Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori.
Activity screening and insertional inactivation of lipopolysaccharide (LPS) biosynthetic genes in Helicobacter pylori have led to the successful characterization of two key enzymes encoded by HP0159 (JHP0147) and HP1105 (JHP1032) open reading frames (ORFs) which are members of the large and diverse carbohydrate active enzymes (CAZY) GT-8 (rfaJ) family of glycosyltransferases. Activity screening of a genomic library led to the identification of the enzyme involved in the biosynthesis of the type 2 N-acetyl-lactosamine O-chain backbone, the beta-1,3-N-acetyl-glucosaminyl transferase. In addition, the activity screening approach led to the identification and characterization of a key core biosynthetic enzyme responsible for the biosynthesis of the alpha-1,6-glucan polymer. This alpha-1,6-glucosyltransferase protein is encoded by the HP0159 ORF. Both enzymes play an integral part in the biosynthesis of LPS, and insertional inactivation leads to the production of a truncated LPS molecule on the bacterial cell surface. The LPS structures were determined by mass spectrometry and chemical analyses. The linkage specificity of each glycosyltransferase was determined by nuclear magnetic resonance (NMR) analysis of model compounds synthesized in vitro. A cryogenic probe was used to structurally characterize nanomole amounts of the product of the HP1105 (JHP1032) enzyme. In contrast to the HP0159 enzyme, which displays the GT-8-predicted retaining stereochemistry for the reaction product, HP1105 (JHP1032) is the first member of this GT-8 family to have been shown to have an inverting stereochemistry in its reaction products.
Logan
Susan M
SM
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1A OR6. susan.logan@nrc-cnrc.gc.ca
Altman
Eleonora
E
Mykytczuk
Oksana
O
Brisson
Jean-Robert
JR
Chandan
Vandana
V
Schur
Melissa J
MJ
St Michael
Frank
F
Masson
Amara
A
Leclerc
Sonia
S
Hiratsuka
Koji
K
Smirnova
Natalia
N
Li
Jianjun
J
Wu
Yuyang
Y
Wakarchuk
Warren W
WW
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D017931:DNA Primers; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019906:Nuclear Magnetic Resonance, Biomolecular; D017386:Sequence Homology, Amino Acid; D016339:Spectrometry, Mass, Fast Atom Bombardment
D017931:DNA Primers; D008070:Lipopolysaccharides; D016695:Glycosyltransferases