Hyaluronate Lyase

Hyaluronate lyase (EC 4.2.2.1), also known as hyaluronidase, hyalurononglucosaminidase, hyaluronoglucuronidase, glucuronoglycosaminoglycan lyase, or mucinase, catalyzes the degradation of hyaluronic acid (HA), which has been described from several pathogenic streptococcal species. Hyaluronate lyase belongs to the family of lyases, specifically those carbon-oxygen lyases acting on polysaccharides. It catalyzes the cleavage of hyaluronate chains at a beta-D-GalNAc-(1->4)-beta-D-GlcA bond, ultimately breaking the polysaccharide down to 3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine.

Classification

Hyaluronidases can be classified into two main groups to date: lyases and hydrolases. The lyases include bacterial hyaluronidase (hyaluronate lyase) secreted by certain strains of Streptococcus, Propionibacterium, Peptostreptococcus, Staphylococcus and Streptomyces genera; the hydrolases include testicular-type hyaluronidase and leech hyaluronidase. There are considerable differences between these two groups. The lyase group of hyaluronidase from bacteria are less stable, which degrade hyaluronic acid at a faster rate and to a greater extent, also produce unsaturated products. Conversely, the hydrolase group of hyaluronidase act by hydrolysis of the same glycosidic linkage, giving oligosaccharide products composed of unchanged repeating disaccharide units.

Substrates

Hyaluronic acid is the primary substrate of hyaluronate lyase, which is a linear glycosaminoglycan consisting of a polymer of a b-1-4 linked N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) disaccharide. It forms a strikingly viscous solution and functions primarily as a joint lubricant and shock absorber. It is present in many mammalian tissues, including synovial fluid, cartilage, skin and brain, and forms a major part of the extracellular matrix. In addition to the structural role in connective tissues, many biologically important molecules are attached to hyaluronic acid, which plays an important role in various physiological processes such as cell proliferation, recognition, and locomotion. To ensure proper cellular activity, HA levels in the body are finely regulated to control HA biosynthesis and degradation. Hyaluronidase is the enzyme responsible for HA degradation in animals and bacteria.

Structure

The crystal structure of the Streptococcus pneumoniae hyaluronate lyase molecule is divided into two distinct structural domains connected by one short linker. The N–terminal α–helical domain contains the first 361 residues (Lys171–Ser531) of the enzyme, and is predominantly composed of 13 α–helices. Ten α–helices (αH3–αH12) are arranged into a twisted (α/α)–barrel structure. The barrel is incomplete, leaving one side open. Every two adjacent helices of these ten form a basic hairpin structure. Five hairpins compose an (α5/α5)–barrel structure. Helices αH2 and αH13 are almost perpendicular to the barrel axis and block the barrel opening. The C-terminal β-domain contains the following 347 residues (Tyr543–Lys889), 24 β-strands are arranged into five β–sheets. All β–sheets are antiparallel in the structure and the five sheets are parallel to each other. Both the two domains are spherical and are of approximately the same size.

Figure 1. Streptococcus pneumoniae hyaluronate lyase complex with the tetrasaccharide hyaluronan substrate. (Jedrzejas, M.J. 2002)

Mechanism

The degradation of hyaluronic acid is thought to be processive with the random initial endolytic enzyme binding to hyaluronan followed by exolytic degradation of the same substrate chain toward its non-reducing end until the whole substrate was degraded. For the pneumococcal hyaluronate lyase, and for several other known bacterial hyaluronate lyases, the final degradation product is an unsaturated disaccharide derivative of hyaluronic acid. The five-step catalytic process responsible for hyaluronic acid degradation was proposed to consist from the following: (i) a hyaluronan substrate binding step, (ii) a catalytic step, (iii) hydrogen exchange with the water microenvironment, (iv) an irreversible product release step, and (v) a translocation of the remaining polymeric substrate step.

In order for the hyaluronan degradation process to be feasible, the enzyme needs to bind to the substrate utilizing its elongated cleft. After the initial binding or docking of the substrate in the cleft, hyaluronan is precisely positioned along the cleft interacting predominantly with charged residues lining the cleft surface. Based on the structure of the native enzyme and its modeled complex structure with the tetra- and hexasaccharide units of HA, it is proposed that the catalytic residues are Asn349, His399, and Tyr408. In the catalytic process, the enzyme molecule loses one hydrogen from Tyr408 and gains another one at His399. As the final step, the hydrogen balance needs to be restored for the enzyme to return to its original state and be ready for the next round of catalysis. For the next round of catalysis to take place, the enzyme also needs to release the generated disaccharide product from the active site.

References

1. Li S, Kelly SJ, et al. Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. EMBO J. 2000;19(6):1228-40.
2. Jedrzejas, M.J. Mechanism of Hyaluronan Degradation by Streptococcus pneumoniae Hyaluronate Lyase. STRUCTURES OF COMPLEXES WITH THE SUBSTRATE. Journal of Biological Chemistry, 2002, 277(31):28287-28297.
3. Allen A G, Lindsay H, et al. Identification and characterisation of hyaluronate lyase from Streptococcus suis. Microbial Pathogenesis, 2004, 36(6):327-335.