endo-1,4-β-Galactanase

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Introduction

The processing of plant biomass is a very important and increasingly valued industrial field. At present, the enzymatic degradation of plant cell walls has attracted considerable interest. The major component of plant cell walls is pectin, which is composed of smooth regions of homogalacturonan and hairy regions of rhamnogalacturonan. Rhamnogalacturonan-I is the most abundant component in the hairy region and it is a polysaccharide constructed from alternating units of α-1,2-linked L-rhamnose and α-1,4-linked D-galacturonic acid. The C4 position of rhamnose can be used as an attachment site for the branches of polysaccharides such as arabinan, galactan, and arabinogalactan, making rhamnogalacturonan highly branched and forming a "hairy region".  

At present, the structural information of rhamnogalacturonase A and rhamnogalacturonan acetyl esterase have been obtained, and they act on the backbone of rhamnogalacturonan I, as well as three β-1,4-galactanases (GAL) acting on the β-1,4 linkages in galactan and arabinogalactan branches. GAL belongs to the family 53 of the glycoside hydrolases, which is a member of clan GH-A. Scientists have determined the fungal GAL structure from Aspergillus aculeatus, which constitutes the first available structure of β-1,4-galactanase and family 53 enzymes.

Overall structure of BLGAL

Bacillus licheniformis galactanase (BLGAL) crystallizes in space group P2₁, and the two non-crystallographically symmetry-related molecules are almost identical. The structure of the native protein and the complexes are very similar, and the overall folding is a regular (βα) ₈-barrel conformation. The loops following β-strands 1 and 3 and the long loop of approximately 65 residues following β-strand 8 contain additional secondary structure elements. There is a calcium ion at the top of the barrel, away from the catalytic residue (>16 Å). It is coordinated with the residues of the two long loops following β-strand 7 (Asp272, Asp274, His276 and Asn278) and 8 (Ser367 and Asp370).

Figure 1. Stereo illustration of the overall structure of BLGAL

The active site and substrate-binding groove

In BLGAL, the catalytic acid/base is Glu165 at the end of the β-strand 4, Glu263 at the end of β-strand 7 is used as a catalytic nucleophile. The galactobiose and galactotriose binding sites identified in the complex include two subsites (22 and 23) and three subsites (22, 23, and 24), respectively. Trp115, Trp3347, and Trp36 residues are involved in substrate binding, and they serve as platforms for the three pyranose units at subsites 22, 23, and 24, respectively, as shown in Figure 2 (a) and (b). The detailed interactions between proteins and galactooligosaccharides are shown in Figure 2 (c) and (d).

Figure 2. Close-up views of substrate binding subsites 24, 23 and 22 in the final models for the galactobiose and galactotriose complexes, respectively

Discussion

Compared with GAL from Bacillus species, the fungal GAL can degrade galactan more completely. The non-productive binding of small oligosaccharides to BLGAL may explain why this enzyme cannot degrade β-1,4-galactan as efficiently as AAGAL. The three tryptophan residues form the aromatic platform of subsites -4, -3, and -2. These three tryptophan residues are basically parallel to the sugar ring and stack against their α side. This extensive parallel interaction is common between β-1,4-linked oligosaccharides and GH-A polysaccharide hydrolase with different specificities. The structure of the BLGAL and oligosaccharide complex provides a deeper understanding of GH-53 galactanase and is the first crystallographic image of a galactan fragment or any y equatorial-axial O-linked oligosaccharide. The shape of the substrate-binding groove and the distribution of aromatic side chains on the surface reflect the shape of the polysaccharide substrate in the Clan GH-A enzymes. In BLGAL, the side chains of Trp115, Trp347, and Trp363 are arranged to bind galactotriose in a helical conformation. It can be seen that the shape complementarity of polysaccharides to polysaccharidases plays a key role in recognition function and specificity.