The structure of CMP:2-keto-3-deoxy-manno-octonic acid synthetase and of its complexes with substrates and substrate analogs.
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.
Jelakovic
S
S
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstr. 21, Freiburg im Breisgau, Germany, 79104.
Schulz
G E
GE
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003565:Cytidine Diphosphate / Q000737:chemistry / Q000378:metabolism; D003568:Cytidine Monophosphate / Q000737:chemistry / Q000378:metabolism; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid
D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D003565:Cytidine Diphosphate; D003570:Cytidine Triphosphate; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase; D000216:N-Acylneuraminate Cytidylyltransferase; D003568:Cytidine Monophosphate