Polysaccharide Lyase Family 42

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• Authors: Alan Cartmell and Harry Gilbert
• Responsible Curator: Alan Cartmell

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Polysaccharide Lyase Family PL42
Clan	None
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/PL42.html

Substrate specificities

Three members of this family (BT3686, BACOVA_0349 and HMPREF9455_02360) have been shown to be exo-α-L-rhamnosidases, targeting rhamnose linked α-1,4 to glucuronic acid (Rha-GlcA) in the complex arabinogalactan protein (AGP) from gum arabic (AGP-GA) [1]. A fungal member of this family (FoRham1 from Fusarium oxysporum 12S) was shown to cleave the Rha-GlcA linkage in AGP-GA harnessing anti-β-elimination chemistry generating L-rhamnose and C4-C5 unsaturated D-glucuronic acid at the new non-reducing end [2]. Three PL42 enzymes were shown to cleave the Rha-GlcA linkage in the highly sulfated AGP from red wine (AGP-Wi) through a β-elimination or exo-lyase mechanism [3]. Although these three enzymes did not display lyase activity against AGP-GA, one of the CAZymes, BT3686, cleaved the glycan through a glycoside hydrolase mechanism. Thus, BT3686 contains two distinct active sites that cleave glycosidic linkages through a hydrolase and lyase mechanism, respectively. The PL42 family was originally assigned to glycoside hydrolase family GH145. It is evident, however, that the catalytic histidine (see below) in the glycoside hydrolase active site is not highly conserved indicating that many of the GH145 enzymes will not catalyse a hydrolytic reaction. In contrast, there is an extremely high degree of sequence conservation in the lyase active site, including invariant catalytic residues. Thus, β-elimination is likely to be the dominant activity displayed by enzymes in this family. GH145 was therefore reassigned to a polysaccharide lyase family; PL42.

Kinetics and Mechanism

The β-elimination catalyzed by PL42 enzymes results in the formation of a C4-C5 unsaturated sugar residue at the new non-reducing end. The first step is the neutralization of the acid group in the +1 subsite by a conserved arginine. A histidine abstracts the labile proton at C5. The same histidine is also believed to act as the catalytic acid, protonating the leaving group (L-Rha) resulting in glycosidic bond cleavage [2].

With respect to the glycoside hydrolase activity displayed by some PL42 enzymes, the catalytic mechanism was explored using BT3686 from Bacteroides thetaiotamicron and AGP-GA as the substrate. NMR analysis of the reaction revealed the family operates via a retaining mechanism [1]. Rather than using a standard double displacement mechanism the enzyme is speculatively predicted to perform catalysis via an epoxide intermediate, similar to GH99 enzymes [4, 5]. BT3686 is proposed to perform catalysis via a substrate assisted mechanism, requiring the carboxyl group of the glucuronic acid and a single catalytic histidine; both acting as an acid/base. This histidine is predicted to deprotonate the O2 of rhamnose, allowing O2 to attack C1 and form an epoxide. Simultaneously the carboxyl group of the glucuronic acid may deprotonate a water molecule generating a hydroxyl group to attach the C1 of rhamnose and allowing protonation of its own O4 thus, leading to glycosidic bond cleavage [1]. Further work is needed to confirm the mechanism by which some GH145 enzymes operate through a glycoside hydrolase mechanism.

Catalytic Residues

With respect to the lyase mechanism, of which FoRham1 is the exemplar. Based on 3D-X-ray crystallography of enzyme-substrate complexes and mutagenesis studies, Arg166 neutralizes the acid group and His85 is proposed to act as the catalytic acid-base [2].

With respect to glycoside hydrolase activity, using BT3686 as the exemplar, a single catalytic histidine, His48, has been shown to be critical for activity [1]. This was the only residue that, when mutated (to Gln, Ala and Gly) caused complete loss of activity. The histidine is thought to act as an acid/base. Several homologues of BT3686 exist which, although >80 %, identical are inactive. These enzymes have a Gln at the equivalent position to His48 in BT3686. Replacing Gln48 with a histidine in the related enzymes BACINT_00347 and BACCELL_00856, from Bacteroides intestinalis and Bacteroides cellulosilyticus, respectively, introduced rhamnosidase activity [1]. No second catalytic residue could be identified and it was tentatively proposed that the glucurnonic acid participates as a second acid/base residue, portonating its own O4 and activating a water molecule (see above).

