RG-I lyases catalyse the exo- and endo-cleavage of L-α-Rhap-1,4-α-D-GalpA bond in RG-I region to release oligosaccharides with α-Δ4,5-unsaturated GalpA at non-reducing ends. PL4 and PL11 families are currently known to possess RG-I lyases according to the CAZy database. PL11 family of RG-I lyases was first described in Pseudomonas cellulosa (McKie et al., 2001). Since then, five PL11 family of RG-I lyases were characterized from bacterial species including B. licheniformis, Bacillus subtilis, Cellvibrio japonicas, and Clostridium cellulolyticum. The acetyl groups and the side chains
made of galactan, arabinan, and arabinogalactan oligosaccharides present on the RG-I backbone affect the catalytic efficiency of RG-I lyases (Silva et al., 2016). Rgl11A from P. cellulosa and Rgl11Y from C. cellulolyticum both showed a preferential activity for cleaving RG-I containing galactose substitutions, indicating galactan side chains are probably required for their enzyme activity (McKie et al., 2001; Pages et al., 2003). The presence of acetyl group appeared to hinder the enzyme efficiency of Rgl11A in that the enzyme did not attack RG-I containing esterified galacturonic acid residues (McKie et al., 2001). These two enzymes were also active on pure galactans from potato pectin, although due to the natural origin of these substrates, the galactans may still have contained a trace amount of RG-I backbone (Silva et al., 2016). PL11 family of YesW and YesX from B. subtilis remained inactive on pure galactans, but showed activity towards potato RG-I substituted with side chains containing 12 % galactose residues (Ochiai et al., 2007). In B. subtilis, the endo-acting YesW was more active towards RG-I, leading to the production of oligosaccharides with differing degrees of polymerization which in turn were cleaved into the unsaturated RG disaccharides by the exo-acting function of YesX (Ochiai et al., 2007). The steric hindrance caused by the presence of acetyl groups and galactose moieties on RG-I backbone may be abated by the co-production of RG-I lyases and auxiliary enzymes such as β-D-galactosidase and rhamnogalacturonan acetylesterases (CE12). The YesW-encoding gene was found adjacent to a gene coding for β-D-galactosidase in B. subtilis, indicating β-D-galactosidase may catalyse the removal of galactan side chains prior to the activity of YesW on RG-I backbone (Ochiai et al., 2007; Silva et al., 2016). Along with Pel4A from C.
cellulovorans, and PL1 and PL9 from R. flavefaciens, Rgl11Y is one of few lyases found incorporated in the cellulosome complexes, suggesting their involvement in the plant cell wall degradation (Pages et al., 2003; Venditto et al., 2016). Unlike PL4 which does not require divalent cations for activity, PL11 RG-I lyases either have an essential requirement for calcium, or show enhanced enzymatic activities in the presence of calcium (Silva et al., 2016).
1.6.2.2 RG-I hydrolases
RG-I hydrolases catalyse exo- and-endo hydrolysis of α-D-GalpA-1,2-α-L-Rhap bond in RG-I backbone. Currently, seven endo-acting and two exo-acting RG-I hydrolases of GH28 family are
registered in the CAZy database, all of them from a fungal origin of the genus Aspergillus (Silva et al., 2016). No bacterial RG-I hydrolases of GH28 family have been reported so far, suggesting bacteria may prefer to use the β-elimination mechanism of RG-I lyases to cleave the RG-I backbone. The RG-I hydrolases from GH28 and GH105 families are structurally different in that GH28
possesses a β-helix structure consisting of 13 turns of parallel β-strands arranged in a helical pattern, while GH105 has an (α/α) double toroid structure with six α-hairpins forming a double helical barrel (Silva et al., 2016). Unsaturated rhamnogalacturonyl hydrolases (URGH) of GH105 has shown to cleave the unsaturated uronic acid at the non-reducing end of two- and three-chain unsaturated polysaccharides from plant and algal origins, as well as the non-reducing β-linked end of rhamnose residue to from fully saturated oligomers (Germane et al., 2015, Collen et al., 2014). So far, two URGHs, YteR and YesR, which belong to the GH105 family have been biochemically characterized from B. subtilis (Itoh et al., 2006). Similar to the PL22 family of oligogalacturonide lyases, YteR and YesR acted specifically on unsaturated RG disaccharides with α-Δ4,5-unsaturated-GalpA at the non- reducing end generated by the activity of pectate lyases (Itoh et al., 2006). Recently, a structural analysis of URGH of GH105 family was carried out in Clostridium acetobutylicum (Germane et al., 2015). In C. acetobutylicum, GH105 was found to possess an active binding pocket for a trisaccharide containing an unsaturated galacturonate (Germane et al., 2015). In contrast to PL22 which produces two monomeric end-products galacturonate and 5-keto-4-deoxyuronate (DKI), the resultant products of GH105 are either a galacturonate monomer and a rhamnose residue, or a galacturonate or rhamnose monomer and saturated dimers of Rhap-GalpA, Rhap-Rhap or GalpA-GalpA (Itoh et al., 2006). The dimeric products can be further cleaved into monomers by the activities of GH78 and GH106 families of α-rhamnosidases and rhamnogalacturonan-rhamnohydrolase, and GH28 family of
polygalacturonases.
1.6.2.3 RG-I auxiliary enzymes
In this review, auxiliary enzymes refer to the enzymes which act on cleaving the glycosidic linkages present in the side chains of pectin. Auxiliary enzymes such as β-D-galactosidases are often co- produced with the enzymes catalysing the hydrolysis of pectin backbone to facilitate a coordinated
process of pectin depolymerisation (Ochiai et al., 2007; Silva et al., 2016). GH43 family is made of a heterogeneous population of enzymes including endo-acting α-L-arabinanases and exo-acting α-L- arabinofuranosidases. The members of GH43 family are abundantly present in bacteria that can degrade the major components of the plant cell wall, especially arabinose- and xylose-containing polysaccharides such as arabinoxylan, arabinan, and the side chains of RG-I (Cartmell et al., 2011). Endo-acting arabinanases of GH43 family introduced internal cleavages at α-1,5-L-Araf linkages in the linear-α-1,5-L-arabinan side chains of RG-I to release arabino-oligosaccharides and L-arabinose monomers (Inacio and Sa-Nogueira 2008). The structure and function of GH43 arabinanases have been characterized in bacterial species such as B. subtilis, B. licheniformis, B. thetaiotaomicron, Cellvibrio japonicus, and R. champanellensis. The activity of arabinanases can be affected by the presence of additional branching within the arabinan side chains, as shown in the two endo- arabinanases Bt0360 and Bt0367 from B. thetaiotaomicron displaying differing preferences for branched and linear arabinan substrates, respectively (Cartmell et al., 2011). The activity of exo- arabinanases that release a terminal arabinose or arabino-oligosaccharides from α-1,5-L-arabinan has been reported in GH93 family, although so far, all exo-acting arabinases have been described in fungi (Carapito et al., 2009; Sakamoto and Thibault, 2001). The exo-acting α-L-arabinofuranosidases catalyse the hydrolysis of α-1,2, α-1,3, and α-1,5 terminal arabinofuranose residues from arabinan (Carapito et al., 2009; Miyanaga et al., 2004; Paes et al., 2008; Taylor et al., 2006; Shi et al., 2014). Currently, α-L-arabinofuranosidases are found in GH2, GH3, GH43, GH51, GH54, and GH62 families. Despite their abundance in bacterial communities, relatively few α-L-arabinofuranosidases have been biochemically characterized. Furthermore, enzymes of the same family often display varying substrate specificities, making it difficult to assign a defined function for each family (Taylor et al., 2006). For example, α-L-arabinofuranosidases of GH3 family usually occur as a bifunctional β- D-xylosidase/α-L-arabinosidase that can cleave both β-1,4,-xylose-xylose and β-1,3-xylose-arabinose linkages (Mai et al., 2000). GH43 family of α-L-arabinofuranosidases typically target the α-1,5-Araf linkages present in arabino-oligosaccharides (Carapito et al., 2009; Nurizzo et al., 2002; Sakamoto et al., 2001). GH43 also includes bifunctional enzymes displaying β-D-xylosidase and α-L-
1,3-Araf substituted with a single β-D-Xylp residue (Wang et al., 2014; Viborg et al., 2014; Bourgois et al., 2007). GH51 and GH54 share a similar broad range of substrate specificities in that both enzymes act on α-1,2-, α-1,3-, and α-1,5-L-Araf linkages, although GH51 shows preferential activities on short and unsubstituted arabino-oligosaccharides, whereas GH54 can act on both short substrates and arabinose-containing polysaccharides such as arabinan and arabinoxylan (Miyanaga et al., 2004; Ioannes et al., 2000; Koseki et al., 2003; Beldman et al., 1993; Taylor et al., 2006; Hoffmam et al., 2013). GH62 showed an exceptionally high activity on arabinoxylan. GH62 was able to cleave Araf substituents that are α-1,2- and α-1,3-linked to xylopyranosyl (Xylp) backbone in arabinoxylan, and also showed a low activity against α-1,5-L-Araf linkages in debranched arabinans (Wang et al., 2014; Maehara et al., 2014; Wilkens et al., 2016; Sakamoto et al., 2011; Kaur et al., 2014).
