A prototypical cytidylyltransferase: CTP:glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis

Weber, Christian; Park, Young-Seo; Sanker, Subramaniam; Kent, Claudia; Ludwig, Martha

doi:10.1016/s0969-2126(99)80178-6

Cited by 95 publications

(126 citation statements)

References 61 publications

Supporting

Mentioning

121

Contrasting

Order By: Relevance

“…Domain C is an ␣/␤ protein fold composed of five ␤-strands (␤1-␤5), and six ␣-helices (␣A, ␣B, ␣C, ␣D, ␣L, and ␣E). This nomenclature strives to be compatible with the nomenclature of structurally analogous helices and strands in the previously solved structures of glycerolphosphate CT (GCT) from B. subtilis (15,16) and Staphylococcus aureus (40). The ␣L-helix in CCT236 replaces the 3 10 segments in GCT.…”

Section: Resultsmentioning

confidence: 99%

“…The 140 RYVD 143 motif, located in loop L3 following helix C is a key contact point in the dimer interface. This motif is a signature sequence of the CT family (15) and likely serves the role of dimer stabilization in all CT folds. The two monomer chains come within 3.6 Å at the C␣ of Arg-140.…”

Section: Resultsmentioning

confidence: 99%

“…Crystal structures of GCT with bound CTP (PDB code 1COZ) (15) or with bound CDP-glycerol (PDB code 1N1D) (16) reveal a dimer of an ␣/␤ nucleotide binding fold composed of a twisted fivestranded parallel ␤-sheet flanked by five helices. The ligands are found in a pocket at the base of the ␤-sheet, in contact with the conserved HXGH and RTEGISTT motifs that are signatures of the GCT family of nucleotidyltransferases (15). This family includes ethanolamine-phosphate cytidylyltransferases (ECT), phosphopantetheine adenylyltransferase (PPAT), and the class I aminoacyl tRNA synthetases (aa-tRS) (10,(15)(16)(17).…”

mentioning

confidence: 99%

“…The ligands are found in a pocket at the base of the ␤-sheet, in contact with the conserved HXGH and RTEGISTT motifs that are signatures of the GCT family of nucleotidyltransferases (15). This family includes ethanolamine-phosphate cytidylyltransferases (ECT), phosphopantetheine adenylyltransferase (PPAT), and the class I aminoacyl tRNA synthetases (aa-tRS) (10,(15)(16)(17). Multiple solved structures of PPAT and aa-tRS enzymes have been analyzed, and one as yet unanalyzed structure of human ECT was recently deposited in the Protein Data Bank (code 3ELB).…”

mentioning

confidence: 99%

See 3 more Smart Citations

Crystal Structure of a Mammalian CTP: Phosphocholine Cytidylyltransferase Catalytic Domain Reveals Novel Active Site Residues within a Highly Conserved Nucleotidyltransferase Fold

Lee

Johnson

Ding

et al. 2009

Journal of Biological Chemistry

104

View full text Add to dashboard Cite

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 CCT␣ spanning residues 1-236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-Å 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 ␤-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.A key rate-limiting step in the synthesis of phosphatidylcholine in animal cells is the formation of the headgroup donor, CDP-choline, by transfer of a cytidylyl group from CTP to phosphocholine. This reaction is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT 4 ; EC 2.7.7.15), an enzyme subject to many layers of regulation (1-4). The ubiquitous and best studied isoform of mammalian CCT (CCT␣, 367 residues) has been described as having four domains (Fig. 1A). An N-terminal domain (ϳ75 residues) housing its nuclear localization signal (NLS) sequence is followed by an ϳ150-residue catalytic domain, an ϳ60-residue membrane binding domain (domain M), and an unstructured phosphorylated tail (ϳ50 residues) (2, 4). CCT functions as a homodimer (5).CCT activation requires transformation of the enzyme from a soluble form to a membrane lipid-bound form. When the full-length soluble CCT interacts with anionic membrane surfaces, domain M transforms from a mixture of structural elements into an amphipathic ␣-helix (6 -8). Domain M appears to act as an autoinhibitory device, whose interaction with phosphatidylcholine-deficient membranes releases an inhibitory constraint at the active site to enhance k cat by 2 orders of magnitude (9). The primary evidence for this model is the constitutive activity of a construct lacking domain M, CCT236 (9).To elucidate the mechanism whereby membrane binding activates this important re...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Crystal Structure of a Mammalian CTP: Phosphocholine Cytidylyltransferase Catalytic Domain Reveals Novel Active Site Residues within a Highly Conserved Nucleotidyltransferase Fold

