Polysaccharide Lyase Family 6

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Author: Emil G.P. Stender
Responsible Curator: ^^^Birte Svensson^^^

Polysaccharide Lyase Family 6
3D structure	parallel β-helix
Mechanism	β-elimination
Charge neutralizer	calcium or water
Active site residues	known
CAZy DB link
https://www.cazy.org/PL6.html

Substrate specificities

PL6 contains 3 subfamilies [1] all of which contain members catalyzing the depolymerisation of alginate [2]. Alginate consist of 1,4 linked β-D-mannuronic acid and α-L-guluronic acid arranged in poly-mannuronic acid blocks, poly-guluronic acid blocks or poly-mannuronic/guluronic acid blocks [3, 4]. Subfamily 2 and 3 have so far only shown specificity for poly-mannuronic/guluronic acid blocks [2], while subfamily 1 has been demonstrated to depolymerize poly-guluronic acid [5, 6], poly-mannuronic acid [7, 8], poly-mannuronic/guluronic acid [2] as well as dermatan sulfate (formerly chrondroitin B) [2, 9, 10]. Dermatan sulfate consist of N-acetyl galactosamine (GalNAc) and glucuronic acid (GlcA) joined by β 1,4 or 1,3 linkages respectively with a variable sulfation pattern [11].

Kinetics and Mechanism

Figure 1. Syn – or anti – β-elimination catalyzed by PL6 enzymes acting on alginate. M represents mannuronic acid and G guluronic acid. n represents the continued sugar chain. In both cases the catalytic base abstracts the C5 proton and an acid donates one resulting in the β-elimination of the 1,4 glycosidic linkage.

The β-elimination catalyzed by the PL6 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 calcium [6, 10] or by water [5]. This lowers the pK_a value of the C5-proton allowing for abstraction by the catalytic base (Figure 1). A catalytic acid then donates a proton to the glycosidic linkage resulting in the β-elimination. This can be done in syn with the acid and base on the same side of the sugar ring in the transition state (the case for D-mannuronic acid) or anti where they are on opposite sides of the sugar ring (the case for L-guluronic acid) [12, 13].

Catalytic Residues

After charge neutralization a lysine functions as the catalytic base and an arginine as the acid. They were originally identified as K253 and R273 in chondroitinase B from Pedobacter heparinus [9]. PL6 is so far the only discovered alginate lyase family that uses K/R as a catalytic base/acid pair [13].

Three-dimensional structures

Figure 2. Crystal structures of the monomeric chrondroitinase B and AlyF as well as the dimeric AlyGC. Catalytic residues and substrates are in green, the neutralizing calcium is the red sphere.

PL6 catalytic domain adopts a parallel β-helix fold with the activesite located on the surface of one of the β-sheets (Figure 2). The first PL6 structure solved was the chrondoitinase B from Pedobacter heparinus (1.7 Å) [9] later it was shown that this enzyme is calcium dependent [10]. The first alginate lyase structure solved was the exolytic, guluronic acid-specific, homo-dimeric AlyGC in complex with tetra-mannuronic acid (2.6 Å) [6]. The first monomeric alginate lyase structure solved was the guluronic acid-specific AlyF in complex with tetra-guluronic acid (1.8 Å) [5]. The first mannuronic acid specific alginate lyase structure was BcelPL6 (1.3Å) from human gut Bacteroides cellulosilyticus [8]. All four structure belong to subfamily 1. There are no available crystal structures from subfamilies 2 and 3.

Family Firsts

First catalytic activity: OS-ALG-9 from Pseudomonas sp. on non-purified recombinant enzyme by the thiobarbituric acid method [7]
First catalytic base/acid: The catalytic arginine was originally identified in Chondroitinase B from Pedobacter heparinus based on the crystal structure, concervation, mutagenesis and activity analysis (R271E no activity, R271K 0.09 % activity) [10]. The catalytic lysine was identified later based on conservation, mutagenesis and activity analysis [6]
First charge neutralizer: Calcium in Chondroitinase B from Pedobacter Heparinus by by crystallography and assaying the effect of calcium on enzyme activity [10].
First 3-D structure: Chondroitinase B from Pedobacter Heparinus [9].

References

All Medline abstracts: PubMed