Linked Life Data

12578366

Source:http://linkedlifedata.com/resource/pubmed/id/12578366

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
2003-2-11
pubmed:abstractText
Folylpolyglutamate synthetase (FPGS) catalyzes the addition of glutamate to folate derivatives to form folate polyglutamates. FPGS is essential for folate biosynthesis in bacteria and retention of folate pools in eukaryotes. X-ray crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS suggest that binding of folate triggers a conformational change that activates FPGS. We used EPR and CD spectroscopy to further characterize the conformational change in the FPGS reaction. For EPR spectroscopy, two cysteine residues were introduced into FPGS by site-directed mutagenesis, K172C in the N-terminal domain and D345C in the C-terminal domain. The mutant protein was expressed, purified, and labeled with methanethiosulfonate. Addition of ATP, tetrahydrofolate, or 5,10-methylenetetrahydrofolate but not glutamate to FPGS showed broadening of EPR spectra, which is due to stronger spin-spin interactions, suggesting that both ATP and tetrahydrofolates cause a conformational change. ATP binding had an EPR spectrum distinct from that of tetrahydrofolate binding, indicating that it caused a different conformational change. When both ATP and THF were bound, the spectrum was identical to that seen when THF alone bound to the enzyme, showing that the THF-induced conformation was dominant. The spectral broadening suggests that the conformation change involves the two domains moving closer together, which is consistent with the rigid-body rotation of the C-terminal domain observed in the FPGS crystal structure with AMPPCP and 5,10-methylenetetrahydrofolate bound. No changes in the CD spectra were observed with the addition of FPGS substrates, suggesting that the conformational changes did not affect the secondary structure elements of the enzyme. These studies confirm the conformational change seen in the crystal structure by an independent method but also show that ATP binds to the free enzyme and affects its conformation.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/(1-oxyl-2,2,5,5-tetramethylpyrroline..., http://linkedlifedata.com/resource/pubmed/chemical/5,6,7,8-tetrahydrofolic acid, http://linkedlifedata.com/resource/pubmed/chemical/Adenosine Triphosphate, http://linkedlifedata.com/resource/pubmed/chemical/Aspartic Acid, http://linkedlifedata.com/resource/pubmed/chemical/Cyclic N-Oxides, http://linkedlifedata.com/resource/pubmed/chemical/Cysteine, http://linkedlifedata.com/resource/pubmed/chemical/Lysine, http://linkedlifedata.com/resource/pubmed/chemical/Mesylates, http://linkedlifedata.com/resource/pubmed/chemical/Peptide Synthases, http://linkedlifedata.com/resource/pubmed/chemical/Solutions, http://linkedlifedata.com/resource/pubmed/chemical/Spin Labels, http://linkedlifedata.com/resource/pubmed/chemical/Tetrahydrofolates, http://linkedlifedata.com/resource/pubmed/chemical/folylpolyglutamate synthetase
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
18
pubmed:volume
42
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1537-43
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Binding of ATP as well as tetrahydrofolate induces conformational changes in Lactobacillus casei folylpolyglutamate synthetase in solution.
pubmed:affiliation
Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't