Glycoside Hydrolase Family 9/Plant endoglucanases

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• Author: ^^^Breeanna Urbanowicz^^^
• Responsible Curator: ^^^David Wilson^^^

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Note: This page is an extension of the Glycoside Hydrolase Family 9 page, which is focussed on a key subgroup enzymes from plants. Please see the main GH9 page for full information on the functional and structural properties of these enzymes.

Introduction

Early reports described the existence of plant "cellulases" or EGases [1]. Subsequently, cellulases have been shown to be associated with plant cell wall restructuring during cell expansion, the wall disassembly that accompanies processes such as fruit ripening and abscission (reviewed in [2, 3, 4]) and cellulose biosynthesis [5, 6, 7]. The amino acid sequences of the first plant "cellulases"/endo-ß-1,4-glucanases revealed that these enzymes belong to the CAZy family GH9 glycoside hydrolases [8].

Most plant GH9 glycoside hydrolases are endoglucanases ("cellulases", EC 3.2.1.4) with low or no activity on crystalline cellulose, but with discernible activity on soluble cellulose derivatives, including carboxymethyl cellulose (CMC), phosphoric acid swollen non-crystalline cellulose, and numerous plant polysaccharides including xylan, 1,3-1,4-ß-glucan, xyloglucan, and glucomannan [9, 10, 11, 12, 13]. The inability of plant “cellulases” to hydrolyze crystaline cellulose is distinct from microbial cellulases, whose modular structure and synergistic action with other enzymes facilitates effective degradation of crystalline cellulose. In muro, the substrates of plant cellulases may include xyloglucan, xylans, and non-crystalline cellulose, especially amorphous regions of cellulose where the microfibrils may be interwoven with xyloglucan.

Plant GH9 subfamilies

In the model plant Arabidopsis thaliana, 25 different GH9 coding regions have been identified. Phylogenic analysis of the deduced amino acid sequences group the proteins into nine classes or three subfamilies [4, 13, 14, 15]. Three distinct types of GH9 proteins are present in plants. Class A proteins are membrane-anchored, Class B proteins are secreted, and Class C proteins are also secreted but contain a family 49 carbohydrate binding module (CBM49) [13]. Class A plant EGases have been reported to lack tryptophans corresponding to substrate binding at subsites -4, -3, and -2 in T. fusca Cel9A [9]. Class C EGases are the only plant EGases to date that contain a tryptophan residue corresponding to the one in subsite -2 in TfCel9A [9, 13]. This tryptophan has been shown to be important for hydrolysis in TfCel9A, and the enzyme retains less than 10% of its normal activity on polymeric cellulose substrates, and less than 1% of wild type activity on cellohexaose when the Trp is replaced by another amino acid [9, 16].

Class A

The Class A EGases are integral type II membrane proteins with a GH9 catalytic core that lack a canonical secretion signal sequence. These enzymes are predicted to have a high degree of N-glycosylation and a long amino-terminal extension with a membrane-spanning domain that anchors the protein to the plasma membrane and/or to intracellular organelles [4, 17]. Membrane anchored EGases were first described in studies of the KORRIGAN (KOR) genes in Arabidopsis thaliana, which showed that they encode EGases that are required for normal cellulose synthesis or assembly. Plants with mutant alleles of the KOR1 gene are dwarfed, with decreased cellulose content and crystallinity [4, 18, 19]. The role of the Class A EGases in plants is not known. However, the KOR proteins have been proposed to cleave sitosterol-b-glucoside primers from the growing cellulose polymer, or may have a role in editing incorrectly formed growing microfibrils [20]. More recently, it has been shown that during cell expansion, KOR1 is cycled from the plasma membrane through intracellular compartments, comprising both the Golgi apparatus and early endosomes; however the role of KOR1 in cellulose biosynthesis remains to be determined [21]. The catalytic domain of PttCel9A, a Class A GH9 enzyme that is upregulated during secondary cell wall synthesis in Populus tremula x tremuloides, has been biochemically characterized and shown to hydrolyse a narrow range of substrates in vitro including CMC, phosphoric acid swollen cellulose and cellulose oligosaccharides (DP≥5) [9, 22].

Class B

Class B proteins are the most common form of plant EGases and are associated with virtually all stages of plant growth and development. These enzymes have a GH9 catalytic domain and a signal sequence for ER targeting and secretion. Different isoforms are expressed during fruit ripening, in abscission zones, in reproductive organ development, and in expanding cells [23, 24, 25, 26]. Numerous studies, especially in tomato, have also shown that many class B EGases are under hormonal control [17, 27, 28].

Class C

Plant Class C GH9 enzymes are the least studied. These proteins are predicted to have a signal sequence followed by a GH9 catalytic domain and a long carboxyl-terminal extension, which contains a CBM49 that has been shown to bind to crystalline cellulose in vitro [13, 15]. CBMs are necessary for activity on crystalline substrates and may promote hydrolysis by increasing the local enzyme concentration at the substrate surface as well as modifying cellulose microfibril structure (for review see [29]). The catalytic domain (CD) SlGH9C1 from tomato is promiscuous and can effectively hydrolyze artificial cellulosic polymers, cellulose oligosaccharides, and several plant cell wall polysaccharides [13]. Nevertheless, the activity of the full length, modular enzyme has still not been characterized. A Class C EGase from rice, OsCel9A, has been shown to be post-translationaly modified at the linker region to yield a 51 kDa GH9 CD and a CBM49, and it was suggested that the cleavage is necessary for function [30]. The OsCel9A CD also displays a broad substrate range and was able to hydrolyze CMC, phosphoric acid-swollen cellulose, mixed linkage 1,3-1,4-ß-glucan, xylan, glucomannan, cellooligosaccharides (DP≥4) and 1,4-ß-xylohexaose [10]. For Information regarding nomenclature of plant GH9 enzymes please see Urbanowicz et al 2007 [15].

References

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