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Crystal structure of a mammalian CTP: phosphocholine cytidylyltransferase catalytic domain reveals novel active site residues within a highly conserved nucleotidyltransferase fold.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2009 Nov 27; Vol. 284 (48), pp. 33535-48. Date of Electronic Publication: 2009 Sep 25. - Publication Year :
- 2009
-
Abstract
- CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCTalpha spanning residues 1-236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-A structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the beta-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism.
- Subjects :
- Animals
Catalysis
Choline-Phosphate Cytidylyltransferase genetics
Choline-Phosphate Cytidylyltransferase metabolism
Crystallization
Crystallography, X-Ray
Cytidine Triphosphate chemistry
Cytidine Triphosphate metabolism
Histidine chemistry
Histidine genetics
Histidine metabolism
Kinetics
Models, Molecular
Mutation
Nucleotidyltransferases genetics
Nucleotidyltransferases metabolism
Phosphorylcholine chemistry
Phosphorylcholine metabolism
Protein Binding
Protein Multimerization
Rats
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Structure-Activity Relationship
Substrate Specificity
Tyrosine chemistry
Tyrosine genetics
Tyrosine metabolism
Catalytic Domain
Choline-Phosphate Cytidylyltransferase chemistry
Nucleotidyltransferases chemistry
Protein Structure, Tertiary
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 284
- Issue :
- 48
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 19783652
- Full Text :
- https://doi.org/10.1074/jbc.M109.053363