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The activation of the sugar 2-keto-3-deoxy-manno-octonic acid (Kdo) is catalyzed by CMP-Kdo synthetase (EC 2.7.7.38) and results in a monophosphate diester with CMP. The enzyme is a pharmaceutical target because CMP-Kdo is required for the biosynthesis of lipopolysaccharides that are vital for Gram-negative bacteria. We have established the structures of an enzyme complex with the educt CTP and of a complex with the product CMP-Kdo by X-ray diffraction analyses at 100 K, both at 2.6 A resolution. The N-terminal domains of the dimeric enzyme bind CTP in a peculiar nucleotide-binding fold with the beta- and gamma-phosphates located at the so-called "PP-loop", whereas the C-terminal domains participate in Kdo binding and in the dimer interface. The unstable nucleotide-sugar CMP-Kdo was produced in a crystal and stabilized by freezing to 100 K. Its formation is accompanied by an induced fit involving mainchain displacements in the 2 A range. The observed binding conformations together with the amino acid conservation pattern during evolution and the putative location of the required Mg(2+) ion suggest a reaction pathway. The enzyme is structurally homologous to the CMP-N-acetylneuraminic acid synthetases in all parts except for the dimer interface. Moreover, the chainfold and the substrate-binding positions resemble those of other enzymes processing nucleotide sugars.

CMP-Kdo synthetases from Gram-negative bacteria activate Kdo for incorporation into lipo-and capsule-polysaccharities. Here we report the crystal structure of the capsulespecific synthetase from E. coli at 2.3 A resolution. The enzyme is a dimer of 2 x 245 amino acid residues assuming C2 symmetry. It contains a central predominantly parallel [~-sheet with surrounding helices. The chain fold is novel; it is remotely related to a double Rossmaun fold. A large pocket at the carboxyl terminal ends of the central ~-strands most likely accommodates the catalytic center. A putative phosphate binding site at the loop between the first ~strand and the following helix is indicated by a bound iridium hexachloride anion.Key words: Capsular polysaccharide; CMP-Kdo synthetase (Escherichia coli); X-ray analysis; Crystal structure; Saccharide activation which 44% are identical with L-CKS. The kinetic data of the two isozymes differ distinctly [16].The rapid spread of antibiotic resistance among bacterial strains and the fact that Kdo is absent from mammalian cells [17] make K-CKS an attractive target molecule for drug design as an anti-infective measure. Cloning of the kpsU gene from the K5 antigen gene cluster [16] permitted structural analysis of this enzyme. Here, we describe the overexpression, purification, crystallization and X-ray structure determination of K-CKS. To our knowledge this is the first actually presented structure of a sugar-activating enzyme. A short note announcing an L-CKS crystal structure has been given earlier [18]. The AMP-transferring enzyme kanamycin nucleotidyltransferase [19] and the sugar phosphate-transferring enzyme galactose-l-phosphate uridylyltransferase [20] use similar substrates, but they do not activate sugars and they differ structurally from K-CKS.

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