4.2 Formas de vuelo
4.2.2 Trayectoria preprogramada
HG-degrading enzymes are mostly active on the HG backbone of pectin, and catalyse the specific cleavages of D-GalpA-α-1,4-D-GalpA linkages. Currently, the majority of HG-degrading enzymes belong to various families and sub-families of pectate lyases which have been extensively studied from bacterial (e.g. Dickeya and Pectobacterium spp.) and fungal pathogens that are known to have devastating effects on crop plants (Hugouvieux-Cotte-Pattat et al., 2014). As a result, despite the small number of families, PLs constitute the highest proportion of biochemically characterized enzymes in the CAZyme database (Cantarel et al., 2008). Depending on the cleavage pattern and substrate specificities, pectate lyases can be grouped into exo- or endo-acting pectate lyases, exo- or endo-acting pectin lyase, and exo- or endo-acting rhamnogalacturonan lyases. Endo-acting lyases cleave randomly inside polygalacturonates or RG-I chain to create products varying in sizes, whereas exo-acting lyases cleave the terminal end of polygalacturonates or RG-I chain to generate 4,5-
unsaturated di- or trigalacturonates (Hugouvieux-Cotte-Pattat et al., 2014). Pectin lyases (also known as polymethyl galacturonate lyases) prefer to attack highly methylated (~98 %) regions of
polygalacturonate. Although pectin lyases are capable of splitting pectin polymers independent of the degree of methylation, such lyases are mostly found in fungi (Dongowski et al., 2000). Only few bacterial species such as Bacillus spp. and Pseudomonas marginalis are currently known to produce pectin lyases (Sakiyama et al., 2001; Kashyap 2000; Hayashi 1997). According to the CAZy database, HG-degrading pectate lyases are divided into five families, PL1, PL2, PL3, PL9, and PL10, with the addition of PL22 which exclusively acts on short galacturonate oligomers. Families of PL fall into different protein-folding categories, such as parallel β-helix (PL1, PL3, and PL9), (α/α)7 barrel (PL2), (α/α)3 barrel (PL10), or β-propeller (PL22) (Hugouvieux-Cotte-Pattat et al., 2014). Although pectate lyases generally prefer unmethylated or partially methylated polygalacturonates, some families or subfamilies of pectate lyases recognize and act on polygalacturonate with varying degrees of
methylation (Hugouvieux-Cotte-Pattat et al., 2014). Aside from being the cause of steric hindrance to pectin-degrading enzymes, methyl groups on HG also alter the overall surface charge of pectin by neutralizing the negative charge on individual galacturonate monomers (Herron et al., 2000). Plant
Figure 1.7 Activities of pectin-degrading enzymes on HG, RG-I, and RG-II. The diagram of RG-II structure was adopted from Ndeh et al., 2017. Copyright permission has been granted for the use of this figure.
pathogens produce a heterogeneous population of pectate lyases with varying substrate specificities to orchestrate the degradation of HG which significantly changes its degree of esterification over the course of plant developmental stages. Unlike most enzyme families that display a high degree of structural conservation in the active sites and the regions surrounding the active sites, the variations in the length, amino acid sequences, and protein-folding topology found within the immediate region around the catalytic sites of pectate lyases were proposed as structural determinants for distinguishing methylated and non-methylated substrates (Herron et al., 2000). The comparison of the substrate- binding sites in pectin and pectate lyases using 3D structural modelling showed that pectin lyases possessed a hydrophobic pocket primarily consisting of an unusual cluster of tryptophan and tyrosine residues to accommodate a methyl group added to an uronic acid moiety (Herron et al., 2000). With the exception of PL2 which uses Co2+, Mn2+, and Ni2+, most families of pectate lyases require Ca2+ as an essential cofactor to mediate the interaction between protein and sugar substrate (Creze et al., 2008). Ca2+ is readily obtainable from the middle lamella and the plant cell wall in which the localized calcium concentration may reach up to 1.0 mM (Herron et al., 2000). Ca2+ ions confer a positive charge to the surface of the groove surrounding the Ca2+-binding sites, possibly contributing to the stabilization of a negative intermediate, or to the transfer of a proton to the target glycosidic bond to promote the acidification at C-5 position (Herron et al., 2000; Hugouvieux-Cotte-Pattat et al., 2014). In contrast to pectate lyases, pectin lyases that target fully methylated pectate do not usually require cations for activity as the positive charge is conferred by Arg-176 residue conserved within the pectin lyase subfamilies (Herron et al., 2000; Hugouvieux-Cotte-Pattat et al., 2014).
PL1
PL1 represents the largest pectate lyase family which is divided into 11 subfamilies. PL1 preferably targets low-methylated HG (Hugouvieux-Cotte-Pattat et al., 2014; Shevchik et al., 1997). PL1 pectate lyases from Dickeya dadantii have been extensively characterized. D. dadantii produces 6 secreted PL1s encoded by genes organized in two gene clusters, pelA-pelE-pelD and pelB-pelC-pelZ, and an outer membrane-associated PL1 encoded by pnlH (Hugouvieux-Cotte-Pattat et al., 2014; Ferrandez and Condemine, 2008). The 5 major PL1s, PelA, PelB, PelC, PelD, and PelE have endo-acting activities with an absolute requirement for Ca2+ ions (Tardy et al., 1997). PelB and PelC which share 84 % sequence identity are most active on partially methylated pectin with up to 22 % DE (Tardy et al., 1997). On the other hand, PelA, PelD, and PelE that share 60 – 80 % sequence homology retain 80 % of enzymatic activities towards low-methoxyl pectin with up to 7 % DE (Tardy et al., 1997). D. dadantii also produces secondary endo-pectate lyases PelL and PelZ which belong to PL9 and PL1 families, respectively. Similar to PelB and PelC, PelZ is an endo-acting extracellular pectate lyase mostly targeting low-methoxyl pectin, although PelZ shows a very low catalytic activity compared to the major PL1s (Pissavin et al., 1998). Each pel gene is regulated under an individual transcriptional control, indicating PL1s with varying substrate specificities may be differentially expressed to adapt to the changes in pectin structures (Tardy et al., 1997). PL1 family of pectate lyases are abundantly present in a wide range of ecological niches including plant pathogens such as D. dadantii (8 PL1s) and Pectobacterium atrosepticum (6 PL1s), a plant symbiont Paenibacillus mucilaginosus (12 PL1s), a soil bacterium Sorangium cellulosum (6 PL1s), and the gut symbionts B. thetaiotaomicron (5 PL1s) and B. ovatus (9 PL1s) (Hugouvieux-Cotte-Pattat et al., 2014).
PL9
PL9 family includes both endo- and exo-acting pectate lyases found in D. dadantii, Bacillus sp. and few fungi (Hassan et al., 2013; Jenkins et al., 2004; Hla et al., 2015). Some environmental isolates of cellulolytic Clostridium spp. such as Clostridium stercorarium, C. thermocellum, and Clostridium cellulovorans are also known to produce small numbers of PL1 and PL9 pectate lyases possibly to facilitate the extraction of cellulose polymers by disrupting the cellulose-pectin interactions in the
plant cell wall (Hugouvieux-Cotte-Pattat et al., 2014). Currently, 10 bacterial PL9 have been biochemically characterized. D. dadantii is known to produce three PL9 pectate lyases, including endo-acting PelL, PelN, and exo-acting PelX (Hassan et al., 2013). The secreted endopectate lyase PelL is a low active, Ca2+-dependent enzyme which converts polygalacturonate with a degree of polymerization of > 5 GalA residues to produce di- and trigalacturonates (Roy et al., 1999). PelN prefers to act on polygalacturonate, although it retains > 50 % of catalytic activity against pectin with 91 % methylation (Hassan et al., 2013). As opposed to most pectate lyases with an absolute
requirement for Ca2+, PelN activity becomes stimulated in the presence of an unusual cation cofactor Fe2+ (Hassan et al., 2013). As suggested by its periplasmic localization, PelX showed maximum activity on GalA oligomers with 4 – 6 residues, liberating unsaturated digalacturonate as the end- product (Shevchik et al., 1999b). PelX activities against polygalacturonate and pectin with up to 22 % methylation were observed, although reaction rates were slower compared to oligomers (Shevchik et al., 1999b). While PelX activity was weakly stimulated using Ca2+, the inhibitory bivalent cations such as Mn2+, Co2+, Ni2+, and Cu2+ also weakly activated PelX, indicating the enzyme had unusual cofactor requirements (Shevchik et al., 1999b).
