Structural and functional characterization of a 5,10‐methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087)

Abstract

Mycoplasma pneumoniae 5,10-methenyltetrahydrofolate synthetase [MTHFS; also known as 5-formyltetrahydrofolate cycloligase; Enzyme Commission (EC) 6.3.3.2] belongs to a large cycloligase protein family with 97 sequence homologues from bacteria to human. To help define the molecular (biochemical and biophysical) function of the M. pneumoniae MTHFS, we have previously determined its crystal structure at 2.2 A resolution (Chen et al., Proteins 2004;56:839-843). In this current study, activity assays confirmed the functionality of the recombinant protein, with K(m) = 165 microM for 5-formyltetrahydrofolate (5-FTHF) and K(m) = 166 microM for MgATP. The methenyltetrahydrofolate activity of M. pneumoniae MTHFS has a requirement for divalent metal ions with Mg2+ being most effective, and an absolute requirement for nucleoside 5'-triphosphates with adenosine triphosphate (ATP) being most effective. Crystallization in the presence of substrates (MgATP, with or without 5-FTHF) produced the complex structures of the protein with adenosine diphosphate (ADP) and phosphate at 2.2 A resolution; with ADP, phosphate, and 5-FTHF at 2.5 A resolution. These structures directly demonstrated that the role of Mg2+ in the reaction is to form the ATP--Mg2+-enzyme complex.

Structure of 5-formyltetrahydrofolate cyclo-ligase fromBacillus anthracis(BA4489)

“…Based on NMR-labelling studies, a catalytic mechanism has been proposed in which 5-formyl-THF becomes transiently phosphorylated by ATP to form a phospho-enol intermediate (Chen et al, 2005; Fig. 3c), which is entirely consistent with our observations.…”

“…It has been suggested that 5-formyl-THF cyclo-ligase forms dimers in solution and that this oligomeric state is related to the cooperative binding of its substrate 5-formyl-THF (Chen et al, 2005). While the structure presented here is, in principle, consistent with a dimeric protein, an analysis (using PISA; Krissinel & Henrick, 2005) suggests that no biologically significant interfaces are present in our structure.…”

“…All the potential interface regions in the crystal consist exclusively of hydrogen bonds and salt bridges and the largest possible interface area comprises only 500 Å 2 ($5% of the total surface of the protein monomer), which is significantly less than for most biological dimers (Lesk, 2004). However, it is conceivable that dimerization occurs transiently in solution, especially at higher concentrations (Chen et al, 2005).…”

Regulation of de Novo Purine Biosynthesis by Methenyltetrahydrofolate Synthetase in Neuroblastoma

“…Homology Model Construction-A model of the mouse MTHFS protein was constructed using the crystal structure of Mycoplasma pneumoniae MTHFS with ADP, phosphate, and 5-formylTHF bound (PDB code 1U3G; Fig. 3) (25,26). A BLAST search of the Swiss-Prot data base (27), using the SIB BLAST Network Service (28), revealed a number of sequences related to mouse MTHFS.…”

Structural Basis for the Inhibition of Human 5,10-Methenyltetrahydrofolate Synthetase by N10-Substituted Folate Analogues

“…To date, relatively little is known about the structural basis for the mechanism of catalysis. The crystal structures of a ternary complex of MTHFS from Mycoplasma pneumoniae (mpMTHFS) with ADP and 5-formyltetrahydrofolate (20) and a binary complex of MTHFS from Bacillus anthracis (bMTHFS) with ADP (21) have been reported. Although the ATP has been depicted as hydrolyzed and modeled as ADP and inorganic phosphate in the mpMTHFS structure, the substrate 5-formyltetrahydrofolate, rather than the product 5,10-methenyltetrahydrofolate, has been modeled in the active site.…”