Characterisation and substrate binding modes of exopolygalacturonase PGQ1 from Saccharobesus litoralis

Abstract Among the enzymes required for the efficient utilisation of pectin is polygalacturonase. Saccharobesus litoralis harbours two polygalacturonases belonging to glycoside hydrolase family 28 (GH28). One of them, PGQ1, cleaved polygalacturonate exolytically at the non-reducing end into monomeric units. It was most active at 60 °C and pH 8, with K m and k cat values of 2.3 mg/ml and 6.4 s−1 respectively. Its homology model of a right-handed parallel β-helix core consisted of Asp297 as the general acid and either Asp276 or Asp298 as the general base. By inferring the substrate binding modes at the −1 and +1 subsites from known crystal structures, a hexagalacturonate could be docked into the highly electropositive binding cleft. Interestingly, while no residues were present in the vicinity to make up the +2 and +4 subsites, Arg361 and Arg430 could readily bind to the carboxyl groups of the galacturonates at the +3 and +5 subsites respectively. Structural comparison suggested that this binding pattern with missing subsites might be unique to closely related exopolygalacturonases. As S. litoralis grew much more slowly on extracellular galacturonate due to the lack of a transporter for the monosaccharide, PGQ1 probably functioned in the periplasm to help degrade oligopectates completely. Communicated by Ramaswamy H. Sarma


Introduction
Pectin is a complex polysaccharide that constitutes a major component of terrestrial plant cell walls.It is composed of backbones of 1,4-linked a-D-galacturonate which are partially methylated and acetylated, and decorated by a variety of neutral sugars such as rhamnose, galactose, arabinose and xylose.Pectin degradation involves a combination of enzymes including pectin methylesterase, pectin acetylesterase, pectate lyase and polygalacturonase (Abbott & Boraston, 2008).Both pectate lyase and polygalacturonase cleave the 1,4 glycosidic bonds between two galacturonate molecules but through different mechanisms: pectate lyase employs b-elimination to produce 1,4-unsaturated galacturonate at the non-reducing end, whereas polygalacturonase exploits inverting hydrolysis to generate b-galacturonate at the reducing end.Polygalacturonases are classified as either endo-acting (EC 3.2.1.15),which are normally found in the extracellular environment, or exo-acting (EC 3.2.1.67),which are usually present in the periplasm and cleave pectic substrates from the non-reducing end into short oligomers and monomers (Abbott & Boraston, 2008).
We have previously isolated a marine bacterium from the surface of an algal turf, Catenovulum sp.CCB-QB4, whose genome encodes numerous carbohydrate-active enzymes (CAZymes) (Lau et al., 2019).The bacterium, which has since been identified as a novel species from a new genus and thus renamed Saccharobesus litoralis (Amrina et al., 2021), is capable of utilising pectin as well as agar, alginate and ulvan as carbon sources, with complete sets of the necessary genes encoded in its genome (Furusawa et al., 2021;Lau et al., 2019).Marine bacteria capable of feeding on terrestrial pectin have been increasingly discovered; in S. litoralis, interestingly, the polysaccharide utilisation loci (PULs) associated with pectin metabolism are found on the bacterium's sole plasmid, indicating that they had been acquired horizontally.The pectin PULs encode at least seven pectate lyases and two polygalacturonases, which is bemusing as to why S. litoralis requires so many enzymes to degrade pectin.The two polygalacturonases, designated PGQ1 and PGQ2, are 26% identical and belong to glycoside hydrolase family 28 (GH28) according to the CAZy database (Drula et al., 2022).
The inverting mechanism of GH28 polygalacturonases involves a catalytic triad of Asp, one for protonating the glycosidic oxygen while the other two for activating the nucleophilic water molecule (van Santen et al., 1999).The crystal structures of an endopolygalacturonase from Stereum purpureum, endoPG I (PDB: 1KCD) (Shimizu et al., 2002), and an exopolygalacturonase from Yersinia enterocolitica in complex with digalacturonate, YeGH28 (PDB: 2UVF) (Abbott & Boraston, 2007), have revealed the binding modes at the À 2, À 1 and þ1 subsites.The pyranose ring at the À 1 subsite is distorted into the 4 H 3 half-chair conformation, one of a handful of conformations with the C2, C1, O5 and C5 atoms being coplanar to favour the oxocarbenium-like transition state during glycoside hydrolysis (Davies et al., 2003;Franconetti et al., 2021).Studies on the exopolygalacturonase from Thermotoga CONTACT Aik-Hong Teh aikhong@usm.mySupplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2023.2167111.maritima, Tm ExoPG, further indicate the presence of at least four subsites, i.e.À 1 to þ3 with a high-affinity þ1 subsite, in the catalytic cleft (Kluskens et al., 2005), but the binding modes at the þ2 and þ3 subsites are unclear.In order to further investigate the binding mechanism of polygalacturonases, we have characterised PGQ1 and performed a docking analysis with its homology model, which has revealed that the exogalacturonase's binding cleft could accommodate at least a hexagalacturonate.

