Molecular characterization of hypothetical scaffolding-like protein S1 in multienzyme complex produced by Paenibacillus curdlanolyticus B-6

Patthra Pason,Khanok Ratanakhanokchai,Rattiya Waeonukul,Chakrit Tachaapaikoon,Sirilak Baramee,Junjarus Sermsathanaswadi,Akihiko Kosugi

doi:10.1186/s13568-019-0896-0

Abstract

Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a hypothetical scaffolding-like protein and several xylanases and cellulases. The largest (280-kDa) component protein, called S1, has cellulose-binding ability and xylanase activity, thus was considered to function like the scaffolding proteins found in cellulosomes. S1 consists of 863 amino acid residues with predicted molecular mass 91,029 Da and includes two N-terminal surface layer homology (SLH) domains, but most of its sequence shows no homology with proteins of known function. Native S1 (nS1) was highly glycosylated. Purified nS1 and recombinant Xyn11A (rXyn11A) as a major xylanase subunit could assemble in a complex, but recombinant S1 (rS1) could not interact with rXyn11A, indicating that S1 glycosylation is necessary for assembly of the multienzyme complex. nS1 and rS1 showed weak, typical endo-xylanase activity, even though they have no homology with known glycosyl hydrolase family enzymes. S1 and its SLH domains bound tightly to the peptide-glycan layer of P. curdlanolyticus B-6, microcrystalline cellulose, and insoluble xylan, indicating that the SLHs of S1 bind to carbohydrate polymers and the cell surface. When nS1 and rXyn11A were co-incubated with birchwood xylan, the degradation ability was synergistically increased compared with that for each protein; however synergy was not observed for rS1 and rXynA. These results indicate that S1 may have a scaffolding protein-like function by interaction with enzyme subunits and polysaccharides through its glycosylated sites and SLH domains.

Highlights

Plant biomass, which has potential as a renewable resource, contains a complex mixture of polysaccharides, such as cellulose, hemicellulose, pectic substances, other polysaccharides(Caffall and Mohnen 2009), and lignin, which is a complex polymer of phenylpropane units
S1 from P. curdlanolyticus B‐6 To determine whether the scaffolding-like S1 protein has a structure similar to that of the cohesins that are normally observed in cellulosomes from cellulolytic clostridia, the S1 gene was cloned from P. curdlanolyticus B-6
According to a BLAST analysis, the first 125 amino acids of S1 were identified as two surface-layer homology (SLH) domain sequences (Fig. 1a)

Summary

Introduction

Plant biomass, which has potential as a renewable resource, contains a complex mixture of polysaccharides, such as cellulose, hemicellulose (xylan and galactomannan), pectic substances (polysaccharides comprising mainly 1,4-linked α-d-galactosyluronic acid such as galacturonan), other polysaccharides (e.g., fuco-xyloglucan)(Caffall and Mohnen 2009), and lignin, which is a complex polymer of phenylpropane units. P. curdlanolyticus B-6 produces a cellulosome-like unique multienzyme complex system of at least 11 protein subunits associated in a 1450-kDa complex by distinct cohesin–dockerin interactions (Pason et al 2006). This extracellular complex is composed of a 280-kDa scaffolding-like core protein (S1), several minor xylanases and cellulases, and major xylanases of ~ 40 kDa (Pason et al 2010). Protein S1 of the multienzyme complex was isolated through four chromatographic steps and has a xylan-degrading ability (Pason et al 2010)

Methods

Results

Conclusion