The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

Abstract

bThe genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical Cterminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley ␤-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/␤-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages. P lant cell wall polysaccharides (PCWPs), composed mainly of cellulose and hemicellulose, are a promising rich resource for renewable biofuel development (1). The complete deconstruction of PCWPs into fermentable, simple mono-or oligosaccharides requires the concerted action of a complex array of glycoside hydrolases (GHs), including cellulases and hemicellulases (2, 3). The genomes of Gram-positive bacteria of the genus Caldicellulosiruptor encode an arsenal of thermophilic plant cell wall polysaccharide-degrading enzymes (3-5), which are appealing candidates in the design of novel, robust enzyme cocktails for PCWP deconstruction.In the genome of Caldicellulosiruptor bescii, there is a gene cluster containing three genes which encode proteins harboring two tandemly linked CBM3b modules and a GH family 48 (GH48) cellobiohydrolase in the C terminus. These CBM3b and GH48 modules, as well as their linker sequences, are extremely similar at the level of the amino acid sequence (see Fig. S1 in the supplemental material). Notably, however, the three multimodular enzymes differ much in their N termini: CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) ha...

Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins (Tāpirins) from Extremely Thermophilic Caldicellulosiruptor Species

“…While not directly responsible for lignocellulose (switchgrass) degradation, the noncatalytic domains in CelA also play an important role. In general, CBMs improve the efficacy of GHs by ensuring proximity to the substrate, as well as contributing to thermostability in some cases (16,17). CBM3s, in particular, are specific to cellulose and allow enzymes such as CelA to attach to their substrates such that their GH domains are proximate to their substrate (9).…”

Novel multidomain, multifunctional glycoside hydrolases from highly lignocellulolytic Caldicellulosiruptor species

“…Cdan_2053 is also similar to sections of Athe_1857 (GH10‐(CBM3) 2 ‐GH48) from C. bescii (88% amino acid identity GH10‐CBM3, 98% amino acid identity GH48). The GH10 of Athe_1857 has been previously characterized and showed activity both on xylan, the canonical substrate for GHs from family 10, and glucan substrates including cellulose. Cmor_0543 is nearly identical (100% coverage, 94% amino acid identity) to a GH identified as “CelE” from Caldicellulosiruptor sp.…”

“…Cdan_2053 was about twice as active as Cmor_0943 or Cmor_0545 on birch wood xylan, likely because of its GH10 domain. The homologous GH10 from Athe_1857 degrades xylans as well as β‐glucans . Cmor_0543 alone was active on locust bean gum galactomannan and 1,4‐β‐D‐mannan, likely because of its GH5 domain.…”

“…The GHs responsible for the majority of extracellular cellulose and lignocellulose degradation are concentrated in a single genomic region, termed the glucan degradation locus (GDL) . Several full‐length enzymes or individual GH domains from the GDL of species, including C. bescii and C. saccharolyticus , have previously been characterized . These GHs in the GDL contain multiple carbohydrate binding module Family 3 (CBM3) domains, interspersed among catalytic domains.…”

Caldicellulosiruptor besciiregulates pilus expression in response to the polysaccharide, xylan

“…Notably, C. bescii is able to degrade insoluble cellulose along with various other plant polysaccharides like xylan (5) and pectin (6), and can grow efficiently on untreated plant 45 biomass with high lignin content (7)(8)(9). Modular enzymes with multiple catalytic domains, found primarily in the glucan degradation locus (GDL) (10) diversifies the substrates that these enzymes can hydrolyze (11)(12)(13)(14)(15).…”