title: 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.
abstract: 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.
authors.affiliation: Division of Nephrology, Department of Medicine, Center for Matrix Biology, Vanderbilt University Medical Center, Nashville, TN 37232-2372, USA.
authors.forename: Jijun
authors.initials: J
authors.lastname: Hao
authors.forename: Willie F
authors.initials: WF
authors.lastname: Vann
authors.forename: Stephan
authors.initials: S
authors.lastname: Hinderlich
authors.forename: Munirathinam
authors.initials: M
authors.lastname: Sundaramoorthy
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004926:Escherichia coli; D006801:Humans; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D008715:Methionine; C018767:N-acylneuraminate-9-phosphate synthase; C120800:2-keto-3-deoxy-glycero-galacto-nononic acid 9-phosphate synthetase; D007652:Oxo-Acid-Lyases; D019158:N-Acetylneuraminic Acid