Endo-galactanases catalyse the hydrolysis of β-1,4-Galp linkages present in type I arabinogalactan and galactan side chains of RG-I. Currently, endo-acting β-galactanases with pectin-associated activities are found only in GH53 family. Endo-β-1,4-galactanses of family GH53 are predominantly found in bacteria, and the enzymes have been characterized in fibrolytic bacterial species such as B. subtilis, B. licheniformis, B. thetaiotaomicron, D. dadantii, C. japonicus, and R. thermocellum. GH53 showed a limited activity on galactan oligomers made of less than three Galp residues, indicating exo- acting β-galactosidases were required to process D-Galp-β-1,4-D-Galp dimers into galactose
monomers (Sakamoto et al., 2013; van Bueren et al., 2016). Exo-acting β-galactosidases catalyse the hydrolysis of terminal β-D-galactosyl residues from non-reducing ends of galacto-oligosaccharides. Exo-acting β-galactosidases have been divided into five different families: GH1, GH2, GH35, GH42, and GH59. The majority of microbial β-galactosidases has been classified into GH1 and GH2
families, whereas some enzymes active in extreme environments such as thermophilic hot springs, hypersaline brines, and psychrophilic soils are found in family GH42 (Gupta et al., 2012; Shipkowski et al., 2006; Karan et al., 2013). GH35 family has been found in all domains of life, mostly from plants and fungi (Gupta et al., 2012; Lee et al., 2017). The β-galactosidases of GH1 and GH2 are best represented by lactose-hydrolysing enzymes, most notably the lacZ gene of Escherichia coli (Talens- Perales et al., 2016; Lee et al., 2017). On the other hand, β-galactosidases belonging GH35 and GH42
families are often associated with galactose-containing polysaccharides such as galactan side chains of RG-I (Gamauf et al., 2007; Talens-Perales et al., 2016). GH35 family of β-galactosidases have demonstrated substrate specificities towards β-1,3-, β-1,6- or β-1,4-galactosidic linkages, of which β- 1,4-D-Galp bonds are predominantly found in pectic galactans (Kim et al., 2006; Gamauf et al., 2007). In family GH42, lactase functions are not always found in all members, as several GH42 enzymes are highly active on galacto-oligosaccharides and galactans, suggesting a function in the degradation of pectic polysaccharides (Shipkowski et al., 2006; Kosugi et al., 2002). In these studies, GH42 β-galactosidases from B. subtilis and C. cellulovorans were found to catalyse the hydrolysis of β-1,4-D-Galp linkages in galacto-oligosaccharides from the galactan backbone of type I
arabinogalactan.
Along with pectin acetylesterases, RG acetylesterases belong to CE12 family. RG acetylesterases facilitate the action of RG-I lyases and hydrolases by removing the acetyl groups at C-2 and C-3 positions of galacturonic acid residues in RG-I (Navarro-Fernandez et al., 2008). Bacterial RG acetylesterases have been studied little, and only two enzymes of this classification have been characterized from Bacillus halodurans (BhRgae) and R. thermocellum (Rgae12A) (Navarro-
Fernandez et al., 2008). The substrate specificities of BhRgae and Rgae12A on pectic polysaccharides are currently unknown; BhRgae has shown activity towards acetylated xylan (Navarro-Fernandez et al., 2008).