Cloning, expression and purification of PGQ1
Full-length PGQ1 excluding the Tat lipoprotein signal peptide (Cys28-Leu476; NCBI: WP_108605238) was cloned by homologous recombination.PCR was run with a pair of primers containing termini overlapping with the pET-21a vector (Novagen) and a His-tag sequence before the stop codon.The PCR product and the linearised vector were mixed and transformed into Escherichia coli BL21 (DE3).Cells were grown in a LB medium supplemented with 100 lg/ml ampicillin at 37 � C until an OD 600 of 0.5-0.6.Enzyme expression was induced with 0.1 mM IPTG and incubated overnight at 25 � C.After harvest, the cells were suspended in 20 mM Tris-HCl, 300 mM NaCl, pH 8, lysed by sonication and centrifuged.The His-tagged enzyme was purified by affinity chromatography using a Ni 2þ -charged Protino Ni-IDA packed column (Macherey-Nagel) with stepwise elution with up to 500 mM imidazole.Fractions containing pure PGQ1 were pooled for characterisation.

Assays of polygalacturonase activity
The activity of the PGQ1 was examined in triplicate using the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959).The optimum temperature was determined by mixing 10 ml of diluted PGQ1 with 90 ml of 2 mg/ml polygalacturonate in the Tris-HCl buffer.After the mixture was incubated at 30-70 � C for 5 min, 200 ml of DNS was added and boiled at 100 � C for 10 min, cooled and measured at 540 nm.Temperature stability was carried out by measuring the residual activity after incubating the enzyme for 1 h.pH effects were evaluated using 20 mM phosphate buffer (pH 6.0 and 7.0), Tris-HCl buffer (pH 8.0 and 9.0) and glycine-sodium hydroxide buffer (pH 10.0) at 60 � C, and pH stability was examined by measuring the residual activity after 24 h of incubation.The kinetic parameters K m and k cat were determined from the Hanes-Woolf plot using 1-12 mg/ml polygalacturonate and 6 lM PGQ1.Degraded products were detected by thin layer chromatography (TLC) using a solvent system of butanol, acetic acid and water in a 4:2:3 ratio.After 2 h of development the TLC plate was dried at 60 � C for 5 min, stained with 10% HCl in ethanol and heated at 90 � C for 20 min.

Homology modelling, docking analysis and molecular dynamics simulation
Structure-based sequence alignment was performed with Expresso (Armougom et al., 2006), manually edited and presented with ESPript (Robert & Gouet, 2014).A homology model of PGQ1 was generated with SWISS-MODEL (Waterhouse et al., 2018) using the crystal structure of the exopolygalacturonase ExoPG from T. maritima (PDB: 3JUR) (Pijning et al., 2009).Electrostatic potential of the PGQ1 model was calculated with the PBEQ Solver (Jo et al., 2008).The structure of the endopolygalacturonase endoPG I from S. purpureum in complex with galacturonate (PDB: 1KCD) (Shimizu et al., 2002) was used as the basis for manually docking a hexagalacturonate into the PGQ1 model.The catalytic acid Asp297, as well as Asp279 which interacted with the base Asp276, were modelled in the protonated state.The complex was regularised using the geometry minimisation tool of Phenix (Liebschner et al., 2019), solvated with 150 mM KCl using CHARMM-GUI (Lee et al., 2016), minimised and equilibrated using GROMACS (Van Der Spoel et al., 2005) with the CHARMM36m force field (Huang et al., 2017), and simulated for 10 ns.Several cycles were repeated to refine the docking.The model was validated with the Ramachandran plot using RAMPAGE (Lovell et al., 2003).Structural figures were generated using PyMOL (Schr€ odinger).

Determination of bacterial growth
Cells of S. litoralis were cultured in a high nutrient artificial seawater medium (H-ASWM) containing 0.5% tryptone, 2.4% artificial sea salt mix (Marine Enterprises International), 10 mM HEPES, pH 7.6.The cells were inoculated in 10 ml H-ASWM with 0.2% glucose.After overnight incubation at 30 � C, 0.1 ml of the cells was transferred into 100 ml fresh H-ASWM containing either 0.2% glucose or galacturonate, and cell growth was monitored at OD 600 for 72 h.

