Structure and function of a novel GH8 endoglucanase from the bacterial cellulose synthase complex of Raoultella ornithinolytica

Abstract

Cellulose synthesis in bacteria is a complex process involving the concerted action of several enzymes whose genes are often organized in operons. This process influences many fundamental physiological aspects such as bacteria and host interaction, biofilm formation, among others. Although it might sound contradictory, the participation of cellulose-degrading enzymes is critical to this process. The presence of endoglucanases from family 8 of glycosyl hydrolases (GH8) in bacterial cellulose synthase (Bcs) complex has been described in different bacteria, including the model organism Komagataeibacter xylinus; however, their role in this process is not completely understood. In this study, we describe the biochemical characterization and three-dimensional structure of a novel GH8 member from Raoultella ornithinolytica, named AfmE1, which was previously identified by our group from the metagenomic analysis of the giant snail Achatina fulica. Our results demonstrated that AfmE1 is an endo-β-1,4-glucanase, with maximum activity in acidic to neutral pH over a wide temperature range. This enzyme cleaves cello-oligosaccharides with a degree of polymerization ≥ 5 and presents six glucosyl-binding subsites. The structural comparison of AfmE1 with other GH8 endoglucanases showed significant structural dissimilarities in the catalytic cleft, particularly in the subsite +3, which correlate with different functional mechanisms, such as the recognition of substrate molecules having different arrangements and crystallinities. Together, these findings provide new insights into molecular and structural features of evolutionarily conserved endoglucanases from the bacterial cellulose biosynthetic machinery.

Succession of Gut Microbial Structure in Twin Giant Pandas During the Dietary Change Stage and Its Role in Polysaccharide Metabolism

“…It is unexpected that the E. coli providing GH8 may become a main contributor of putative cellulase (EC 3.2.1.4) in cubs. Actually, as generally considered, the E. coli cannot degrade cellulose, and more than 75% of the endoglucanases from GH8 produced by bacterial taxa such as E. coli at Proteobacteria are as a component of the bacterial cellulose synthesis (bcs) system (Berlemont and Martiny, 2013), but interestingly, most of them are potentially required to correct packing of cellulose microfibrils (Mazur and Zimmer, 2011); that is, GH8 may retain its activity of endoglucanase to cello-oligosaccharides in the bcs system (Scapin et al, 2017). Under the product enrichment conditions, such as microfibril-like structured cellulose, GH8 will be secreted extracellularly, and out of the bcs system, it can hydrolyze soluble cellulose and cello-oligosaccharides in the environment (Pang et al, 2019), and structure-specific GH8 subfamily can directly hydrolyze amorphous CMC and crystalline cellulose (Attigani et al, 2016).…”

Synergic activity of Cel8Pa β-1,4 endoglucanase and Bg1Pa β-glucosidase from Paenibacillus xylanivorans A59 in beta-glucan conversion

“…GH8 are bacterial enzymes which have been characterized mainly as endoglucanases and chitosanases [ 19 ] as well as xylanases [ 31 ]. GH8 endoglucanases have been widely studied as part of Bacterial Cellulose Synthase (Bcs) complexes involved in production and export of the glucan extracellular matrix in different bacteria [ 27 , 32 ] and also as part of polysaccharide degrading systems, with CelA from the anaerobic bacterium Clostridium thermocellum as one of the most studied ones [ 33 ]. In this regard, Cel8Pa had high identity with Paenibacillus sp.…”

Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome

“…The mesophilic/thermophilic characteristic displayed by AFase-D3 has only been reported for AFases that belong to the GH43 family previously isolated from Paenibacillus species [34] with no characterized GH51 family enzyme displaying this thermal profile. Broad temperature optima have been observed for several different enzymes isolated from a range of organisms including plants [35–38]. Of particular interest are enzymes (phophoribosyl anthranilate isomerase, indoleglycerol phosphate synthase and l-isoaspartyl (D-aspatyl) O-methyltransferase) from T. maritima that show highest catalytic efficiency at 25 °C as opposed to the organism’s optimum growth temperature [39–41].…”