Conformational flexibility of PL12 family heparinases: structure and substrate specificity of heparinase III from Bacteroides thetaiotaomicron (BT4657).
Glycosaminoglycans (GAGs) are linear polysaccharides comprised of disaccharide repeat units, a hexuronic acid, glucuronic acid or iduronic acid, linked to a hexosamine, N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine. GAGs undergo further modification such as epimerization and sulfation. These polysaccharides are abundant in the extracellular matrix and connective tissues. GAGs function in stabilization of the fibrillar extracellular matrix, control of hydration, regulation of tissue, organism development by controlling cell cycle, cell behavior and differentiation. Niche adapted bacteria express enzymes called polysaccharide lyases (PL), which degrade GAGs for their nutrient content. PL have been classified into 24 sequence-related families. Comparison of 3D structures of the prototypic members of these families allowed identification of distant evolutionary relationships between lyases that were unrecognized at the sequence level, and identified occurrences of convergent evolution. We have characterized structurally and enzymatically heparinase III from Bacteroides thetaiotaomicron (BtHepIII; gene BT4657), which is classified within the PL12 family. BtHepIII is a 72.5 kDa protein. We present the X-ray structures of two crystal forms of BtHepIII at resolution 1.8 and 2.4 Å. BtHepIII contains two domains, the N-terminal α-helical domain forming a toroid and the C-terminal β-sheet domain. Comparison with recently determined structures of two other heparinases from the same PL12 family allowed us to identify structural flexibility in the arrangement of the domains indicating open-close movement. Based on comparison with other GAG lyases, we identified Tyr301 as the main catalytic residue and confirmed this by site-directed mutagenesis. We have characterized substrate preference of BtHepIII toward sulfate-poor heparan sulfate substrate.
Department of Biochemistry, University of Saskatchewan, Saskatoon, S7N 5E5 Saskatchewan, Canada.
ThirumalaiSelvi
T
Ulaganathan
Département de Biochimie, de Microbiologie et de Bio-informatique, PROTEO, and Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Pavillon Charles-Eugène-Marchand, Québec City, QC G1V 0A6, Canada.
Rong
R
Shi
National Center for Protein Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China and Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai 201204, China.
Deqiang
D
Yao
Department of Biological Sciences and Centre for Molecular Simulation, University of Calgary, 2500 University Dr NW, Calgary, AB T2N 1N4, Canada H4P 2R2, Canada.
Ruo-Xu
RX
Gu
the Architecture et Fonction des Macromolécules Biologiques, UMR7257 CNRS, Aix-Marseille University, F-13288 Marseille, France, the INRA, USC1408 Architecture et Fonction des Macromolécules Biologiques, F-13288 Marseille, France.
Marie-Line
ML
Garron
Department of Biochemistry, University of Saskatchewan, Saskatoon, S7N 5E5 Saskatchewan, Canada.
Maia
M
Cherney
Department of Biological Sciences and Centre for Molecular Simulation, University of Calgary, 2500 University Dr NW, Calgary, AB T2N 1N4, Canada H4P 2R2, Canada.
D Peter
DP
Tieleman
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Eric
E
Sterner
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Guoyun
G
Li
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Lingyun
L
Li
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Robert J
RJ
Linhardt
Department of Biochemistry, University of Saskatchewan, Saskatoon, S7N 5E5 Saskatchewan, Canada miroslaw.cygler@usask.ca.
Miroslaw
M
Cygler
D000070077:Bacteroides thetaiotaomicron / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D006025:Glycosaminoglycans / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase
active site; crystal structure; heparin lyase; protein dynamics; substrate specificity