Characterisation of PGQ1
The single-domained PGQ1 of 476 residues consists of a Tat lipoprotein signal peptide (Met1-Ala27) as predicted with SignalP (Teufel et al., 2022), and a GH28 catalytic domain.Characterised GH28 polygalacturonases that share the highest identity with PGQ1 include the exo-acting polygalacturonase PsGH28 from the marine bacterium Pseudoalteromonas sp.PS47 at 56% (Hobbs et al., 2019); PecJKR01, which was isolated from a hot spring soil metagenome sample, at 49% (Singh et al., 2012); the exopolygalacturonase from the hyperthermophilic bacterium Thermotoga maritima, Tm ExoPG, at 46% (Kluskens et al., 2005;Pijning et al., 2009); as well as BT1018 and BT4155 from the human colonic bacterium Bacteroides thetaiotaomicron at 41% and 39% respectively (Luis et al., 2018;Ndeh et al., 2017).Sequence alignments with these enzymes showed that residues that form an active site with a closed end such as Tm ExoPG's Trp214, Lys266 and Glu304 are also conserved in PGQ1 as Trp251, Lys303 and Glu339 (Figure 1), indicating that PGQ1 might similarly function as an exopolygalacturonase.This was confirmed with the TLC analysis, which detected the presence of only galacturonate but not other oligosaccharides irrespective of the length of reaction time (Figure 2A).Although S. litoralis was a mesophile, PGQ1 exhibited an optimum temperature of 60 � C (Figure 2B), which was lower than that of the thermostable PecJKR01 (70 � C) (Singh et al., 2012) and Tm ExoPG (80 � C) (Kluskens et al., 2005).PGQ1 was relatively stable up to 50 � C, but its activity declined sharply after an hour of incubation at 60 and 70 � C (Figure 2B).PecJKR01 similarly lost about 60% activity after treatment at its optimum temperature, 70 � C, for 30 min (Singh et al., 2012).Meanwhile, PGQ1 was most active at pH 8.0 (Figure 2C), and both PecJKR01 (Singh et al., 2012) and Tm ExoPG (Kluskens et al., 2005) also display comparable optimum pH at pH 7.0 and 6.5 respectively.PGQ1 retained nearly 100% relative activity over the range of pH tested after incubation for 24 h (Figure 2C).PGQ1's K m and k cat values obtained from the Hanes-Woolf plot were 2.3 mg/ml and 6.4 s À 1 respectively (Figure 2D).

