Chemoenzymatic synthesis with distinct Pasteurella heparosan synthases: monodisperse polymers and unnatural structures.
Heparosan (-GlcUA-beta1,4-GlcNAc-alpha1,4-)(n) is a member of the glycosaminoglycan polysaccharide family found in the capsule of certain pathogenic bacteria as well as the precursor for the vertebrate polymers, heparin and heparan sulfate. The two heparosan synthases from the Gram-negative bacteria Pasteurella multocida, PmHS1 and PmHS2, were efficiently expressed and purified using maltose-binding protein fusion constructs. These relatively homologous synthases displayed distinct catalytic characteristics. PmHS1, but not PmHS2, was able to produce large molecular mass (100-800 kDa) monodisperse polymers in synchronized, stoichiometrically controlled reactions in vitro. PmHS2, but not PmHS1, was able to utilize many unnatural UDP-sugar analogs (including substrates with acetamido-containing uronic acids or longer acyl chain hexosamine derivatives) in vitro. Overall these findings reveal potential differences in the active sites of these two Pasteurella enzymes. In the future, these catalysts should allow the creation of a variety of heparosan and heparinoids with utility for medical applications.
Sismey-Ragatz
Alison E
AE
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
Green
Dixy E
DE
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
Otto
Nigel J
NJ
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
Rejzek
Martin
M
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, United Kingdom.
Field
Robert A
RA
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, United Kingdom.
DeAngelis
Paul L
PL
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104. Electronic address: paul-deangelis@ouhsc.edu.
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D006025:Glycosaminoglycans / Q000138:chemical synthesis* / Q000737:chemistry; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics; D058619:Maltose-Binding Proteins; D016979:Pasteurella multocida / Q000201:enzymology* / Q000235:genetics; D011108:Polymers / Q000138:chemical synthesis* / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D014539:Uridine Diphosphate Sugars / Q000737:chemistry; D014574:Uronic Acids / Q000737:chemistry
D001426:Bacterial Proteins; D002352:Carrier Proteins; D006025:Glycosaminoglycans; D058619:Maltose-Binding Proteins; D011108:Polymers; D011993:Recombinant Fusion Proteins; D014539:Uridine Diphosphate Sugars; D014574:Uronic Acids; D016695:Glycosyltransferases; C450833:heparosan synthase