Structural and functional characterization of a 5,10-methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087).
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.
Chen
Shengfeng
S
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Yakunin
Alexander F
AF
Proudfoot
Michael
M
Kim
Rosalind
R
Kim
Sung-Hou
SH
D000244:Adenosine Diphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D002955:Leucovorin / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009177:Mycoplasma pneumoniae / Q000201:enzymology*; D010710:Phosphates / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D010710:Phosphates; D000244:Adenosine Diphosphate; D019731:Carbon-Nitrogen Ligases; C021557:5,10-methenyltetrahydrofolate synthetase; D008274:Magnesium; D002955:Leucovorin