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Biochem J 2006 Jul 1;397(1):195-201

Elimination of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activity from human N-acetylneuraminic acid 9-phosphate synthase by a single mutation.

Hao J, Vann WF, Hinderlich S, Sundaramoorthy M

Sundaramoorthy M, Vanderbilt Univ, Med Ctr, Div Nephrol, Dept Med,Ctr Matrix Biol, Nashville, TN 37232 USA Vanderbilt Univ, Med Ctr, Div Nephrol, Dept Med,Ctr Matrix Biol, Nashville, TN 37232 USA Vanderbilt Univ, Med Ctr, Dept Biochem, Nashville, TN 37232 USA US FDA, Lab Bacterial Toxins, Ctr Biol Evaluat & Res, Bethesda, MD 20892 USA Charite Univ Med Berlin, Inst Biochem & Mol Biol, D-14195 Berlin, Germany


The most commonly occurring sialic acid Neu5Ac (N-acetylneuraminic acid) and its deaminated form, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), participate in many biological functions. The human Neu5Ac-9-P (Neu5Ac 9-phosphate) synthase has the unique ability to catalyse the synthesis of not only Neu5Ac-9-P but also KDN-9-P (KDN 9-phosphate). Both reactions are catalysed by the mechanism of aldol condensation of PEP (phosphoenolpyruvate) with sugar substrates, ManNAc-6-P (N-acetylmannosamine 6-phosphate) or Man-6-P (mannose 6-phosphate). Mouse and putative rat Neu5Ac-9-P synthases, however, do not show KDN-9-P synthase activity, despite sharing high sequence identity (>95%) with the human enzyme. Here, we demonstrate that a single mutation, M42T, in human Neu5Ac-9-P synthase can abolish the KDN-9-P synthase activity completely without compromising the Neu5Ac-9-P synthase activity. Saturation mutagenesis of Met42 of the human Neu5Ac-9-P synthase showed that the substitution with all amino acids except leucine retains only the Neu5Ac-9-P synthase activity at levels comparable with the wild-type enzyme. The M42L mutant, like the wild-type enzyme, showed the additional KDN-9-P synthase activity. In the homology model of human Neu5Ac-9-P synthase, Met42 is located 22 A (1 A=0.1 nm) away from the substrate-binding site and the impact of this distant residue on the enzyme functions is discussed.

Category: Journal Article
PubMed ID: #16503877
Includes FDA Authors from Scientific Area(s): Biologics
Entry Created: 2011-10-04 Entry Last Modified: 2012-08-29