RESUMO
The enzyme CMP-Kdo synthetase (CKS) catalyzes the activation of the sugar Kdo (2-keto-3-deoxy-manno-octonic acid) by forming a monophosphate diester. CKS is a pharmaceutical target because CMP-Kdo is used in the biosynthesis of lipopolysaccharides that are vital for Gram-negative bacteria. We have refined the structure of the unligated capsule-specific CKS from Escherichia coli at 1.8 A resolution (1 A=0.1 nm) and we have established the structures of its complexes with the substrate CTP, with CDP and CMP as well as with the product analog CMP-NeuAc (CMP-sialate) by X-ray diffraction analyses at resolutions between 2.1 A and 2.5 A. The N-terminal domains of the dimeric enzyme bind CTP in a peculiar nucleotide-binding fold, whereas the C-terminal domains form the dimer interface. The observed binding geometries together with the amino acid variabilities during evolution and the locations of a putative Mg(2+) and of a very strongly bound water molecule suggest a pathway for the catalysis. The N-terminal domain shows sequence homology with the CMP-NeuAc synthetases. Moreover, the chain fold and the substrate-binding position of CKS resemble those of other enzymes processing nucleotide-sugars.
Assuntos
Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Cistina Difosfato/química , Cistina Difosfato/metabolismo , Monofosfato de Citidina/química , Monofosfato de Citidina/metabolismo , Ácido N-Acetilneuramínico do Monofosfato de Citidina/química , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , Dimerização , Escherichia coli/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , N-Acilneuraminato Citidililtransferase/química , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de AminoácidosRESUMO
CMP-Kdo synthetases from Gram-negative bacteria activate Kdo for incorporation into lipo- and capsule-polysaccharides. Here we report the crystal structure of the capsule-specific 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 beta-sheet with surrounding helices. The chain fold is novel; it is remotely related to a double Rossmann fold. A large pocket at the carboxyl terminal ends of the central. beta-strands most likely accommodates the catalytic center. A putative phosphate binding site at the loop between the first beta-strand and the following helix is indicated by a bound iridium hexachloride anion.
