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Polysaccharide Lyase Family 8
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|Polysaccharide Lyase Family PL8|
|3D Structure||(α/α)6 barrel + anti-parallel β-sheet|
|Charge neutraliser||His399 (S. pneumoniae)|
|Active site residues||Known|
|CAZy DB link|
Activities and Substrate Specificities
PL8s are active on a variety of uronic acid-containing polysaccharides including: hyaluronan (EC 184.108.40.206) [4)-β-D-Glucuronate-1,3-β-D-N-Acetyl-Glucosamine(1]n, chondroitin AC (EC 220.127.116.11) [4)-β-D-Glucuronate-1,3-β-D-N-Acetyl-GalactosamineΔ4,6S(1]n, xanthan (EC 18.104.22.168) [4)-β-D-Glucuronate-1,4-β-D-Glucuronate (1]n, and chondroitin ABC (EC 22.214.171.124) [chondroitin AC and chondroitin B (aka. dermatan sulfate: 4)-β-L-Iduronate2S-1,3-β-D-N-Acetyl-Galactosamine4S(1]n.
Kinetics and Mechanism
One of the major unresolved controversies around the PL8 catalytic mechanism is the candidate of the general base. Jedrzejas et al. proposed that in the Streptococcus pneumoniae hyaluronidase, His399 acts as the general base, Asn349 acts to neutralize the C5-carboxylate group, and Tyr408 is the proton donor [1, 2]. However, for two other PL8 family members: the Bacillus sp. GL1 xanthanase and Streptomyces coelicolor A3 hyaluronidase, it was suggested that an equivalent tyrosine residue served as the general acid and general base throughout the reaction [3, 4]. Combined quantum mechanical and molecular mechanical (QM/MM) simulations of the S. pneumoniae enzyme suggests the latter hypothesis is favored, with His399 participating in the neutralization of the C5-carboxylate group . Molecular dynamic simulations of the pneumococcal hyaluronidase with hyaluronan fragments suggest that a combination of opening/closing and twisting domain motions of the (α/α)6 barrel with respect to the anti-parallel β-sheet domain underlies processive substrate translocation .
In S. pneumoniae, mutagenesis and kinetic analysis of the HysA mutant suggested three residues were involved in catalysis Asn249, His399, Tyr408 and that two residues, Arg243 and Asn580 were responsible for substrate binding and translocation . However, there is some question over what the identity is over the general base (please see Elmabrouk or Zheng et al. for discussions [4, 5]. With respect to chondroitin AC and chondroitin ABC substrate specificity; structural comparisons of the Flavobacterium heparinum chondroitin AC lyase with the Proteus vulgaris chondroitin ABC lyase suggests that an Asp444 for an Asn differential in the Proteus vulgaris active centre provides the mechanism for enzymatic distinguishing between the two epimers [7, 8].
Three dimensional structure
The enzymatic PL8 domain is comprised by an N-terminal α-helical and C-terminal β-sheet domain, which constitute incomplete α5/α5-barrel and anti-parallel β-sheet structures, respectively. A deep cleft is located in the N-terminal α-helical domain facing the interface between the two domains that accommodates the substrate.
Three dimensional structures by activity:
Chondroitin AC lyase (EC 126.96.36.199) - F. heparinum (PDB 1CB8).
Chondroitin ABC lyase (EC 188.8.131.52) – P. vulgaris (PDB 1HN0).
Hyaluronan lyase (EC 184.108.40.206) – S. pneumoniae R6 (PDB 1OJM).
Xanthan lyase (EC 220.127.116.11) – Bacillus sp GL1 (PDB 1J0M).
- First catalytic activity
- The hydrolysis of hyaluronan was attributed to a pneumococcal hyaluronidase from Pneumococcus type II strain D39R .
- First catalytic base correctly identified
- Xanthanase Y315 from 'Bacillus sp. GL1 .
- First 3-D structure
- Chondroitin AC lyase – F. heparinum (PDB 1CB8).
- Kelly SJ, Taylor KB, Li S, and Jedrzejas MJ. (2001) Kinetic properties of Streptococcus pneumoniae hyaluronate lyase. Glycobiology. 11, 297-304. DOI:10.1093/glycob/11.4.297 |
- Li S, Kelly SJ, Lamani E, Ferraroni M, and Jedrzejas MJ. (2000) Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. EMBO J. 19, 1228-40. DOI:10.1093/emboj/19.6.1228 |
- Maruyama Y, Hashimoto W, Mikami B, and Murata K. (2005) Crystal structure of Bacillus sp. GL1 xanthan lyase complexed with a substrate: insights into the enzyme reaction mechanism. J Mol Biol. 350, 974-86. DOI:10.1016/j.jmb.2005.05.055 |
- Elmabrouk ZH, Vincent F, Zhang M, Smith NL, Turkenburg JP, Charnock SJ, Black GW, and Taylor EJ. (2011) Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations. Proteins. 79, 965-74. DOI:10.1002/prot.22938 |
- Zheng M and Xu D. (2013) Catalytic mechanism of hyaluronate lyase from Streptococcus pneumonia [corrected] : quantum mechanical/molecular mechanical and density functional theory studies. J Phys Chem B. 117, 10161-72. DOI:10.1021/jp406206s |
- Joshi HV, Jedrzejas MJ, and de Groot BL. (2009) Domain motions of hyaluronan lyase underlying processive hyaluronan translocation. Proteins. 76, 30-46. DOI:10.1002/prot.22316 |
- Féthière J, Eggimann B, and Cygler M. (1999) Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes. J Mol Biol. 288, 635-47. DOI:10.1006/jmbi.1999.2698 |
- Huang W, Lunin VV, Li Y, Suzuki S, Sugiura N, Miyazono H, and Cygler M. (2003) Crystal structure of Proteus vulgaris chondroitin sulfate ABC lyase I at 1.9A resolution. J Mol Biol. 328, 623-34.
- Ahlgren JA (1991) Purification and characterization of a pyruvated-mannose-specific xanthan lyase from heat-stable, salt-tolerant bacteria. Appl Environ Microbiol. 57, 2523-8. DOI:10113/24406 |
- RAPPORT MM, LINKER A, and MEYER K. (1951) The hydrolysis of hyaluronic acid by pneumococcal hyaluronidase. J Biol Chem. 192, 283-91.
- Sato N, Shimada M, Nakajima H, Oda H, and Kimura S. (1994) Cloning and expression in Escherichia coli of the gene encoding the Proteus vulgaris chondroitin ABC lyase. Appl Microbiol Biotechnol. 41, 39-46.
- Hashimoto W, Miki H, Tsuchiya N, Nankai H, and Murata K. (1998) Xanthan lyase of Bacillus sp. strain GL1 liberates pyruvylated mannose from xanthan side chains. Appl Environ Microbiol. 64, 3765-8.