Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability.
Sialic acids participate in many important biological recognition events, yet eukaryotic sialic acid biosynthetic genes are not well characterized. In this study, we have identified a novel human gene based on homology to the Escherichia coli sialic acid synthase gene (neuB). The human gene is ubiquitously expressed and encodes a 40-kDa enzyme. The gene partially restores sialic acid synthase activity in a neuB-negative mutant of E. coli and results in N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) production in insect cells upon recombinant baculovirus infection. In vitro the human enzyme uses N-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates to generate phosphorylated forms of Neu5Ac and KDN, respectively, but exhibits much higher activity toward the Neu5Ac phosphate product.
Lawrence
S M
SM
Departments of Chemical Engineering and Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Huddleston
K A
KA
Pitts
L R
LR
Nguyen
N
N
Lee
Y C
YC
Vann
W F
WF
Coleman
T A
TA
Betenbaugh
M J
MJ
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013400:Sugar Acids / Q000378:metabolism*
D011994:Recombinant Proteins; D013400:Sugar Acids; C050008:3-deoxyglycero-galacto-nonulosonic acid; C018730:N-acetylneuraminate synthase; C018767:N-acylneuraminate-9-phosphate synthase; D007652:Oxo-Acid-Lyases