Location of the Pteroylpolyglutamate-binding Site on Rabbit Cytosolic Serine Hydroxymethyltransferase

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-␥-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 3 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.

Crystal Structures of Cobalamin-independent Methionine Synthase Complexed with Zinc, Homocysteine, and Methyltetrahydrofolate

“…At this position, the distance between the methyl to be transferred from CH 3 -H 4 PteGlu 5 to the sulfur of Hcy is about 7 Å. As observed for AtMetE, most of the crystal structures of folaterequiring enzymes have been determined without the polyglutamate chain or having only a single glutamyl residue (23). This deficiency is not due to a hydrolysis of folate but could be inherent of the nature of the anionic polyglutamate chain.…”

“…This deficiency is not due to a hydrolysis of folate but could be inherent of the nature of the anionic polyglutamate chain. Indeed, the enzymes are often crystallized in high salt concentrations, and the anions of these salts compete for binding with the anionic polyglutamate chain (23). In addition, it is also possible that the polyglutamate chain of folate has several conformations preventing its determination from an electron density map (23).…”

Catalytic and Thermodynamic Properties of Tetrahydromethanopterin-dependent Serine Hydroxymethyltransferase from Methanococcus jannaschii

“…This suggests that significant differences between H 4 MPT-dependent and H 4 PteGlu-dependent SHMTs may be limited to only the pteridine binding site. Crystallographic studies have shown that the pteridine substrate binds to the enzyme from different eubacterial and eukaryotic sources with similar modalities, although the stoichiometry and subunit occupancy for the binding is different for each of the four structures solved so far (34,35,36,38). Asn 347 , which binds to N-1 and N-8 of H 4 PteGlu, is probably the most important structural element in the pteridine ring recognition.…”

A three-dimensional structure of Plasmodium falciparum serine hydroxymethyltransferase in complex with glycine and 5-formyl-tetrahydrofolate. Homology modeling and molecular dynamics

“…At first hand, it seems that it would be very difficult to design selective inhibitors for pfSHMT, however, if this is possible, we expect that those compounds would be more durable as efficient antimalarial drugs, once the close similarity between the SHMT active sites of the different species is an indication of the low potential for mutation of this enzyme. Furthermore, Fu et al [52] propose that the canonical SHMT structure supports the inference that the folate interactions site evolved from a type I PLP precursor enzyme through sequence insertions and not by domain swapping from other folate-requiring enzymes. This may be the reason why antifolate compounds developed as chemotherapeutic agents are ineffective as inhibitors of SHMT and suggests that an effective antifolate inhibitor of SHMT may not inhibit other folate enzymes.…”