3k8d
From Proteopedia
Crystal structure of E. coli lipopolysaccharide specific CMP-KDO synthetase in complex with CTP and 2-deoxy-Kdo
Structural highlights
FunctionKDSB_ECOLI Activates KDO (a required 8-carbon sugar) for incorporation into bacterial lipopolysaccharide in Gram-negative bacteria (By similarity). Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe 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. Structure-based mechanism of CMP-2-keto-3-deoxymanno-octulonic acid synthetase: convergent evolution of a sugar-activating enzyme with DNA/RNA polymerases.,Heyes DJ, Levy C, Lafite P, Roberts IS, Goldrick M, Stachulski AV, Rossington SB, Stanford D, Rigby SE, Scrutton NS, Leys D J Biol Chem. 2009 Dec 18;284(51):35514-23. Epub . PMID:19815542[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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