Glucuronyltransferase activity of KfiC from Escherichia coli strain K5 requires association of KfiA: KfiC and KfiA are essential enzymes for production of K5 polysaccharide, N-acetylheparosan.
Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA beta1-4 and GlcNAc alpha1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.
Sugiura
Nobuo
N
Institute for Molecular Science of Medicine, Aichi Medical University, Yazako, Nagakute, Aichi 480-1195, Japan. nsugiura@aichi-med-u.ac.jp
Baba
Yuichi
Y
Kawaguchi
Yoshirou
Y
Iwatani
Toru
T
Suzuki
Kiyoshi
K
Kusakabe
Takahiro
T
Yamagishi
Kiwamu
K
Kimata
Koji
K
Kakuta
Yoshimitsu
Y
Watanabe
Hideto
H
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis; D057005:Bioengineering; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D016695:Glycosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000117:Acetylglucosamine; D000942:Antigens, Bacterial; D029968:Escherichia coli Proteins; D014453:Glucuronosyltransferase; D006025:Glycosaminoglycans; D016695:Glycosyltransferases; D006497:Heparan Sulfate; D017351:N-Acetylglucosaminyltransferases; D011108:Polymers; D011994:Recombinant Proteins; C527794:N-acetylheparosan; C413756:KfiA protein, E coli; C097521:KfiC protein, E coli