Binding modes of polygalacturonate
The PGQ1 model showed a core of right-handed parallel b-helix architecture typical of the GH28 family (Figure 3A).The well conserved active site was enclosed at one end as seen in the exopolygalacturonases Tm ExoPG (Pijning et al., 2009) and YeGH28 (Abbott & Boraston, 2007), which would allow only the non-reducing end of a sugar chain to bind.Validation with the Ramachandran plot showed that the model was of reasonable quality with 95.8% of its residues in the favoured region, 4.2% in the allowed region and none in the outlier region (Figure S1).Calculation of the model's electrostatic potential further revealed the presence of an elongated electropositive patch stretching from the catalytic site, indicating that the binding cleft probably contained several subsites (Figure 3B).In an attempt to delineate all the possible subsites that PGQ1 possessed, a docking analysis with oligogalacturonates was performed on the model.The galacturonate molecules at the À 1 and þ1 subsites were docked by analogy to the complex structure of S. purpureum endoPG I with galacturonate (PDB: 1KCD) (Shimizu et al., 2002), with the pyranose ring in the À 1 subsite modelled into the 4 H 3 half-chair conformation (Figure 3C).The Asp297 acted as the general acid to protonate the glycosidic O atom between the À 1 and þ1 subsites, while either Asp276 or Asp298 as the general base to activate a conserved water molecule for a nucleophilic attack on the anomeric C1 atom at the À 1 subsite.
Characterisation of the related Tm ExoPG with a series of oligouronates of increasing degrees of polymerisation has revealed the existence of four subsites (Kluskens et al., 2005), and PGQ1's elongated electropositive catalytic cleft likewise suggested the presence of additional subsites besides the À 1 and þ1 subsites.The digalacturonate was hence extended by adding a third galacturonate antiparallel to the one at the þ1 subsite, with their carboxyl groups opposite to one another in the most stable arrangement.No residue, however, was in close proximity to interact with the galacturonate at this putative þ2 subsite.Adding a fourth galacturonate antiparallel to the third one, meanwhile, showed that the strictly conserved Arg361 could readily interact with the sugar's carboxyl group at this putative þ3 subsite (Figures 1 and 3C).Although the absence of the þ2 subsite was rather unexpected, it was observed that in the Tm ExoPG study, the same K m value, 0.34 mM, was obtained for both digalacturonate and trigalacturonate in contrast to the successively decreasing K m values for the other oligouronates (Kluskens et al., 2005), which might be due to the lack of a third subsite for trigalacturonate.Also, Tm ExoPG's K m continued to decrease with longer oligomers until hexagalacturonate when its value approached that for polygalacturonate (Kluskens et al., 2005), suggesting the exopolygalacturonase might harbour more than four subsites.As PGQ1's electropositive patch also stretched beyond the þ3 subsite, attempts to dock further galacturonates were made.While Arg430 could possibly interact with the sugar's O2 atom at the putative þ4 subsite, the hydrogen bond was weak and broke immediately during MD simulation; the þ4 subsite therefore might not exist too.Nevertheless, Arg430 as well as Arg425 were readily available to interact with the carboxyl group of the galacturonate at the putative þ5 subsite (Figure 3C), while no other residues were within reach to bind beyond it.Accordingly, PGQ1's binding cleft possibly contained up to the þ5 subsite and ran the full length of the electropositive patch.This binding pattern was further verified when the hexagalacturonate remained bound to the subsites during the MD simulation (Figure 3D).
Comparison with the related exopolygalacturonases Tm ExoPG (PDB: 3JUR) (Pijning et al., 2009) and B. thetaiotaomicron BT4155 (PDB: 5OLP) (Luis et al., 2018) showed that, similarly, no available residue could bind at the þ2 and þ4 subsites, while their respective Arg324 and Arg333, corresponding to PGQ1's Arg361 and conserved in all the related exopolygalacturonases (Figure 1), could interact with the þ3 subsite.At the þ5 subsite, PGQ1's Arg430 was likewise conserved as Lys408 in BT4155 (Figure 1).Although in Tm ExoPG it was replaced by Glu393, the preceding Arg391, also conserved in PecJKR01 and BT1018 (Figure 1), was close to the galacturonate and could interact with its carboxyl group.Meanwhile, PGQ1's Arg425 was conserved as Lys433 in PsGH28, which could interact with the subsite þ5 (Figure 1).In summary, such a binding cleft for a hexasaccharide with two missing subsites, i.e. þ2 and þ4, might be a unique feature for this subgroup of GH28 exopolygalacturonases.The additional þ3 and þ5 subsites could help the enzyme to capture the polysaccharide more efficiently, while the absence of the þ2 and þ4 subsites might facilitate the release of the galacturonate from the À 1 subsite and subsequent movement of the remaining polysaccharide into the vacated subsite for successive cleavage.

Physiological role of PQG1
Saccharobesus is a new genus of literally 'fat sugar-eating' bacteria, and the type species S. litoralis obligately requires a sugar such as glucose, alginate, pectin or ulvan for growth (Amrina et al., 2021;Furusawa et al., 2021).The bacterium's plasmid encodes a complete set of enzymes for pectin utilisation including polygalacturonases, lyases and esterases that work in concert to cleave pectin into either saturated or 4,5-unsaturated galacturonates, which are finally converted to pyruvate and glyceraldehyde 3-phosphate through separate pathways.Saturated galacturonate is metabolised via the isomerase pathway, and in fact S. litoralis even encodes the two alternative routes available for this pathway -first turning galacturonate into tagaturonate and then either reducing it to altronate or epimerising it to fructuronate (Figure 4A) -while some bacteria habour only either one.
Meanwhile, PGQ2 (NCBI: WP_108605277), the other GH28 polygalacturonase encoded by S. litoralis with a Sec/SPI signal peptide and 26% identity to PGQ1, might be extracellular and endo-acting like the 48% identical endopolygalacturonase from Ralstonia solanacearum (Schell et al., 1988).While the exo-acting PGQ1 degraded polygalacturonate into galacturonate, interestingly S. litoralis hardly grew in a 30 h of culture containing the monosaccharide (Furusawa et al., 2021) despite possessing the two galacturonate pathways.Upon extending the culture to 72 h, the bacterium finally started entering the exponential phase only at 24 h, and reached maximum growth with an OD 600 of about 0.6 at 56 h (Figure 4B).In contrast, it grew more than twice as fast and as much on glucose, already reaching maximum growth at 24 h with an OD 600 of 1.3.These results indicated that S. litoralis did not utilise efficiently extracellular galacturonate.
The pectin-utilising bacterium Pseudoalteromonas sp.PS47 that harbours PsGH28, a periplasmic exopolygalacturonase, similarly does not grow on extracellular galacturonate in a 30 h of culture due to its inability to transport the monosaccharide across the outer membrane (Hobbs et al., 2019).With a Tat lipoprotein signal peptide, PGQ1 is likewise expected to be translocated to the periplasm and hence does not generate galacturonate extracellularly.Currently the only known transporter in bacteria for extracellular galacturonate is ExuT (Nemoz et al., 1976; San Francisco & Keenan, 1993).Although S. litoralis does encode two similar major facilitator superfamily (MFS) transporters, one of them (NCBI: WP_108601501) is encoded in an operon together with two alginate lyases, indicating that it may function instead in alginate transport.The other (NCBI: WP_108602313) has its gene clustered in a putative operon for galactonate utilisation and resembles more E. coli galactonate transporter (DgoT) (Leano et al., 2019) with 47% identity, which instead suggests that S. litoralis may also feed on this sugar.In short, the slow growth of S. litoralis on extracellular galacturonate may be due to the lack of a dedicated uptake system for this monosaccharide, and similar to PsGH28, PGQ1 may likewise function to completely depolymerise oligogalacturonates within the bacterium for consumption.

