The structure of serine hydroxymethyltransferase as modeled by homology and validated by site‐directed mutagenesis

Abstract

We describe a model for the three-dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of the a-family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of site-directed mutants. New active site features are also proposed for further experimental testing.Keywords: aspartate aminotransferase; catalysis; evolution; homology modeling; profile analysis; pyridoxal phosphate; serine hydroxymethyltransferase; site-directed mutagenesis Serine hydroxymethyltransferase (SHMT: E.C. 2.1.2. I ) catalyzes the reversible conversion of serine and tetrahydropteroyglutamate (H,PteGlu) to glycine and 5, IO-methylene-H4PteG1u (Schirch, 1982). This reaction is the major source of one-carbon groups required in the biosynthesis of methionine, choline, thymidylate, and purines. The SHMT enzyme is widely distributed in nature, and found in both prokaryotic and eukaryotic cells with the latter containing both cytosolic and mitochondrial forms. In addition to H4PteGlu, the enzyme also requires pyridoxal 5'-phosphate (PLP) as a coenzyme. It is one of a group of PLP enzymes that cleave one of the bonds at the a-carbon of their amino acid substrate. These are referred to as the a-family of PLP enzymes and include the transaminases and amino acid decarboxylases (Alexander et al.. 1 994).Three-dimensional structures have been determined for several members of the a-family of vitamin B6-dependent enzymes. Amongst them are aspartate aminotransferase (AAT; McPhalen et al., 1992;Okamoto et al., 1994;Malashkevich et al., 1995). tyrosine phenol-lyase (ITPL) (Antson et al., 1993), w-amino acid aminotransferase (Watanabe et al., 1989), 2,2-dialkylglycine decarboxylase (2DKB) (Toney et al., 1995), ornithine aminotransferase (Shen et al., 1994), glutamate-1-semialdehyde aminomutase (Henning et al., 1997), and ornithine decarboxylase (IORD) (Momany et al., 1995a). Despite the widely differing reaction speciReprint requests to: Francesco Bossa, Dipartimento di Scienze Biochimiche, Universita La Sapienza, P. le A. Moro, 5, 00185 Rorna, Italy; e-mail: BOSSA@axrma.uniromal .it. ficity and weak sequence similarity, these proteins share the same basic folding pattern, which was assumed also for SHMT (Pascarella et al., 1993). The prolonged unavailability of the SHMT atomic structure prompted the construction of a three-dimensional "homology" model that integrated knowledge regarding the enzyme acquired through site-directed mutagenesis experiments and other experimental measures. Escherichia coli SHMT was modeled because experimental data are readily available for this enzyme. New features of the active site are proposed for further experimental testing. Results and discussionHomology modeling was based on the multiple sequence alignment displayed in Figure 1.Comparison between ...

Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

“…all show a good hydrogen bond between the Lys257 ε -amino group and the hydroxyl group of the Thr254 side chain, as originally suggested by Pascarella et al [16]. The nucleophilicity of the ε -amino group and the NH 2 of the substrate must be balanced so that transaldimination can occur rapidly from either direction.…”

“…Prior to the availability of crystallographic structure information on SHMT, it was noted that the amino acid sequences of SHMTs from diverse species have a conserved run of 4 threonine residues terminating 2 residues upstream of the active site lysine: V-V-T-T-T 254 -T-H-K 257 (PLP)-T (numbering is that for rabbit cytosolic serine hydroxymethyltransferase (rcSHMT)) [16]. To determine the possible roles of this conserved sequence in SHMT catalysis, each threonine of this active site stretch was mutated in ecSHMT to an alanine, and the effects of the changes on the spectral and kinetic properties were investigated [17].…”

Role of Proline Residues in the Folding of Serine Hydroxymethyltransferase

“…It is unlikely to be a hinge point, but because it is exposed in the intermediate, the hinge point must precede this residue. Domain 2 most likely starts at a small Pascarella et al (22) have defined a special algorithm for comparing sequence alignments of SHMT proteins. Using their alignments in 53 SHMT structures from a wide variety of organisms, we found that our putative first hinge point Gly 61 is conserved in 39 of 53 known structures of SHMT and its preceding Pro 60 is conserved in 49 of the 53 structures.…”

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

“…The conversion of the amino acid substrates and substrate analogues to the external aldimine is a slow, rate-determining step (40). Thr-226 is conserved as either a Thr or Ser in all known SHMTs, and in modeling the active site of E. coli SHMT, Pascarella et al (21) remarked that in the external aldimine the released Lys-229 -amino group could hydrogen bond to Oγ of Thr-226. Our structure confirms that rotations at χ 3 and χ 4 of the expelled Lys-229 side chain bring the -amino group within hydrogen bonding distance of Thr-226.…”