Three-dimensional structures

PL42 enzymes comprise a single catalytic domain displaying a seven bladed β-propeller fold. Each blade is composed of four anti parallel β-strands that extend out radially from the central core. The final blade is formed by strands from both the N- and C-terminus of the protein, which is termed as 'molecular velcro' and is believed to add considerable stability to the fold. The glycoside hydrolase active site is located on the posterior surface, while the anterior surface houses the b-elimination or lyase catalytic apparatus. PL42 is distantly related to PL25 and PL24, in which the anterior surface houses the catalytic apparatus [6, 7].

Family Firsts

Polysaccharide lyase activity

First demonstration of lyase activity

FoRham1 from Fusarium oxysporum 12S. Activity shown against AGP-GA derived oligosaccharides by UV absorbance spectroscopy and HPAEC [2].

First catalytic base/acid identification

FoRham1. His85 was suggested as the acid/base based upon the crystal structure of the substrate complex, residue conservation, mutagenesis and activity analysis (H311A: inactive) remaining)[2].

First charge neutralizer identification

FoRham1 Arg166 was suggested based on the crystal structure, whiccarboxylate group at the +1 subsite its conservation, mutagenesis and activity analysis (R166A was inactive).;First 3-D structure: FoRham1 from Fusarium oxysporum 12S [2].

Glycoside hydrolase activity

First stereochemistry determination

Determined for the Bacteroides thetaiotaomicron enzyme BT3686 [1].

First catalytic acid/base residue identification

Predicted to be a histidine [1].

Second general acid/base residue identification

Predicted to be provided by the substrate [1].

First 3-D structure

BT3686, BACINT_00347 and BACCELL_00856 were the first enzymes to have their structures solved from the organisms Bacteroides thetaiotaomicron, bacteroides intestinalis and bacteroides cellulosilyticus, respectively. [1].

References

1. Munoz-Munoz J, Cartmell A, Terrapon N, Henrissat B, and Gilbert HJ. (2017). Unusual active site location and catalytic apparatus in a glycoside hydrolase family. Proc Natl Acad Sci U S A. 2017;114(19):4936-4941. DOI:10.1073/pnas.1701130114 |
2. Kondo T, Kichijo M, Maruta A, Nakaya M, Takenaka S, Arakawa T, Fushinobu S, and Sakamoto T. (2021). Structural and functional analysis of gum arabic l-rhamnose-α-1,4-d-glucuronate lyase establishes a novel polysaccharide lyase family. J Biol Chem. 2021;297(3):101001. DOI:10.1016/j.jbc.2021.101001 |
3. Munoz-Munoz J, Ndeh D, Fernandez-Julia P, Walton G, Henrissat B, and Gilbert HJ. (2021). Sulfation of Arabinogalactan Proteins Confers Privileged Nutrient Status to Bacteroides plebeius. mBio. 2021;12(4):e0136821. DOI:10.1128/mBio.01368-21 |
4. Thompson AJ, Williams RJ, Hakki Z, Alonzi DS, Wennekes T, Gloster TM, Songsrirote K, Thomas-Oates JE, Wrodnigg TM, Spreitz J, Stütz AE, Butters TD, Williams SJ, and Davies GJ. (2012). Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase. Proc Natl Acad Sci U S A. 2012;109(3):781-6. DOI:10.1073/pnas.1111482109 |
5. Fernandes PZ, Petricevic M, Sobala L, Davies GJ, and Williams SJ. (2018). Exploration of Strategies for Mechanism-Based Inhibitor Design for Family GH99 endo-α-1,2-Mannanases. Chemistry. 2018;24(29):7464-7473. DOI:10.1002/chem.201800435 |
6. Ulaganathan T, Boniecki MT, Foran E, Buravenkov V, Mizrachi N, Banin E, Helbert W, and Cygler M. (2017). New Ulvan-Degrading Polysaccharide Lyase Family: Structure and Catalytic Mechanism Suggests Convergent Evolution of Active Site Architecture. ACS Chem Biol. 2017;12(5):1269-1280. DOI:10.1021/acschembio.7b00126 |
7. Ulaganathan T, Helbert W, Kopel M, Banin E, and Cygler M. (2018). Structure-function analyses of a PL24 family ulvan lyase reveal key features and suggest its catalytic mechanism. J Biol Chem. 2018;293(11):4026-4036. DOI:10.1074/jbc.RA117.001642 |