Location of the pteroylpolyglutamate-binding site on rabbit cytosolic serine hydroxymethyltransferase.
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.
Fu
Tzu-Fun
TF
Department of Biochemistry and the Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond 23219-1570, USA.
Scarsdale
J Neel
JN
Kazanina
Galina
G
Schirch
Verne
V
Wright
H Tonie
HT
D000818:Animals; D001665:Binding Sites; D002412:Cations; D018360:Crystallography, X-Ray; D003600:Cytosol / Q000201:enzymology*; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011624:Pteroylpolyglutamic Acids / Q000737:chemistry*; D011817:Rabbits
D002412:Cations; D008024:Ligands; D011624:Pteroylpolyglutamic Acids; D005973:Glutamine; D018698:Glutamic Acid; D012696:Glycine Hydroxymethyltransferase; D008239:Lysine