3K8E

Crystal structure of E. coli lipopolysaccharide specific CMP-KDO synthetase

Summary for 3K8E

• Entry DOI: 10.2210/pdb3k8e/pdb
• Related: 3K8D
• Descriptor: 3-deoxy-manno-octulosonate cytidylyltransferase (2 entities in total)
• Functional Keywords: kdsb synthetase deoxy kdo complex, lipopolysaccharide biosynthesis, magnesium, nucleotidyltransferase, transferase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm (Potential): P04951
• Total number of polymer chains: 4
• Total formula weight: 117825.51

Authors

Heyes, D.J.,Levy, C.W.,Lafite, P.,Scrutton, N.S.,Leys, D. (deposition date: 2009-10-14, release date: 2009-11-10, Last modification date: 2023-11-01)

Primary citation

Heyes, D.J.,Levy, C.,Lafite, P.,Roberts, I.S.,Goldrick, M.,Stachulski, A.V.,Rossington, S.B.,Stanford, D.,Rigby, S.E.J.,Scrutton, N.S.,Leys, D.
Structure-based mechanism of CMP-2-keto-3-deoxymanno-octulonic acid synthetase: convergent evolution of a sugar-activating enzyme with DNA/RNA polymerases
J.Biol.Chem., 284:35514-35523, 2009

PubMed 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.

PubMed: 19815542
DOI: 10.1074/jbc.M109.056630

Experimental method

X-RAY DIFFRACTION (2.51 Å)