1.6.1.2 Glycoside hydrolases GH28
GH28 is found in all domains of life and is one of the largest family of glycoside hydrolases described to date (Abbott and Boraston, 2007b). GH28 belongs to the GH-N clan characterized by the right- handed (β)3 solenoid fold composed of four parallel sheets connected by ten complete turns (Abbott and Boraston, 2007b; Naumoff 2011). In Enterobacteriaceae, GH28s are divided into either exo- acting or endo-acting classes depending on the mode of action. The active site of endo-acting enzymes forms an open-ended channel structure in which the reducing ends of polygalacturonates become randomly cleaved to release n > 3 oligogalacturonates (Abbott and Boraston, 2007b). A GH28 endo-polygalacturonase PehA from Pectobacterium caratovorum adopted an open-ended active site to attack the internal galacturonic acid residues within the polygalacturonate chain protruding out from the catalytic site at either end (Abbott and Boraston, 2007b; Pickersgill et al.,
1998). In contrast, the periplasmic exo-acting GH28 uses a pocket-like active site to hydrolyse the terminal non-reducing ends of oligogalacturonates to liberate mono-, di-, or trigalacturonate residues that are suitable for intracellular transport (Abbott and Boraston, 2007b). D. dadantii produces three GH28 exo-polygalacturonases encoded by pehV, pehW, and pehX organized in a single gene cluster (Nasser et al., 1999). The high sequence similarities between pehV, pehW, and pehX indicated that these genes arose from gene duplication events (Nasser et al., 1999). While the exo-acting PL9 PelX cleaves 6-residue polygalacturonate to produce a tetragalacturonate and an unsaturated
digalacturonate, PehX uses the same substrate to produce a tetragalacturonate and a saturated digalacturonate as the sole end-product (Shevchik et al., 1999b). Although both PelX and PehX cleaved GalA oligomers with 4 – 6 residues, the presence of unsaturation at the non-reducing end of polygalacturonate adversely affected the activity of PehX, indicating PehX preferred to act on the saturated oligomeric products from endo-polygalacturonases (Shevchik et al., 1999b). Using a modified hexogalacturonate with an artificially reduced end, PehX produced a digalacturonate and reduced tetragalacturonate, indicating PehX attacks at the non-reducing end of the polygalacturonate (Shevchik et al., 1999b). In contrast, the modification of the reducing end inhibited PelX activity on hexogalacturonate, indicating PelX attacked from the reducing end of polygalacturonate (Shevchik et al., 1999b).
Rhamnogalacturonase and xylogalacturonan hydrolases are also pectin-associated hydrolases of GH28 family. However, these enzymes have been exclusively found and characterized from fungi, therefore will not be discussed in this review.