Conclusion
PGQ1 is a GH28 exopolygalacturonase from S. litoralis that cleaves polygalacturonate into monosaccharides.It contained an elongated electropositive binding cleft made up of the catalytic triad Asp276, Asp297 and Asp298 (Figure 3A and B).In addition to the well conserved À 1 and þ1 subsites, PGQ1 might also accommodate a hexagalacturonate occupying up to the þ5 subsite (Figure 3C).Although Arg361 could bind to the sugar's carboxyl group at the þ3 subsite while Arg425 and Arg430 to that at the þ5 subsite (Figure 3C), interestingly no residues were available nearby to interact with the putative þ2 and þ4 subsites.Such a binding cleft with missing subsites, possibly a unique feature for the closely related GH28 exopolygalacturonases, might facilitate substrate transition into the À 1 subsite for successive cleavage.The inability of S. litoralis to utilise extracellular galacturonate efficiently indicated that PGQ1, which harbours a Tat lipoprotein signal peptide, acted to degrade oligopectates in the periplasm.The new insights into PGQ1's substrate binding may facilitate engineering of improved polygalacturonases for industrial applications, such as bioscouring of cotton fabrics (Zhang et al., 2021) and generation of galacturonate as raw materials for biorefineries (Jeong et al., 2021).

Disclosure statement
No potential conflict of interest was reported by the authors.

Figure 1 .
Figure1.Structure-based sequence alignment.While GH28 polygalacturonases generally employ a catalytic triad of Asp (red triangles) for hydrolysing the 1,4 glycosidic bond, PGQ1 and closely related exopolygalacturonases (Group 1) further maintain well conserved À 1 and þ1 subsites (blue triangles) that constitute acatalytic site with a closed end.The þ3 subsite was also formed by a well conserved Arg361 (cyan triangle), whereas the þ5 subsite was probably formed by basic residues conserved at three different but nearby positions (cyan highlight).

Figure 2 .
Figure 2. Characterisation of PGQ1.(A) Only spots that corresponded to galacturonate (G) were detected with TLC during the degradation of polygalacturonate (PGA) by PGQ1.(B) Although PGQ1 had the highest activity at 60 � C (circle), it was more stable at lower temperatures (square).(C) PGQ1 was most active at pH 8 (circle), but was relatively stable at all the pH tested (square).(D) The Hanes-Woolf plot for PGQ1 kinetics.

Figure 3 .
Figure 3. Modelling and docking analysis.(A) The PGQ1 model consisted of right-handed parallel b-helices, with a closed-end active site that could accommodate a hexagalacturonate (pink).(B) The substrate binding cleft was highly electropositive throughout the full length of the hexagalacturonate.(C) The À 1 and þ1, as well as the þ3, subsites were well conserved, while the þ2 and þ4 subsite (grey) were characteristically absent.The þ5 subsite was made up of Arg425 and Arg430.(D) The hexagalacturonate remained stably bound to the subsites during the MD simulation.

Figure 4 .
Figure 4. Galacturonate utilisation in S. litoralis.(A) The bacterium harbours the genes for both the two alternative routes in the isomerase pathway, which breaks down galacturonate to pyruvate and glyceraldehyde 3-phosphate through the formation of either altronate or fructuronate.(B) S. litoralis achieved maximum growth only at 56 h on galacturonate (solid circles), whereas it achieved more than double that at 24 h on glucose (open circles).