by beta-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 mu mol 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.

Enzymatic hydrolysis of cellulosic biomass is a complex process involving many factors, including multiple enzymes, heterogeneous substrates, and multi-step enzyme reactions. Cellulase researchers have conventionally used a double-exponential equation to fit the experimental time course of product formation, but no theoretical basis for this has been established. Here we present a mechanism-based equation that fits well the progress curves of cellulase reaction, incorporating the concepts of non-productive and productive binding on the cellulose surface and processivity. The derived equation is double exponential. Our findings indicate that the reaction mechanism of cellulase itself can account for the double- exponential nature of the progress curve independently of other factors that may contribute, such as substrate heterogeneity and involvement of other enzymes.

Glycoside hydrolase family 6 cellobiohydrolase (GH6 CBH) is a group of cellulases capable of hydrolyzing crystalline cellulose. However, the synergistic reaction of GH6 CBH with other cellulases is hindered by its relatively low thermotolerance. We previously obtained a thermotolerant double mutant, C240S/C393S, of GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCel6A) by replacing the two free cysteine (Cys) residues, C240 and C393, with serine (Yamaguchi et al., J Appl Glycosci. 2020; 67;79-86). In the accompanying paper (Part I; Yamaguchi et al., J Appl Glycosci. 2024; 71: 55-62), we measured the temperature dependence of the activity and folding of C240S/C393S and its single mutants, C240S and C393S, and found that replacement of C393 was the major contributor to the increased thermotolerance of C240S/C393S. Here, in order to investigate the mechanism involved, we crystallized the wild-type and the mutant enzymes and compared their X-ray crystal structures. The overall structures of the wild-type and the three mutant enzymes were similar. However, C240S/C393S had the lowest relative B-factor at both the N-terminal loop (residues 172-177) and the C-terminal loop (residues 390-425). This result suggests that reduced structural fluctuation of the substrate-enclosing loops, possibly due to stronger hydrogen bonding involving C393, could account for the increased thermotolerance of C240S/C393S.

Endo-type xylanases are key enzymes in microbial xylanolytic systems, and xylanases belonging to glycoside hydrolase (GH) families 10 or 11 are the major enzymes degrading xylan in nature. These enzymes have typically been characterized using xylan prepared by alkaline extraction, which removes acetyl sidechains from the substrate, and thus the effect of acetyl groups on xylan degradation remains unclear. Here, we compare the ability of GH10 and 11 xylanases, PcXyn10A and PcXyn11B, from the white-rot basidiomycete Phanerochaete chrysosporium to degrade acetylated and deacetylated xylan from various plants. Product quantification revealed that PcXyn10A effectively degraded both acetylated xylan extracted from Arabidopsis thaliana and the deacetylated xylan obtained by alkaline treatment, generating xylooligosaccharides. In contrast, PcXyn11B showed limited activity towards acetyl xylan, but showed significantly increased activity after deacetylation of the xylan. Polysaccharide analysis using carbohydrate gel electrophoresis showed that PcXyn11B generated a broad range of products from native acetylated xylans extracted from birch wood and rice straw, including large residual xylooligosaccharides, while non-acetylated xylan from Japanese cedar was readily degraded into xylooligosaccharides. These results suggest that the degradability of native xylan by GH11 xylanases is highly dependent on the extent of acetyl group substitution. Analysis of 31 fungal genomes in the Carbohydrate-Active enZymes database indicated that the presence of GH11 xylanases is correlated to that of carbohydrate esterase (CE) family 1 acetyl xylan esterases (AXEs), while this is not the case for GH10 xylanases. These findings may imply coevolution of GH11 xylanases and CE1 AXEs.

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) β-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the KM values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X2), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.

Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from Phanerochaete chrysosporium. Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D. MD simulation suggests that the result of AA9D action to produce chain breaks in crystalline cellulose can oxidatively disturb the crystalline surface by disrupting hydrogen bonds. HS-AFM observations showed that AA9D increased the number of Cel7D molecules moving on the substrate surface and increased the processivity of Cel7D, thereby increasing the depolymerization performance, suggesting that AA9D not only generates chain ends but also amorphizes the crystalline surface, thereby increasing the activity of CBHs.

Cellobiohydrolase I from Trichoderma reesei ( Tr Cel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of Tr Cel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of Tr Cel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of Tr Cel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of Tr Cel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake ( k on for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel.

Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are a kind of cellulase. In general, CBHs belonging to glycoside hydrolase (GH) family 6 (Cel6) act synergistically with CBHs of GH family 7 (Cel7) and other carbohydrate-active enzymes during the degradation of cellulosic biomass. However, while the catalytic rate of enzymes generally becomes faster at higher temperatures, Cel6 CBHs are inactivated at lower temperatures than Cel7 CBHs, and this represents a limiting factor for industrial utilization. In this study, we produced a series of mutants of the glycoside hydrolase family 6 cellobiohydrolase Pc Cel6A from the fungus Phanerochaete chrysosporium , and compared their thermal stability. Eight mutants from a random mutagenesis library and one rationally designed mutant were selected as candidate thermostable mutants and produced by heterologous expression in the yeast Pichia pastoris . Comparison of the hydrolytic activities at 50 and 60 °C indicated that the thermal stability of Pc Cel6A is influenced by the number and position of cysteine residues that are not involved in disulfide bonds.