1.6.1.3 Carbohydrate esterases Pectin methylesterases (CE8)
Pectin methylesterases facilitate the action of pectate lyases by catalysing the removal of methyl esters from the pectin backbone to release methanol, leading to the formation of polygalacturonate (Hugouvieux-Cotte-Pattat et al., 1996). An extensive de-methylation of pectin causes the cross- linking of HG regions via the chelation of Ca2+ ions as the removal of methyl esters creates negatively charged uronic acid residues (Øbro et al., 2009). The majority of pectin methylesterases have been
studied from plant enzymes that play significant roles during the regulation of plant development and fruit ripening processes. Pectin methylesterases can be described with three general patterns of polysaccharide modification: (1) a single-chain mechanism in which pectin methylesterases move in a processive fashion to remove all methyl groups from a single pectin chain before dissociating into the solvent; (2) a multi-chain or distributive mechanism in which the enzyme randomly removes a single methyl ester before dissociating; and (3) a multi-attack mechanism where pectin methylesterases carry out a number of reaction cycles before dissociation (Øbro et al., 2009; Fries et al., 2007; Duvetter et al., 2006). Plant pectin methylesterases usually operate in a processive manner by removing methyl esters in a block-wise arrangement while fungal enzymes generate randomly distributed de-esterified galacturonic acids using multi-chain mechanism (Duvetter et al., 2006). Bacterial pectin
methylesterases use all three mechanisms, although environmental factors such as pH and cation concentrations may affect the enzyme activities (Øbro et al., 2009). Few CE8 pectin methylesterases from bacterial sources have been characterized, including PemA and PemB from D. dadantii. Despite the presence of PemA and PemB, D. dadantii cannot grow on high methyl pectin (98 %) (Shevchik et al., 1996). PemA is an extracellular pectin methylesterase encoded by pemA gene located at the downstream of the pelA-pelE-pelD locus (Hugouvieux-Cotte-Pattat et al., 1996). PemB is an outer membrane-associated lipoprotein that carries out the periplasmic de-esterification of methylated oligomeric products (Shevchik et al., 1996). While PemB was able to remove ester groups from 98 % DE pectin, the activity of PemB was 100-fold higher on methylated oligogalacturonate (Shevchik et al., 1996). PemA was also more active on methylated oligogalacturonate than natural pectin, but only by 5-fold (Shevchik et al., 1996).
Pectin acetylesterases (CE12)
In general, pectin acetylesterases cleave acetyl groups esterified to the O-2 and/or O-3 positions of galacturonic acid residues present in HG to liberate acetate (Shevchik et al., 1997). The biochemical properties of pectin acetylesterases from D. dadantii (PaeY and PaeX), and Bacillus licheniformis (BliPAE) have been characterized (Shevchik et al., 1997; Shevchik et al., 2003; Remoroza et al., 2014). The paeY gene is present in the pelA-pelE-pelD-paeY-pemA locus which encodes various
types of pectinases acting in a synergetic manner for an efficient breakdown of pectin in plant tissues (Shevchik et al., 1996; Shevchik et al., 1997). The paeX gene is present in the pelW-togMNAB- kdgM-paeX operon which contains a cluster of genes involved in the catabolism and transport of pectic oligomers (Condemine and Ghazi, 2007). PaeY possesses a secretory signal peptide sequence, whereas PaeX becomes localized at the periplasm (Shevchik et al., 1997; Shevchik et al., 2003). It was proposed that PaeY and PaeX have different substrate preferences for acetyl esters linked to pectin either at O-2 or O-3 configurations, although the exact mode of action is currently not well understood (Shevchik et al., 2003). Despite these differences, both PaeY and PaeX showed a hierarchical deacetylation of sugar beet pectin in which the highest deacetylation activities were observed with pectin pre-treated with PemA and pectate lyases, followed by de-methylated pectin (Shevchik et al., 1997; Shevchik et al., 2003). PaeY and PaeX removed only 15 % and 3 % of acetyl groups from untreated natural pectin, respectively, indicating non-methylated oligogalacturonates were the best substrates for pectin acetylesterases (Shevchik et al., 1997; Shevchik et al., 2003). BliPAE from B. licheniformis showed a similar preference for low methoxyl pectin in that a 2-fold increase in the BliPAE activity was observed when sugar beet pectin was pre-treated with pectin methylesterases (Romoroza et al., 2014). Pre-treating pectin with PemA and PaeY significantly improved the pectate lyase activities, suggesting the synergetic action of pectin methylesterases, pectin acetylesterases, and pectate lyases facilitated the efficient plant cell wall degradation by D. dadantii (Shevchik et al., 1997).
1.6.2 Rhamnogalacturonan-I (RG-I)-degrading enzymes