Computation 2015, 3, 336-353; doi:10.3390/computation3030336 OPEN ACCESS computation ISSN 2079-3197 www.mdpi.com/journal/computation Article In Silico Comparison of the Hemicelluloses Xyloglucan and Glucuronoarabinoxylan in Protecting Cellulose from Degradation Indrakumar Vetharaniam 1,*, Martin Upsdell 1, William J. Kelly 2, Graeme T. Attwood 2, Christina D. Moon 2 and Philip J. Harris 3 1 AgResearch Limited, Ruakura Research Centre, Private Bag 3123, Hamilton 3240, New Zealand; E-Mail: [email protected] 2 AgResearch Limited, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand; E-Mails: [email protected] (W.J.K.); [email protected] (G.T.A.); [email protected] (C.D.M.) 3 School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +64-7-838-5314; Fax: +64-7-838-5012. Academic Editor: Rainer Breitling Received: 10 April 2015/ Accepted: 17 June 2015 / Published: 6 July 2015 Abstract: We used a previously developed simulation model of a plant cell wall and its enzymatic degradation to compare the abilities of two hemicelluloses, glucuronoarabinoxylan (GAX) and xyloglucan (XG), to protect cellulose microfibrils (CMFs) from attack by cellulose-degrading enzymes. Additionally, we investigated the effect of XG abundance on the degradation rate of CMFs in the presence of the same enzymes. Simulations were run using hypothetical cell-wall compositions in which the numbers and arrangement of CMFs and (1,3;1,4)-β-glucan were kept constant, but the proportions of GAX and XG were altered. Scenarios considered walls with low and equal proportions of either GAX or XG, and also low, medium and high proportions of XG in the absence of GAX. The rate of CMF degradation was much lower in walls with GAX than walls with XG, except for early in the simulation when the reverse held, suggesting that XGs were protecting CMFs by competitive inhibition. Increasing XG content reduced both the degradation rate of CMFs and the percent of XG degraded, indicating that activity of enzymes decreased with XG density despite XG being degradable. Glucose oligosaccharide breakdown products were analysed on the basis of the originating polysaccharide and their Computation 2015, 3 337 degree of polymerisation (DP). The presence of GAX as opposed to equal amounts of XG had some significant effects on the amount and profile of breakdown products from XG and CMFs. Keywords: plant cell wall; lignocellulose; enzymatic degradation; enzymes; accessibility; competitive inhibition; rumen; second-generation biofuels, agent-based modelling; simulation model 1. Introduction Hemicelluloses are a group of plant cell-wall polysaccharides characterised by having a 1,4-β-linked backbone with an equatorial configuration [1]. They include heteroxylans, xyloglucans, 1,3;1,4-β-glucans and heteromannans, and often interact with cellulose (1,4-β-glucan), which is the most abundant polymer in plant cell walls [2] and occurs as crystalline cellulose microfibrils (CMFs) formed by the lateral association of parallel cellulose molecules [3,4]. The hemicellulose xyloglucan (XG) can account for 20%–25% of primary cell walls in eudicotyledons, such as forage legumes (family Fabaceae) and brassicas (family Brassicaceae), but accounts for only a small proportion (~2%–5%) of primary cell walls in the grass family (Poaceae) [2,5]. The predominant hemicellulose in both primary and secondary cell walls in this family is instead the heteroxylan glucuronoarabinoxylan (GAX), with the primary cell wall also containing varying, but often small proportions of 1,3;1,4-β-glucan [2]. Another heteroxylan, 4-O-methylglucuronoxylan (4-O-MeX), is the predominant hemicellulose in the secondary cell walls of the eudicotyledons. Despite the structural differences between the different hemicelluloses, they have traditionally all been assumed to have the same main role of providing structural strength to the cell wall by tethering the CMFs [1] as well as protecting CMFs from degradation by enzymes secreted by pathogenic microorganisms. This protective role of hemicelluloses is also probably important in reducing the enzymatic degradation of cellulose in plant cell walls (lignocellulose) in two applied contexts. First, in the rumens of ruminant animals which are populated by microorganisms that produce cell-wall degrading enzymes, and second, in the saccharification step in the production of second generation biofuels from plant biomass rich in cell walls, where enzyme mixtures including cellulases are used, particularly from the cellulolytic brown-rot fungus Trichoderma reesei (recently re-named Hypocrea jecorina). In the latter context, it has been found that pre-treatments of biomass that remove hemicelluloses (and lignin) can enhance the enzymatic hydrolysis of cellulose [6–8]. However, recent in vitro work with composites of