Synthesis of complex carbohydrates and glyconjugates: enzymatic synthesis of globotetraose using alpha-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus infuenzae strain Rd.
The lipopolysaccharide of capsule-deficient Haemophilus infuenzae strain Rd contains an N-acetylgalactosamine residue attached to the terminal globotriose moiety in the Hex5 glycoform. Genome analysis identified an open reading frame, HI1578, referred to as LgtD, whose amino acid sequence shows a significant level of similarity to those of a number of bacterial glycosyltransferases involved in lipopolysaccharide biosynthesis. To investigate its function, overexpression and biochemical characterization were performed. Most of the protein was obtained in a highly soluble and active form. Standard glycosyltransferase assay, high-performance liquid chromatography (HPLC), and liquid chromatography (LC)/mass spectrometry (MS) show that LgtD is an N-acetylgalactosaminyltransferase with high donor substrate specificity, and globotriose is a highly preferred acceptor substrate for the enzyme.
Ryu
Kang
K
Department of Biochemistry, The Ohio State University, Columbus, USA.
Lin
Steven
S
Shao
Jun
J
Song
Jing
J
Chen
Min
M
Wang
Wei
W
Li
Hanfen
H
Yi
Wen
W
Wang
Peng George
PG
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D005915:Globosides / Q000138:chemical synthesis*; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D005915:Globosides; D008070:Lipopolysaccharides; C035968:globotetraose; D017350:N-Acetylgalactosaminyltransferases