In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2.
In vertebrates and bacteria, heparosan the precursor of heparin is synthesized by glycosyltransferases via the stepwise addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. As heparin-like molecules represent a great interest in the pharmaceutical area, the cryptic Pasteurella multocida heparosan synthase PmHS2 found to catalyze heparosan synthesis using substrate analogs has been studied. In this paper, we report an efficient way to purify PmHS2 and to maintain its activity stable during 6 months storage at -80 degrees Celsius using His-tag purification and a desalting step. In the presence of 1 mM of each nucleotide sugar, purified PmHS2 synthesized polymers up to an average molecular weight of 130 kDa. With 5 mM of UDP-GlcUA and 5 mM of UDP-GlcNAc, an optimal specific activity, from 3 to 6 h of incubation, was found to be about 0.145 nmol/microg/min, and polymers up to an average of 102 kDa were synthesized in 24 h. In this study, we show that the chain length distribution of heparosan polymers can be controlled by change of the initial nucleotide sugar concentration. It was observed that low substrate concentration favors the formation of high molecular weight heparosan polymer with a low polydispersity while high substrate concentration did the opposite. Similarities in the polymerization mechanism between PmHS2, PmHS1, and PmHAS are discussed.
Chavaroche
Anaïs A E
AA
Bioprocess Engineering Group, Wageningen University and Research Centre, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
Springer
Jan
J
Kooy
Floor
F
Boeriu
Carmen
C
Eggink
Gerrit
G
D001426:Bacterial Proteins / Q000737:chemistry*; D004795:Enzyme Stability; D016695:Glycosyltransferases / Q000737:chemistry* / Q000378:metabolism; D016979:Pasteurella multocida / Q000201:enzymology*; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000737:chemistry*
D001426:Bacterial Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D014539:Uridine Diphosphate Sugars; C083732:uridine diphosphate N-acetyllactosamine; D016695:Glycosyltransferases; C450833:heparosan synthase