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PL38 is a multispecific family whose characterised members act on one of two distinct uronic acid polysaccharides: Glucuronates (β-1,4-glucuronan, EC 4.2.2.14) or alginate (β-1,4-mannuronate/α-1,4-guluronate, EC 4.2.2.3, 4.2.2.11, 4.2.2.-). CUL-I, from Brevundimonas sp. SH203, the founding member of PL38 (distantly related to PL5), specifically depolymerises the β-1,4-glycoside linkages of β-D-1,4-glucuronan prepared by TEMPO-mediated oxidation from regenerated cellulose, and does not act on the structurally related α-1,4-polyglucuronate or β-1,3-polyglucuronate. Its mode of action is endo-type, consistent with production of a distribution of oligosaccharide products [1].
TpPL38A from Trichoderma parareesei is a glucuronan lyase (EC 4.2.2.14) acting on β-1,4-polyglucuronic acid, and displays an exo-lytic mode of action [2].
The discovery of Aly38A from Agarivorans sp. B2Z047 revealed that PL38 is not restricted to glucuronan substrates. Aly38A was the first alginate lyase belonging to the PL38 family, as demonstrated by TLC analysis, which showed it degrades sodium alginate, polyM (β-D-polymannuronate), and polyG (α-L-polyguluronate) [3].
BoPL38 from Bacteroides ovatus CP926 was found to degrade well-characterised alginates, polyM-, polyG-, and polyMG-substrates in endo-mode, with the ability to cleave G-G, M-M, and M-G/G-M glycosidic linkages. The most prominent products of BoPL38 action are oligosaccharides of DP5–6, and hardly any DP1 (unsaturated monouronate) is formed, consistent with a strict endo-lytic mode. Notably, BoPL38 did not degrade β-1,4-glucuronan, indicating that alginate specificity and glucuronan specificity represent distinct substrate preferences within the family [4].
Polysaccharides cleaved by PL38 enzymes
β-D-1,4-polyglucuronic acid
Alginate β-D-1,4-mannuronate (M) and α-L-1,4-guluronate arranged in blocks of M, G, or MG/GM.
Mechanism
PL38 enzymes operate via a β-elimination mechanism, producing oligosaccharides with a new non-reducing end with a 4-deoxy-L-erythro-hex-4-en pyranosyl uronate residue (Δ). This unsaturated uronate is characteristic of polysaccharide lyases acting on uronic acid-containing substrates.
The mechanism traditionally follows three steps: (I) charge neutralisation of the uronate carboxyl, which in some PL families is assisted by metal cations, such as Ca2+. In PL38, the function of charge neutralisation is taken by an Asn residue. (II) general base-catalyzed abstraction of the proton on the C5 and (III) β-elimination of the 4-O-glycosidic bond [5].
The mechanistic analysis of BoPL38 revealed that a single active site architecture can facilitate both syn- and anti-β-elimination. C5 proton abstraction at subsite +1 is carried out by Y298 (enabling syn-β-elimination) and H243 (enabling anti-β-elimination), and the two pathways proceed through distinct transition states. In the syn reaction, the general base abstracts the H5 proton from the uronate moiety at subsite +1 on the same side as the glycosidic oxygen, while in the anti-reaction they are on opposite sides. This dual mechanism allows BoPL38 to process both mannuronate and guluronate blocks, which differ in the relative orientation of the C5 hydrogen and the glycosidic oxygen [6].
R292 plays a critical role in BoPL38, distorting the sugar at subsite +1 into a preactivated conformation, while also stabilising the active site tunnel through a salt bridge. A well-defined residue network mediates substrate recognition, and site-directed mutagenesis confirmed that disruption of this network destabilises the active site architecture [6].
NMR spectroscopy revealed that BoPL38 also catalyses mannuronate-to-guluronate epimerization, a secondary activity that had not previously been reported in any alginate lyase. As in lyase-catalysed β-elimination, the epimerization reaction initiates with H5 abstraction; however, instead of cleaving the glycosidic linkage, a proton is donated back to C5 from the opposite face, inverting the configuration at that center [6].
For the exo-lytic glucuronan lyase TpPL38A, NMR analysis showed that the monomeric unsaturated end-product undergoes spontaneous rearrangement to tautomeric forms, a non-enzymatic phenomenon that follows product release and has also been observed in alginate [2, 7].
Optimal activity conditions differ between characterised members. BoPL38 has an activity optimum at pH 7.5 [4], while TpPL38A displays optimal activity at pH 6 [2].
PL38 mechanisms on alginate
Syn-β-elimination
Anti-β-elimination
Catalytic Residues
The catalytic residues of PL38 have been established through the combined structural, computational, and mutagenesis analysis of BoPL38 from Bacteroides ovatus CP926 [6].
The uronate carboxyl charge neutralising residue is Asn242 (BoPL38 numbering), Y298 (enabling syn-β-elimination) and H243 (enabling anti-β-elimination). Based on the mechanistic study of BoPL38, Arg292 seems to be essential for maintaining Tyr298's protonation state, distorting the uronate in the +1 subsite, and maintaining the tunnel architecture of the enzyme [6]. However, this has only been described for the alginate lyase, BoPL38.
Three-dimensional structures
The first three-dimensional structure of a PL38 protein was determined without associated publication by the Joint Center for Structural Genomics (JCSG) for an uncharacterized Bacteroides ovatus ATCC 8483 protein (PDB: 3NFV and 3NNB). BoPL38 was found to be very similar to this uncharacterized PL38 structure, sharing the residues desctibed above [4]. PL38 is an ( α / α )7 barrel with an electronegative tunnel formed by two loop regions.
Michaelis complexes of BoPL38 with alginate oligosaccharides hexaguluronate (PDB: 9FHT), hexamannuronate (PDB: 9FHU), and a hexameric alternating M-G block (PDB: 9FHV) were resolved by X-ray crystallography at pH 3.5, far from the catalytic optimum. These complexes confirmed the location of the active site in the electropositive tunnel of the (α/α)₇-barrel fold. The alginate oligosaccharides are visible in the electron density, binding at the same subsites, with most M units adopting the canonical ⁴C₁ chair conformation and G units at subsites –1 and +1 found in the ²S₀ skew-boat conformation. The distortion of the +1 sugar into a non-standard ring conformation is consistent with a preactivation mechanism preceding catalysis [6].
No three-dimensional structure has been reported to date for the glucuronan-active PL38 members CUL-I or TpPL38A, and structural comparison between the glucuronan-specific and alginate-specific subfamilies within PL38 remains an open question.
Structural features of PL38
Secondary structure of PL38 ( α / α )7 barrel shown in cartoon representation, colored from N-to-C-terminal in blue to red rainbow.
Surface electrostatic features and active site residues The electronegative tunnel of PL38 in which the catalytic residues are placed.
Family Firsts
First description of catalytic activity
(1,4)-β-D-glucuronan activity by Brevundimonas sp. SH203 cellouronate lyase (CUL-I) [1].
Sodium-alginate, poly-mannuronate and poly-guluronate activity by Agarivorans sp. B2Z047 alginate lyase (Aly38A) [3].
Gacesa P. (1987) Alginate‐modifying enzymes: A proposed unified mechanism of action for the lyases and epimerases. FEBS Letters, 212, 1873-3468. DOI:10.1016/0014-5793(87)81344-3[Gacesa1987]
β-D-1,4-polyglucuronic acid
Alginate
Syn-β-elimination
Anti-β-elimination
Secondary structure of PL38
Surface electrostatic features and active site residues
