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Structure-based mechanism of CMP-Kdo synthetase: convergent evolution of a sugar activating enzyme with DNA-/RNA-polymerases

DOI: 10.1074/jbc.M109.056630 DOI Help
PMID: 19815542 PMID Help

Authors: Derren Heyes (University of Manchester) , Ian Roberts (University of Manchester) , Marie Goldrick (University of Manchester) , Andrew V. Stachulski (University of Liverpool) , Steven Rossington (University of Manchester) , Deborah Stanford (University of Liverpool) , Stephen Rigby (University of Manchester) , Nigel Scrutton (University of Manchester) , Pierre Lafite (University of Manchester) , Colin Levy (Manchester Interdisciplinary Biocentre, The University of Manchester) , David Leys (Manchester Interdisciplinary Biocentre, The University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: October 2009

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 2?-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2?-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg2+ ion (Mg-B). Both ligands occupy conformations compatible with an Sn2-type attack on the ?-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 Mg2+ 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 Asp100 and Asp235 in addition to the CTP ?-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn2+-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 Mg2+ to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the ?-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.

Journal Keywords: X-Ray; Cytidine; DNA-Directed; DNA-Directed; Escherichia; Evolution; Molecular; Nucleotidyltransferases; Protein; Tertiary; Structural; Protein

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography