Crystal Structure of Rabbit Cytosolic Serine Hydroxymethyltransferase at 2.8 Å Resolution:  Mechanistic Implications^,

Abstract

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and single carbon groups that are essential for many biosynthetic pathways. SHMT requires both pyridoxal phosphate (PLP) and tetrahydropteroylpolyglutamate (H4PteGlun) as cofactors, the latter as a carrier of the single carbon group. We describe here the crystal structure at 2.8 A resolution of rabbit cytosolic SHMT (rcSHMT) in two forms: one with the PLP covalently bound as an aldimine to the Nepsilon-amino group of the active site lysine and the other with the aldimine reduced to a secondary amine. The rcSHMT structure closely resembles the structure of human SHMT, confirming its similarity to the alpha-class of PLP enzymes. The structures reported here further permit identification of changes in the PLP group that accompany formation of the geminal diamine complex, the first intermediate in the reaction pathway. On the basis of the current mechanism derived from solution studies and the properties of site mutants, we are able to model the binding of both the serine substrate and the H4PteGlun cofactor. This model explains the properties of several site mutants of SHMT and offers testable hypotheses for a more detailed mechanism of this enzyme.

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

“…This was confirmed by the modelling studies of hcSHMT and rabbit liver cytosolic SHMT [3,4]. A detailed study of the catalytic mechanism of SHMT revealed that the addition of substrate, serine\glycine, generates an external aldimine that undergoes deprotonation at the α-carbon atom to form the quinonoid intermediate.…”

“…This alteration in reaction and\or substrate specificity of an enzyme is brought about by subtle changes in local conformations around the active site owing to changes in the primary structure of the proteins. As mentioned previously, although the active-site geometries of AATase and mammalian SHMT are quite similar [3,4], they catalyse different physiological reactions. AATase uses dicarboxylic amino acids as substrates and catalyses transamination, whereas SHMT is more efficient with monocarboxylic acids and transfers the hydroxymethyl group of serine to H % -folate [11].…”

“…An examination of the X-ray structure of hcSHMT revealed that Pro-297 was not interacting directly with any other residue within a radius of 8 A / [3,4]. Replacement of Pro-297 with Arg showed that the positive charge had not affected the network of interactions at the active site, nor had it provided an additional anchor for interacting with the γ-carboxy group of -Asp.…”

“…SHMTs isolated from both prokaryotic and eukaryotic sources are either homodimers or homotetramers with subunit molecular masses ranging from 45 to 54 kDa. The availability of the X-ray structures of human liver cytosolic SHMT (hcSHMT) [3] and rabbit liver cytosolic SHMT [4], coupled with site-directed mutagenesis studies on Escherichia coli SHMT (eSHMT) and sheep liver recombinant SHMT (rSHMT) [5], have enriched our understanding of the structure and function of this enzyme. Mammalian SHMTs are a ' dimer of dimers ' that are stabilized by the N-terminal arm [3,4,6].…”

“…The availability of the X-ray structures of human liver cytosolic SHMT (hcSHMT) [3] and rabbit liver cytosolic SHMT [4], coupled with site-directed mutagenesis studies on Escherichia coli SHMT (eSHMT) and sheep liver recombinant SHMT (rSHMT) [5], have enriched our understanding of the structure and function of this enzyme. Mammalian SHMTs are a ' dimer of dimers ' that are stabilized by the N-terminal arm [3,4,6]. The active-site lysine residue (Lys-257 in hcSHMT) is present at the interface of tight dimers and binds PLP to form the internal aldimine [3].…”