On the functionality of the N-terminal domain in xylanase 10A from Ruminococcus albus 8

https://doi.org/10.1016/j.enzmictec.2020.109673Get rights and content

Highlights

  • R. albus 8 xylanase 10A presents a distinctive functional N-terminus (N34) motif.

  • The N34-domain confers redox sensitivity for the activity and substrate binding.

  • The N34-motif would be involved with bacterial membrane interaction.

  • The enzyme functionality is related with rumen redox potential.

Abstract

We analyzed the structure to function relationships in Ruminococcus albus 8 xylanase 10A (RalXyn10A) finding that the N-terminus 34-amino acids sequence (N34) in the protein is particularly functional. We performed the recombinant wild type enzyme’s characterization and that of the truncated mutant lacking the N34 extreme (RalΔN34Xyn10A). The truncated enzyme exhibited about half of the activity and reduced affinity for binding to insoluble saccharides. These suggest a (CBM)-like function for the N34 motif. Besides, RalXyn10A activity was diminished by redox agent dithiothreitol, a characteristic absent in RalΔN34Xyn10A. The N34 sequence exhibited a significant similarity with protein components of the ABC transporter of the bacterial membrane, and this motif is present in other proteins of R. albus 8. Data suggest that N34 would confer RalXyn10A the capacity to interact with polysaccharides and components of the cell membrane, enhancing the degradation of the substrate and uptake of the products by the bacterium.

Introduction

Lignocellulose constitutes the primary raw material for the production of secondgeneration biofuels [1,2]. The development of accurate technology for such bioenergy purposes has a critical limitation linked to the complex structure that makes lignocellulosic material recalcitrant to degradation. In this context, the search for more efficient enzymes with the ability to efficiently hydrolyze the polymeric biomass is a significant issue for biochemical studies. Xylans are the hemicellulose’s main components being closely associated with cellulose fibrils by, together with lignin, covering the fiber surfaces [[1], [2], [3], [4]]. In degradative biomass treatments, even low amounts of residual xylans can limit the extent and efficiency of the enzymatic hydrolysis of cellulose. The addition of xylanases can overcome this curtail, as such enzymes hydrolyze hemicellulose, releasing xylan from the substrates, thus improving the degradation of the polymeric material [[3], [4], [5], [6], [7], [8], [9]].

Xylans are polysaccharides made up of a linear backbone of β-(1→4)-D-xylopyranosyl units with side branches at positions 2-O and 3-O of α-L-arabinofuranosyl, 4-O-methyl-glucopyranosyl uronic acid, and acetyl groups [8,10]. Endo β-1,4-xylanases cleave internal β-1,4-glycosyl bonds in the xylans’ main chain, giving rise xylooligosaccharides as products. As reported for cellulases [[11], [12], [13]], many xylanases exhibit a complex modular structure. This latter comprises a catalytic domain (CD), and a non-catalytic polypeptide fused to by either the N- or the C-terminus (or both) by flexible linkers rich in proline, threonine, and serine residues. The non-catalytic domain exerts a function as a carbohydrate-binding module (CBM) or as an enhancer of the enzyme thermostability [[14], [15], [16], [17], [18]]. Currently, 87 different families of CBMs have been categorized based on amino acid sequence similarities (http://www.cazy.org) [14,19].

Ruminococcus albus 8 is widely known as one of the most active lignocellulolytic ruminal microorganisms. It can degrade cellulose and hemicellulose in forages such as alfalfa and grass hays. This bacterium produces a wide range of glycoside hydrolase (GH) proteins, including enzymes that can degrade lignocellulose [[20], [21], [22], [23]]. In this respect, different genes and protein products from R. albus 8 have been characterized as (i) cellulases Cel5G, Cel9B, Cel9C, and Cel48A); (ii) an extracellular α-L-arabinofuranosidase [20]; and (iii) xylanase 11C [24]. Besides, the molecular cloning of the gene xynA from R. albus 7 allowed the production and biochemical study of the respective recombinant xylanase [21].

