21057010
not annotated - annotated - LINNAEUS only
Biochemical characterization of UDP-Gal:GlcNAc-pyrophosphate-lipid Beta-1,4-Galactosyltransferase WfeD, a new enzyme from Shigella boydii type 14 that catalyzes the second step in O-antigen repeating-unit synthesis.
The O antigen is the outer part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria and contains many repeats of an oligosaccharide unit. It contributes to antigenic variability and is essential to the full function and virulence of bacteria. Shigella is a Gram-negative human pathogen that causes diarrhea in humans. The O antigen of Shigella boydii type 14 consists of repeating oligosaccharide units with the structure [->6-d-Galpalpha1->4-d-GlcpABeta1->6-d-GalpBeta1->4-d-GalpBeta1->4-d-GlcpNAcBeta1->]n. The wfeD gene in the O-antigen gene cluster of Shigella boydii type 14 was proposed to encode a galactosyltransferase (GalT) involved in O-antigen synthesis. We confirmed here that the wfeD gene product is a Beta4-GalT that synthesizes the GalBeta1-4GlcNAcalpha-R linkage. WfeD was expressed in Escherichia coli, and the activity was characterized by using UDP-[^3H]Gal as the donor substrate as well as the synthetic acceptor substrate GlcNAcalpha-pyrophosphate-(CH2)11-O-phenyl. The enzyme product was analyzed by liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and galactosidase digestion. The enzyme was shown to be specific for the UDP-Gal donor substrate and required pyrophosphate in the acceptor substrate. Divalent metal ions such as Mn^2(+), Ni^2(+), and, surprisingly, also Pb^2(+) enhanced the enzyme activity. Mutational analysis showed that the Glu101 residue within a DxD motif is essential for activity, possibly by forming the catalytic nucleophile. The Lys211 residue was also shown to be required for activity and may be involved in the binding of the negatively charged acceptor substrate. Our study revealed that the Beta4-GalT WfeD is a novel enzyme that has virtually no sequence similarity to mammalian Beta4-GalT, although it catalyzes a similar reaction.
Ann file
T1 Species 122 137 Shigella boydii
N1 Reference T1 Taxonomy:621
T2 Species 517 522 human
N2 Reference T2 Taxonomy:9606
T3 Species 556 562 humans
N3 Reference T3 Taxonomy:9606
T4 Species 581 596 Shigella boydii
N4 Reference T4 Taxonomy:621
T5 Species 802 817 Shigella boydii
N5 Reference T5 Taxonomy:621
T6 Species 1050 1066 Escherichia coli
N6 Reference T6 Taxonomy:562