title: Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3'-phosphoadenosine-5'-phosphosulfate.
abstract: Heparan sulfate (HS) belongs to a major class of glycans that perform central physiological functions. Heparin is a specialized form of HS and is a clinically used anticoagulant drug. Heparin is a natural product isolated from pig intestine. There is a strong demand to replace natural heparin with a synthetic counterpart. Although a chemoenzymatic approach has been employed to prepare synthetic heparin, the scale of the synthesis is limited by the availability of sulfotransferases and the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Here, we present a novel method to produce secreted forms of sulfotransferases in the yeast cells, Kluyveromyces lactis. Five sulfotransferases including N-sulfotransferase, 2-O-sulfotransferase, 3-O-sulfotransferase 1 and 6-O-sulfotransferases 1 and 3 were expressed using this method. Unlike bacterial-expressed sulfotransferases, the yeast proteins can be directly used to modify polysaccharides without laborious purification. The yeast-expressed sulfotransferases also tend to have higher specific activity and thermostability. Furthermore, we demonstrated the possibility for the gram-scale synthesis of PAPS from adenosine 5'-triphosphate at only 1/5000th of the price purchased from a commercial source. Our results pave the way to conduct the enzymatic synthesis of heparin in large quantities.
authors.affiliation: Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Xianxuan
authors.initials: X
authors.lastname: Zhou
authors.forename: Kasemsiri
authors.initials: K
authors.lastname: Chandarajoti
authors.forename: Truong Quang
authors.initials: TQ
authors.lastname: Pham
authors.forename: Renpeng
authors.initials: R
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
mesh_terms: D002236:Carbohydrate Conformation; D015870:Gene Expression; D007716:Kluyveromyces / Q000201:enzymology*; D010724:Phosphoadenosine Phosphosulfate / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry; D015238:Sulfotransferases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; C021350:heparitin sulfotransferase