Arabinogalactan proteins (AGPs) are functional plant proteoglycans, but their functions are largely unexplored, mainly because of the complexity of the sugar moieties, which are generally analyzed with the aid of glycoside hydrolases. In this study, we solved the apo and liganded structures of exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium ( Pc 1,3Gal43A), which specifically cleaves AGPs. It is composed of a glycoside hydrolase family 43 subfamily 24 (GH43\_sub24) catalytic domain together with a carbohydrate-binding module family (CBM) 35 binding domain. GH43\_sub24 lacks the catalytic base Asp that is conserved among other GH43 subfamilies. Crystal structure and kinetic analyses indicated that the tautomerized imidic acid function of Gln263 serves instead as the catalytic base residue. Pc 1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s. Some of the residues involved in ligand recognition differ from those of galactomannan-binding CBM35, including substitution of Trp for Gly, which affects pyranose stacking, and substitution of Asn for Asp in the lower part of the binding pocket. Pc 1,3Gal43A WT and its mutants at residues involved in substrate recognition are expected to be useful tools for structural analysis of AGPs. Our findings should also be helpful in engineering designer enzymes for efficient utilization of various types of biomass.

Sugarcane bagasse is a useful biomass resource. In the present study, we examined the efficacy of ammonia pretreatment for selective release of hemicellulose from bagasse. Pretreatment of bagasse with aqueous ammonia resulted in significant loss of xylan. In contrast, pretreatment of bagasse with anhydrous ammonia resulted in almost no xylan loss. Aqueous ammonia or anhydrous ammonia-pretreated bagasse was then subjected to enzymatic digestion with a xylanase from the glycoside hydrolase (GH) family 10 or a xylanase from the GH family 11. The hydrolysis rate of xylan in bagasse pretreated with aqueous ammonia was approximately 50 %. In contrast, in the anhydrous ammonia-treated bagasse, xylan hydrolysis was > 80 %. These results suggested that anhydrous ammonia pretreatment would be an effective method for preparation of sugarcane bagasse for enzymatic hydrolysis to recover xylooligosaccharides.

Pyranose 2-oxidases catalyze the oxidation of various pyranose sugars at the C2 position. However, their potential application for detecting sugars other than glucose in blood is hindered by relatively high activity towards glucose. In this study, in order to find a mutant enzyme with enhanced specificity for 1,5-anhydro-D-glucitol (1,5-AG), which is a biomarker for diabetes mellitus, we conducted site-directed mutagenesis of pyranose 2-oxidase from the basidiomycete Phanerochaete chrysosporium ( Pc POX). Considering the three-dimensional structure of the substrate-binding site of Pc POX and the structural difference between glucose and 1,5-AG, we selected alanine 551 of Pc POX as a target residue for mutation. Kinetic studies of the 19 mutants of Pc POX expressed as recombinant proteins in E. coli revealed that the ratio of k cat / K m for 1,5-AG to k cat / K m for glucose was three times higher for the A551L mutant than for wild-type Pc POX. Although the A551L mutant has lower specific activity towards each substrate than the wild-type enzyme, its increased specificity for 1,5-AG makes it a promising lead for the development of POX-based 1,5-AG detection systems.

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.

Toshihisa Kotake, Naohiro Hirata, Yuta Degi, Maki Ishiguro , Kiminari Kitazawa, Ryohei Takata, Hitomi Ichinose**, Satoshi Kaneko**, Kiyohiko Igarashi , Masahiro Samejima , and Yoichi Tsumuraya From the Division of Life Science, Graduate School of Science and Engineering, Institute for Environmental Science and Technology, and Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, the Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, and the **Food Biotechnology Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar K m and k cat values to cellobiose. β-D-Glucopyranosyl-(1→4)-D-xylopyranose (Glc-Xyl) shows a higher K m value, while xylobiose does not serve as a substrate. Trisaccharides show similar behavior; i.e., trisaccharides with cellobiose and Xyl-Glc units at the reducing end show similar kinetics, while the enzyme was less active towards those with Glc-Xyl, and inactive towards those with xylobiose. We also use docking simulation to evaluate substrate recognition of the disaccharides, and we discuss possible molecular mechanisms of substrate recognition by CDH.

BACKGROUND:Mushrooms have been widely considered as health foods as their extracts have anti-hypertensive and anti-tumor activities. After a thorough literature survey, we hypothesized that enzymes in mushroom extracts play an important role in synthesizing functional molecules. Therefore, in this study, proteins extracted from reishi mushroom (Ganoderma lucidum), which is used in oriental medicine, were identified by the proteomic approach, and appropriate extraction methods for improving angiotensin-converting enzyme (ACE) inhibitory activities were investigated.RESULTS:Various glycoside hydrolases (GHs), such as β-N-acetylhexosaminidase (GH family 20), α-1,2-mannosidase (GH family 47), endo-β-1,3-glucanase (GH family 128), and β-1,3-glucanase (GH152), that degrade glycans in the fruiting body were identified. The residual glucanase activities generated β-oligosaccharides. Additionally, the glutamic acid protease of the peptidase G1 family was determined as the major protein in the extract, and the residual peptidase activity of the extracts was found to improve ACE inhibitory activities. Finally, it was observed that extraction at 50 °C is suitable for yielding functional molecules with high ACE inhibitory activities.CONCLUSION:Water extraction is generally believed to extract only functional macromolecules that exist in mushroom fruiting bodies. This study proposed a new concept that describes how functional molecules are produced by enzymes, including proteases and GHs, during extraction. © 2018 Society of Chemical Industry.