title: Structure-based mechanism of CMP-2-keto-3-deoxymanno-octulonic acid synthetase: convergent evolution of a sugar-activating enzyme with DNA/RNA polymerases.
abstract: The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2beta-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2beta-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg(2+) ion (Mg-B). Both ligands occupy conformations compatible with an S(n)2-type attack on the alpha-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg(2+) ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp(100) and Asp(235) in addition to the CTP alpha-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn(2+)-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg(2+) to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the alpha-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.
authors.affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN.
authors.forename: Derren J
authors.initials: DJ
authors.lastname: Heyes
authors.forename: Colin
authors.initials: C
authors.lastname: Levy
authors.forename: Pierre
authors.initials: P
authors.lastname: Lafite
authors.forename: Ian S
authors.initials: IS
authors.lastname: Roberts
authors.forename: Marie
authors.initials: M
authors.lastname: Goldrick
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Stachulski
authors.forename: Steven B
authors.initials: SB
authors.lastname: Rossington
authors.forename: Deborah
authors.initials: D
authors.lastname: Stanford
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
authors.forename: David
authors.initials: D
authors.lastname: Leys
mesh_terms: D018360:Crystallography, X-Ray; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D019143:Evolution, Molecular; D009713:Nucleotidyltransferases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein
chemical_list: D003570:Cytidine Triphosphate; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase