Abstract
Serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) catalyzes the reversible interconversion of serine and glycine with transfer of the serine side chain one-carbon group to tetrahydropteroylglutamate (H(4)PteGlu), and also the conversion of 5,10-methenyl-H(4)PteGlu to 5-formyl-H(4)PteGlu. In the cell, H(4)PteGlu carries a poly-gamma-glutamyl tail of at least 3 glutamyl residues that is required for physiological activity. This study combines solution binding and mutagenesis studies with crystallographic structure determination to identify the extended binding site for tetrahydropteroylpolyglutamate on rabbit cytosolic SHMT. Equilibrium binding and kinetic measurements of H(4)PteGlu(3) and H(4)PteGlu(5) with wild-type and Lys --> Gln or Glu site mutant homotetrameric rabbit cytosolic SHMTs identified lysine residues that contribute to the binding of the polyglutamate tail. The crystal structure of the enzyme in complex with 5-formyl-H(4)PteGlu(3) confirms the solution data and indicates that the conformation of the pteridine ring and its interactions with the enzyme differ slightly from those observed in complexes of the monoglutamate cofactor. The polyglutamate chain, which does not contribute to catalysis, exists in multiple conformations in each of the two occupied binding sites and appears to be bound by the electrostatic field created by the cationic residues, with only limited interactions with specific individual residues.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The pteridine ring of 5-CHO-H4PteGlu is reported to be in a half-chair conformation with the C-6 –C-9 bond axial to the formyltetrahydropyrazine ring [52, 53], whereas H4PteGlu exists as a roughly equal mixture of two half-chair conformations with C-6 –C-9 either axial or equatorial [54]
The data reported in two NMR studies [52, 53] on the conformation of 5-CHO-H4PteGlu in solution are not in complete agreement and are consistent with more than one conformation of the pteridine ring. Such polymorphism is suggested by the pattern of broken density in the formyltetrahydropyrazine ring in both subunits B and D of the soaked rcSHMT1⁄75-CHOH4PteGlu3 complex structure described here
Summary
H4PteGlu, tetrahydropteroylglutamate; H4PteGlun, tetrahydropteroylglutamate with n glutamyl residues; SHMT, serine hydroxymethyltransferase; bsSHMT, B. stearothermophilus serine hydroxymethyltransferase; rcSHMT, rabbit cytosolic serine hydroxymethyltransferase; eSHMT, E. coli serine hydroxymethyltransferase; mocSHMT, mouse cytosolic serine hydroxymethyltransferase; 5-CHO-H4PteGlu, 5-formyltetrahydropteroylglutamate; 5-CHOH4PteGlun, 5-formyltetrahydropteroylglutamate with n glutamyl residues; MES, 2-(N-morpholino)ethanesulfonic acid; PABA, p-aminobenzoic acid; PLP, pyridoxal phosphate; NCS, non-crystallographic symmetry. We have determined the crystal structure at 2.7-Å resolution of a complex of rcSHMT with active site-bound 5-CHO-H4PteGlu obtained by soaking unliganded crystals of rcSHMT in 5-CHO-H4PteGlu3 This is the first crystal structure of a natural polyglutamylated folate bound to an enzyme and is formed under conditions of low ionic strength. It provides new information on the nature of the interaction of this cofactor with proteins and, together with the solution data, provides a consistent picture for the location and properties of the H4PteGlun-binding site of SHMT
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