Lee

Johnson

Ding

et al. 2009

Journal of Biological Chemistry

104

View full text Add to dashboard Cite

show abstract

“…The CCT␣ amino-terminal region contains a cluster of positively charged amino acids that specify nuclear localization (57)(58)(59), whereas the CCT␤ proteins lack this signature sequence (32,36). Thus, CCT␣ is predominantly a nuclear protein in most cell types (58), with the exception of lung cells (48), and a smaller amount of extranuclear CCT␣ is found associated with the endoplasmic reticulum (35).…”

mentioning

confidence: 99%

Disruption of CCTβ2 Expression Leads to Gonadal Dysfunction

Jackowski

Rehg

Zhang³

et al. 2004

Molecular and Cellular Biology

View full text Add to dashboard Cite

There are two mammalian genes that encode isoforms of CTP:phosphocholine cytidylyltransferase (CCT), a key rate-controlling step in membrane phospholipid biogenesis. Quantitative determination of the CCT transcripts reveals that CCT␣ is ubiquitously expressed and is found at the highest levels in the testis and lung, with lower levels in the liver and ovary. CCT␤2 is a very minor isoform in most tissues but is significantly expressed in the brain, lung, and gonads. CCT␤3 is the third isoform recently discovered in mice and is expressed in the same tissues as CCT␤2, with its highest level in testes. We investigated the role(s) of CCT␤2 by generating knockout mice. The brains and lungs of mice lacking CCT␤2 expression did not exhibit any overt defects. On the other hand, a large percentage of the CCT␤2 ؊/؊ females were sterile and their ovaries exhibited defective ovarian follicle development. The proportion of female CCT␤2 ؊/؊ mice with defective ovaries increased as the animals aged. The rare litters born from CCT␤2 ؊/؊ ؋ CCT␤2 ؊/0 matings had the normal number of pups. The abnormal ovarian histopathology was characterized by disorganization of the tissue in young adult mice and absence of follicles and ova in older mice, along with interstitial stromal cell hyperplasia which culminated in the emergence of tubulostromal ovarian tumors by 16 months of age. Grossly defective CCT␤2 ؊/؊ ovaries were associated with high follicle-stimulating (FSH) and luteinizing (LH) hormone levels. Male CCT␤2 ؊/0 mice exhibited progressive multifocal testicular degeneration and reduced fertility but had normal FSH and LH levels. Thus, the most notable phenotype of CCT␤2 knockout mice was gonad degeneration and reproductive deficiency. The results indicate that although CCT␤2 is expressed at very low levels compared to the ␣-isoform, loss of CCT␤2 expression causes a breakdown in the gonadal response to hormonal stimulation.Phosphatidylcholine (PtdCho) is a major component of biological membranes in higher eukaryotes and is also secreted by specialized tissues for important extracellular tasks. CTP: phosphocholine cytidylyltransferase (CCT) is a key rate-controlling step in the major biosynthetic pathway leading to PtdCho in most tissues (for reviews, see references 15, 17, and 36). In mammals there are two genes, Pcyt1a (formerly Cptct), located on murine chromosome 16, and Pcyt1b, located on the X chromosome, that encode proteins termed CCT␣ and CCT␤, respectively (35, 38). The two genes exhibit tissuespecific expression, with CCT␣ predominating in most tissues and CCT␤ being most abundant in brain tissue (32). Two transcripts arise from the Pcyt1a gene that encode the identical CCT␣ protein. Alternate splicing of the X-linked Pcyt1b gene directs the synthesis of two mRNAs that encode the CCT␤2 and CCT␤3 isoforms in mice (32,35,38). CCT␤3 is 28 residues shorter at the amino terminus than CCT␤2 due to transcript initiation at an alternate first exon (32). In humans, intron retention gives rise to a CCT␤1 transcript found in the exp...

show abstract