Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

Abstract

Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (K a ) values of 5.2 ؋ 10 6 to 44 ؋ 10 6 M ؊1 among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.

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

“…In addition to CBMs within modular enzymes, Caldicellulosiruptor species also use noncatalytic proteins to bind to lignocellulosic substrates. Transcriptomic and proteomic analysis of cellulose-bound Caldicellulosiruptor cultures identified the presence of carbohydrate binding proteins (19), including the recently characterized 'tāpirins' (20). Tāpirins typically have a Mr of 105 approximately 70 kDa as a single polypeptide; recombinant versions from C. kronotskyensis have a specific affinity for cellulose fibers in plant material and an affinity for Avicel similar to that of CBM3s, despite no significant structural homology (20).…”

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

“…In addition to CBMs within modular enzymes, Caldicellulosiruptor species also use noncatalytic proteins to bind to lignocellulosic substrates. Transcriptomic and proteomic analysis of cellulose-bound Caldicellulosiruptor cultures identified the presence of carbohydrate binding proteins (20), including the recently characterized "ta pirins" (21). Ta pirins typically have a M r of approximately 70 kDa as a single polypeptide; recombinant versions from C. kronotskyensis have a specific affinity for cellulose fibers in plant material and an affinity for Avicel similar to that of CBM3s, despite the absence of significant structural homology (21).…”

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

“…Some of the modular enzymes from the genus Caldicellulosiruptor are cell-anchored using S-layer homology domains (30) and are hypothesized to mediate cell-substrate proximity. In addition, substrate-binding proteins (in some cases, components of ATP-binding cassette transporters (24,31)) may also play a role in orienting Caldicellulosiruptor species toward carbohydrate moieties in plant biomass, possibly through positively charged amino acid residues interacting with negatively charged areas (32). However, comparative proteogenomics analysis of whole cell, extracellular, and cellulose-bound fractions of Caldicellulosiruptor species revealed that previously annotated hypothetical proteins (referred to here as "ta pirins" taken from the Ma ori verb "to join") bound tightly to cellulose, thereby implicating yet another element in the mechanism by which these bacteria attach to plant biomass.…”