title: Heparosan-glucuronate 5-epimerase: Molecular cloning and characterization of a novel enzyme.
abstract: Iduronic acid (IdoA) is a critical component of heparan sulfate in its interaction with functional proteins. Heparosan-N-sulfate-glucuronate 5-epimerase (HNSG-5epi) converts d-glucuronic acid (GlcA) residues in N-sulfated heparosan (NS-heparosan), as an intermediate in heparan sulfate biosynthesis, to IdoA. In the present study, the authors discovered a different 5-epimerase, designated HG-5epi (heparosan-glucuronate 5-epimerase), that is involved in acharan sulfate biosynthesis and possesses novel substrate specificity. A candidate cDNA of HG-5epi was cloned from the cDNA library of Achatina fulica. The cloned cDNA contained a whole coding region that predicts a type II transmembrane protein composed of 601 amino acid residues. The amino acid sequence of HG-5epi is homologous to that of HNSG-5epi. Recombinant HG-5epi was expressed in insect cells and its enzymatic properties characterized. As expected, HG-5epi epimerizes GlcA residues in heparosan, but not in NS-heparosan. Conversion of IdoA to GlcA was also catalyzed by HG-5epi when completely desulfated N-acetylated heparin was used as the substrate, indicating a reversible reaction mechanism. At equilibrium of the epimerization, the proportion of IdoA in the reaction product reached up to 30% of total hexuronic acid. To our knowledge, this is the first report to describe an enzyme that catalyzes the epimerization of non-sulfated heparosan. This new enzyme may be applied to the study of synthetic heparan sulfate-related polysaccharides having certain biological and pharmacological activities. In addition, a new method using anion-exchange HPLC connected to a post-column fluorescent labeling system was developed for analyzing hexuronic acid isomers.
authors: Central Research Laboratories, Seikagaku Corporation, 3-1253 Tateno, Higashiyamato, Tokyo 207-0021, Japan hideo.mochizuki@seikagaku.co.jp.
authors: Hideo
authors: H
authors: Mochizuki
authors: Central Research Laboratories, Seikagaku Corporation, 3-1253 Tateno, Higashiyamato, Tokyo 207-0021, Japan.
authors: Kiwamu
authors: K
authors: Yamagishi
authors: Central Research Laboratories, Seikagaku Corporation, 3-1253 Tateno, Higashiyamato, Tokyo 207-0021, Japan.
authors: Kiyoshi
authors: K
authors: Suzuki
authors: Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
authors: Yeong Shik
authors: YS
authors: Kim
authors: Advanced Medical Research Center, Aichi Medical University, Nagakute, Aichi 480-1195, Japan.
authors: Koji
authors: K
authors: Kimata
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D002238:Carbohydrate Epimerases; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase
keywords: 5-epimerase; Achatina fulica; acharan sulfate; heparan sulfate; iduronic acid