title: Biosynthesis of colitose: expression, purification, and mechanistic characterization of GDP-4-keto-6-deoxy-D-mannose-3-dehydrase (ColD) and GDP-L-colitose synthase (ColC).
abstract: L-Colitose is a 3,6-dideoxyhexose found in the O-antigen of Gram-negative lipopolysaccharides. To study the biosynthesis of this unusual sugar, we have cloned and sequenced the L-colitose biosynthetic gene cluster from Yersinia pseudotuberculosis VI. The colD and colC genes in this cluster have been overexpressed and each gene product has been purified and characterized. Our results showed that ColD functions as GDP-4-keto-6-deoxy-D-mannose-3-dehydrase responsible for C-3 deoxygenation of GDP-4-keto-6-deoxy-D-mannose. This enzyme is coenzyme B(6)-dependent and its catalysis is initiated by a transamination step in which pyridoxal 5'-phosphate (PLP) is converted to pyridoxamine 5'-phosphate (PMP) in the presene of L-glutamate. This coenzyme forms a Schiff base with the keto sugar substrate and the resulting adduct undergoes a PMP-mediated beta-dehydration reaction to give a sugar enamine intermediate, which after tautomerization and hydrolysis to release ammonia yields GDP-4-keto-3,6-dideoxy-D-mannose as the product. The combined transamination-deoxygenation activity places ColD in a class by itself. Our studies also established ColC as GDP-L-colitose synthase, which is a bifunctional enzyme catalyzing the C-5 epimerization of GDP-4-keto-3,6-dideoxy-D-mannose and the subsequent C-4 keto reduction of the resulting L-epimer to give GDP-L-colitose. Reported herein are the detailed accounts of the overexpression, purification, and characterization of ColD and ColC. Our studies show that their modes of action in the biosynthesis of GDP-L-colitose represent a new deoxygenation paradigm in deoxysugar biosynthesis.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
authors.forename: Jenefer
authors.initials: J
authors.lastname: Alam
authors.forename: Noelle
authors.initials: N
authors.lastname: Beyer
authors.forename: Hung-wen
authors.initials: HW
authors.lastname: Liu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003837:Deoxy Sugars / Q000096:biosynthesis*; D003903:Deuterium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006156:Guanosine Diphosphate Sugars / Q000378:metabolism*; D006602:Hexosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D015011:Yersinia pseudotuberculosis / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003837:Deoxy Sugars; D006156:Guanosine Diphosphate Sugars; C096550:guanosine diphosphate-colitose; D009243:NAD; C025141:colitose; D006859:Hydrogen; D003903:Deuterium; D007658:Ketone Oxidoreductases; C449658:GDP-4-keto-6-deoxy-D-mannose reductase; C495814:GDP-L-colitose synthase, Yersinia pseudotuberculosis; D006602:Hexosyltransferases