Moon et al. [22] reported biochemical analyses of the genome from R. albus 8 with an emphasis on identifying enzymes that degrade the hemicellulose component of the plant cell wall. The authors identified five putative endoxylanases: ORF2725, ORF2882, ORF997, ORF1984, and ORF2008 and characterized the respective recombinant proteins. Based on amino acid sequences, the protein products of ORF2725 and ORF2882 belong to the GH10 family, and they were R. albus 8 Xyn10A and Xyn10B, respectively. According to this study, the structure of Xyn10B comprises a GH10 domain and three carbohydrate-binding modules (CBMs), whereas Xyn10A would be a protein with only endoxylanase ability. We performed a more detailed analysis to find that Xyn10A (377 amino acids) comprises not only a GH10 catalytic module but also an N-terminal domain of 34-amino acids (N34) with no assigned putative function. This latter raised the question about a possible functionality for such an N-term of Xyn10A.

Herein, we report the molecular cloning of the ralxyn10A gene to produce and characterize the entire recombinant protein and the N34 truncated form. Results support differences between the full-length protein and the shortened form of RalXyn10A in the efficiency and redox dependence for hydrolyzing polysaccharides, thus identifying the N-term as a new domain with CBM and regulatory functions.

Section snippets

Bacteria, plasmids, and primers

Escherichia coli Top 10 F′ and E. coli BL21 (DE3) (Invitrogen) served as hosts for cloning purposes and expression of the genes cloned in the pETDuet vector (Novagen). DNA manipulations and E. coli cultures, as well as transformations, were performed according to standard protocols [25]. Table S1 details all the primers (obtained from GenBiotech) utilized in this work.

Endoxylanase constructs

We designed a gene (ralxyn10A) coding for endoxylanase RalXyn10A for de novo synthesis (BIO BASIC INC) based on information from

Characterization of the kinetic and physicochemical properties of recombinant RalXyn10A and RalΔN34Xyn10A from R. albus 8

Two endo-xylanases assigned to the GH10 family (Xyn10A and Xyn10B) are identified from the genome of R. albus 8 (GenBank accession no. ADKM00000000.2). Both proteins, recognized in the proteome project ID UP000004259, were characterized by Moon et al. [22] in a study based on the functional analysis of structural domains. The authors considered that Xyn10A was a protein primarily comprising a xylanase domain, whereas Xyn10B exhibited a higher structural complexity by also having three

Discussion

The production of biofuels from lignocellulosic materials requires the enzymatic hydrolysis of cellulosic substrates (cellulose and hemicellulose) to fermentable sugars. The enzymes hydrolyzing cellulose and hemicellulose usually arrange in a multi-enzymatic complex known as cellulosome, particularly in anaerobic microorganisms [57,58]. Cellulosomes set up the scaffolding of proteins anchored to the cell membrane that coordinate the recruitment of cellulolytic enzymes through the interaction of

Conclusions

Ruminococcus albus 8 endo β-1,4 xylanase 10A (RalXyn10A) presents an N-terminus amino acid motif beside the GH10 catalytic domain that is distinctively functional. This domain plays a critical role in redox modulation of the xylanolytic activity of RalXyn10A in the rumen environment. Besides, our results show that N34 is involved in enzyme binding to polysaccharides and has significant similarity with SBP proteins from ABC transporters of the cell membrane. Previous research already

Authors agreement

All the authors have read and approved the present version of the manuscript. The manuscript reports unpublished work that is not under active consideration for publication elsewhere, it has not been accepted for publication nor been published in full or in part.

Funding

AS is a doctoral fellow from CONICET. AAI and SAG are investigator carrer members from CONICET. This work was supported by grants from CONICET (PUE 2016-0040 to IAL), ANPCyT (PICT-2015-1149 & PICT-2016-1110 to SAG, and PICT-2017-1515 & PICT-2018-00929 to AAI).

CRediT authorship contribution statement

Alem Storani: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing. Sergio A. Guerrero: Conceptualization, Methodology, Validation, Resources, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration, Funding acquisition. Alberto A. Iglesias: Conceptualization, Methodology, Validation, Resources, Writing - original draft, Writing - review &

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