title: Biochemical characterization of a novel bifunctional glycosyl-1-phosphate transferase involved in the exopolysaccharide biosynthesis.
abstract: Ebosin produced by Streptomyces sp. 139 is a novel exopolysaccharide with anti-rheumatic arthritis activity in vivo and its biosynthesis gene cluster (ste) has been previously identified. In our previous research, ste5 gene has been identified as priming glycosyltransferase involved in Ebosin biosynthesis. However, it remains unclear how ste5 initiated Ebosin biosynthesis in molecular level. Here we show that Ebosin derivative produced by ste5 mutant lost the antagonist activities for IL-1R and Overexpression of ste5 in mutant dramatically enhanced the antagonist activities for IL-1R. For biochemical characterization of Ste5, the ste5 gene was cloned and expressed in Escherichia coli BL21. We identified that the recombinant Ste5 can transfer galactose-1-Phosphate (Gal-1-P) or glucose-1-Phosphate (Glc-1-P) from UDP-galactose and UDP-glucose to the lipid carrier located in the cytoplasmic membrane of Streptomyces sp. 139 (ste5(-)) with a continuous coupled spectrophotometric assay. 12.6 μM of Km was for UDP-galactose and 23.9 μM for UDP-glucose respectively. Our results indicate that Ste5 is bifunctional Gal-1-P and Glc-1-P transferase to initiate Ebosin biosynthesis and may be further applied in remoulding carbohydrate compounds.
authors.affiliation: Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, 100050, Tian Tan Xi Li No. 1, Beijing, China.
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Qi
authors.affiliation: Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, 100050, Tian Tan Xi Li No. 1, Beijing, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Ma
authors.affiliation: Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, 100050, Tian Tan Xi Li No. 1, Beijing, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, 100050, Tian Tan Xi Li No. 1, Beijing, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, 100050, Tian Tan Xi Li No. 1, Beijing, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, 100050, Tian Tan Xi Li No. 1, Beijing, China. Electronic address: lipingbai1973@163.com.
authors.forename: Liping
authors.initials: L
authors.lastname: Bai
authors.affiliation: Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, 100050, Tian Tan Xi Li No. 1, Beijing, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005695:Galactosephosphates / Q000378:metabolism; D015870:Gene Expression; D005958:Glucosephosphates / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005695:Galactosephosphates; D005958:Glucosephosphates; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; C029973:galactose-1-phosphate; D014531:Uridine Diphosphate Galactose; C031590:glucose-1-phosphate; D016695:Glycosyltransferases; D014532:Uridine Diphosphate Glucose
keywords: Ebosin biosynthesis; Glycosyl-1-phosphate transferase; Ste5; Streptomyces