hemicelluloses and CMFs has demonstrated that GAX and XG interact differently with CMFs [9], with GAX-CMF composites being mechanically less strong than XG-CMF composites [10]. There may also be differences between GAX and XG in their abilities to protect CMFs from degradation by cellulases. Two mechanisms of protection have been proposed: First, by the hemicellulose itself or oligosaccharides produced by its enzymatic degradation acting as an inhibitor of cellulose degradation, and second, by the hemicellulose forming a physical barrier limiting accessibility of cellulases to the CMFs. There is evidence that GAX and oligosaccharides (xylooligosaccharides) obtained from this by xylanase activity bind to the active sites Computation 2015, 3 338 of cellulases and competitively inhibit them [11–15]. This inhibition was found to be particularly marked for cellobiohydrolases, which are exo-cellulases that successively cleave cellobiose units from the reducing end (type I) or non-reducing end (type II) of cellulose or cellooligosaccharides. Importantly, the most abundant cellulase produced by H. jecorina, Cel7A, is a cellobiohydolase I. X-ray crystallographic studies showed that xylooligosaccharides bind to Cel7A at the entrance to the substrate-binding tunnel [15]. XG is hydrolysed by cellulases, but whether it or the oligosaccharides produced inhibit these cellulases appear not to have been investigated. Furthermore, there is no evidence to indicate whether GAX or XG forms a better physical barrier to the access of cellulases to CMFs. Here we report a modelling study in which we compared the effectiveness of GAX and XG in protecting CMFs from degradation by cellulases, and further investigated the role of XG density in protecting CMFs in cell walls that lack GAX or other heteroxylans. In this study, we created four hypothetical “cell walls”, similar to the cellulose-hemicellulose composites reported in [10] and used simulations of cellulase activity on these. 2. Methods We modelled the four composite cell walls by adapting a previously published 3-D model of a perennial ryegrass cell wall (RGCW) and its enzymatic degradation [16]. All four cell walls had several elements in common with RGCW, namely nine CMFs arranged in parallel (with the cellulose molecules 60 glucosyl residues long) with 35 (1,3;1,4)-β-glucans attached, all in their original configurations. The first model cell wall (Low XG) was created as a control, and contained 93 XGs, which were in the RGCW model [16]. The second model cell wall (Low GAX) had no XGs, but contained 93 GAXs from the RGCW in their original configurations. These two model cell walls allowed a comparison to be made of the abilities of XG and GAX to protect cellulose from degradation. The third and fourth model cell walls were designed to investigate the effect of XG density on cellulose degradation. These walls contained no GAXs and were formed by inserting 76 additional XGs into the Low XG model to obtain a cell wall with 169 XGs (Medium XG) and then a further 76 XGs to obtain a cell wall with 245 XGs (High XG). The number 245 was chosen to match the number of GAXs in the RGCW, with the Medium XG cell wall being intermediate between the two. The GAXs in Low GAX had an average degree of polymerisation (DP) of 27, with standard deviation (SD) of 9, and no glycosidic linkages that could be degraded by a cellulase/β-glucanase. The DP of the XG backbones averaged 29 for both Low and Medium XG and 30 for High XG (with an SD of 7 for all three). Low, Medium and High XG contained respectively 2742, 5104 and 7206 XG-sourced glucoses and respectively 2649, 4936 and 6961 glycosidic bonds degradable by a cellulase/β-glucanase. The CMFs in each cell wall contained 15,120 glucoses and 14,868 glycosidic bonds (1680 glucoses and 1652 glycosidic bonds per CMF). The (1,3;1,4)-β-glucans in each wall contained 1028 glucoses and 993 glycosidic bonds, with average DPs and numbers of glycosidic linkages of 29 and 28 respectively, both with SDs of 3. Dimensions were specified in terms of the radius of a glucose molecule, rg. As a reference, the Stoke’s radius of glucose is 0.365 nm [17]. Each wall had an approximate dimension of 120 rg deep (in the orientation of the CMF direction), 80 rg wide and 80 rg high. The volume not occupied by CMFs Computation 2015, 3 339 3 was approximately 690,000 rg . The total number of residues contained in all hemicelluloses of different types was 5311 for the Low GAX wall, and 5048, 8516 and 11,592 for the Low, Medium and High XG walls. Assuming each residue to be similar in size to a glucose molecule, the non-CMF residues occupied about 3.2% of the volume of the non-CMF space in the Low GAX wall, and about 3.1%, 5.2% and 7.0% of the volume of the non-CMF space in the Low, Medium and High XG walls.