Binding of ATP as Well as Tetrahydrofolate Induces Conformational Changes in Lactobacillus casei Folylpolyglutamate Synthetase in Solution

Abstract

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.

Docking Studies and Ligand Recognition in Folylpolyglutamate Synthetase

“…During the reaction, both the glutamate tail and the attacking glutamate must be present at the same time. The folate binds before the glutamate, , and its presence may bias the location of the attacking glutamate. To verify this view and find the real attacking-glutamate binding mode, we performed another docking calculation in the presence of a “tail glutamate” (a FPGS·ATP·Glu model like Figure A).…”

“…Experiments have shown that glutamate is unable to bind the free enzyme and does not cause any conformational changes, but binding folate (and ATP) results in protein domain movement. 11 This conformational change brings residues H316, S412, and Y414 closer to the active site, forming the binding site of glutamate. We found that, even with the proper protein conformation, it was still difficult to obtain the attackingglutamate binding mode.…”

“…10 Crystal structures, CD spectra, and EPR studies have shown that binding mTHF and ATP results in a conformational change of the protein. 10,11 Our studies have focused on this reactive conformation seen in the ternary crystal structure (FPGS‚mTHF‚ACP 4 ).…”

“…Three crystal structures of L. casei FPGS have been reported. , One contains its preferred substrate 5,10-methylenetetrahydrofolate (mTHF) and an adenosine-5‘-[β,γ-methylene]-tetraphosphate (ACP 4 ), which was formed by phosphorylation of the cofactor mimic adenosine-5‘-[β,γ-methylene]-triphosphate . Crystal structures, CD spectra, and EPR studies have shown that binding mTHF and ATP results in a conformational change of the protein. , Our studies have focused on this reactive conformation seen in the ternary crystal structure (FPGS·mTHF·ACP 4 ).

1 The mechanism of the ligation reaction catalyzed by FPGS.

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CofE Catalyzes the Addition of Two Glutamates to F420-0 in F420 Coenzyme Biosynthesis in Methanococcus jannaschii

“…Initial velocity, product inhibition, and competitive inhibition studies were consistent with the mechanism of MgATP binding first to the enzyme, tetrahydrofolate or other folate anologs second, and glutamate last. However, recent X-ray crystallographic data suggested that although the binding order of the first two substrates (ATP and folate) can be random for FPGS, folate may effectively be the first substrate to bind and causes a conformational change in FPGS that is essential for the initiation of catalysis (7,34,35). Whether F 420 -0 or GTP binds first to CofE is unknown; however, there may be a similar requirement for the conformational change at the active site, so that the third substrate L-glutamate can bind.…”