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Identification and characterization of endo-α-, exo-α-, and exo-β-D-arabinofuranosidases degrading lipoarabinomannan and arabinogalactan of mycobacteria.

ABSTRACT: The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of D-arabinose, the enantiomer of the typical L-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-D-arabinanases, GH172 exo-α-D-arabinofuranosidase, and GH116 exo-β-D-arabinofuranosidase) from Microbacterium arabinogalactanolyticum. These enzymes completely degraded the complex D-arabinan core structure of lipoarabinomannan and arabinogalactan in a concerted manner. Furthermore, through biochemical characterization using synthetic substrates and X-ray crystallography, we elucidated the mechanisms of substrate recognition and anomer-retaining hydrolysis for the α- and β-D-arabinofuranosidic bonds in both endo- and exo-mode reactions. The discovery of these D-arabinan-degrading enzymes, along with the understanding of their structural basis for substrate specificity, provides valuable resources for investigating the intricate glycan architecture of mycobacterial cell wall polysaccharides and their contribution to pathogenicity.

SUBMITTER: Shimokawa M

PROVIDER: S-EPMC10509167 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Identification and characterization of endo-α-, exo-α-, and exo-β-D-arabinofuranosidases degrading lipoarabinomannan and arabinogalactan of mycobacteria.

Shimokawa Michiko M Ishiwata Akihiro A Kashima Toma T Nakashima Chiho C Li Jiaman J Fukushima Riku R Sawai Naomi N Nakamori Miku M Tanaka Yuuki Y Kudo Azusa A Morikami Sae S Iwanaga Nao N Akai Genki G Shimizu Nobutaka N Arakawa Takatoshi T Yamada Chihaya C Kitahara Kanefumi K Tanaka Katsunori K Ito Yukishige Y Fushinobu Shinya S Fujita Kiyotaka K

Nature communications 20230919 1

The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of D-arabinose, the enantiomer of the typical L-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-D-arabinanases, GH172 exo-α-D-arabinofuranosidase, and GH116 exo ...[more]

PMID: 37726269

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Project description:BackgroundImproving the hydrolytic performance of xylanolytic enzymes on arabinoxylan is of importance in the ethanol fermentation industry. Supplementation of debranching (arabinofuranosidase) and depolymerizing (xylanase) enzymes is a way to address the problem. In the present study, we identified a bifunctional α-l-arabinofuranosidase/endo-xylanase (Ac-Abf51A) of glycoside hydrolase family 51 in Alicyclobacillus sp. strain A4. Its biochemical stability and great hydrolysis efficiency against complex biomass make it a potential candidate for the production of biofuels.ResultsThe gene encoding Ac-Abf51A was cloned. The comparison of its sequence with reference proteins having resolved 3D-structures revealed nine key residues involved in catalysis and substrate-binding interaction. Recombinant Ac-Abf51A produced in Escherichia coli showed optimal activity at pH 6.0 and 60 °C with 4-nitrophenyl-α-l-arabinofuranoside as the substrate. The enzyme exhibited an exo-type mode of action on polyarabinosides by catalyzing the cleavage of α-1,2- and α-1,3-linked arabinofuranose side chains in sugar beet arabinan and water-soluble wheat arabinoxylan and α-1,5-linked arabinofuranosidic bonds in debranched sugar beet arabinan. Surprisingly, it had capacity to release xylobiose and xylotriose from wheat arabinoxylan and was active on xylooligosaccharides (xylohexaose 1.2/mM/min, xylopentaose 6.9/mM/min, and xylotetraose 19.7/mM/min), however a lower level of activity. Moreover, Ac-Abf51A showed greater synergistic effect in combination with xylanase (2.92-fold) on wheat arabinoxylan degradation than other reported enzymes, for the amounts of arabinose, xylose, and xylobiose were all increased in comparison to that by the enzymes acting individually.ConclusionsThis study for the first time reports a GH51 enzyme with both exo-α-l-arabinofuranosidase and endo-xylanase activities. It was stable over a broad pH range and at high temperature, and showed greater synergistic effect with xylanase on the degradation of wheat arabinoxylan than other counterparts. The distinguished synergy might be ascribed to its bifunctional α-l-arabinofuranosidase/xylanase activity, which may represent a possible way to degrade biomass at lower enzyme loadings.

Project description:BackgroundCellulase is the one of the largest contributors to the high production costs of the lignocellulose-based biorefineries. As the most widely used cellulase producer, Trichoderma reesei has two weaknesses, deficiencies in β-glucosidase and cellobiohydrolase II. This work aimed at solving this problem by simultaneous enhancement of the beta-exo synergism and exo-exo synergism in T. reesei cellulase to increase the cellulose degrading capability, i.e. enhanced co-expression of the β-glucosidase gene the cellobiohydrolase II gene of T. reesei.ResultsEnhanced co-expression of the β-glucosidase gene and the cellobiohydrolase II gene in T. reesei using the strong promoter Pcbh1 was found successful in overcoming the two weaknesses. Filter paper activities of T. reesei cellulase were greatly elevated, which were 7.21 ± 0.45 (E7, Aabgl1 and Trcbh2) and 7.69 ± 0.42 (F6, Anbgl1 and Trcbh2) FPIU/mL. They were much higher than that of the parental strain Rut-C30, 2.45 ± 0.36 FPIU/mL. Enzymatic hydrolysis yields were also improved, from 67.22 ± 1.61% by Rut-C30 cellulase to 87.98 ± 0.65% by E7 cellulase and 86.50 ± 1.01% by F6 cellulase. The substrate loading for 1 g glucose release from SECS were decreased, from 2.9637 g SECS using Rut-C30 cellulase to 2.0291 g SECS using E7 cellulase and 2.0573 g SECS using F6 cellulase. As a result, the efficiency of the process from SECS to glucose was substantially improved.ConclusionsEnhanced co-expression of the β-glucosidase gene and the cellobiohydrolase II gene in T. reesei using the strong promoter Pcbh1 in T. reesei was proven triumphal in the simultaneous enhancement of the beta-exo synergism and exo-exo synergism in T. reesei cellulase. This strategy also improved the cellulase production, enzymatic hydrolysis yield and the efficiency of the process from SECS to glucose in the context of on-site cellulase production. This work is a commendable attempt in the cellulase composition optimization at the transcriptional level.

Dataset Information

Identification and characterization of endo-α-, exo-α-, and exo-β-D-arabinofuranosidases degrading lipoarabinomannan and arabinogalactan of mycobacteria.

Publications

Identification and characterization of endo-α-, exo-α-, and exo-β-D-arabinofuranosidases degrading lipoarabinomannan and arabinogalactan of mycobacteria.

Similar Datasets

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