- Top of page
- Materials and methods
The three-dimensional structures of human and rabbit liver cytosolic recombinant serine hydroxymethyltransferases (hcSHMT and rcSHMT) revealed that E75 and Y83 (numbering according to hcSHMT) are probable candidates for proton abstraction and Cα-Cβ bond cleavage in the reaction catalyzed by serine hydroxymethyltransferase. Both these residues are completely conserved in all serine hydroxymethyltransferases sequenced to date. In an attempt to decipher the role of these residues in sheep liver cytosolic recombinant serine hydroxymethyltransferase (scSHMT), E74 (corresponding residue is E75 in hcSHMT) was mutated to Q and K, and Y82 (corresponding residue is Y83 in hcSHMT) was mutated to F. The specific activities using serine as the substrate for the E74Q and E74K mutant enzymes were drastically reduced. These mutant enzymes catalyzed the transamination of d-alanine and 5,6,7,8-tetrahydrofolate independent retroaldol cleavage of lallo threonine at rates comparable with wild-type enzyme, suggesting that E74 was not involved directly in the proton abstraction step of catalysis, as predicted earlier from crystal structures of hcSHMT and rcSHMT. There was no change in the apparent Tm value of E74Q upon the addition of l-serine, whereas the apparent Tm value of scSHMT was enhanced by 10 °C. Differential scanning calorimetric data and proteolytic digestion patterns in the presence of l-serine showed that E74Q was different to scSHMT. These results indicated that E74 might be required for the conformational change involved in reaction specificity. It was predicted from the crystal structures of hcSHMT and rcSHMT that Y82 was involved in hemiacetal formation following Cα-Cβ bond cleavage of l-serine and mutation of this residue to F could lead to a rapid release of HCHO. However, the Y82F mutant had only 5% of the activity and failed to form a quinonoid intermediate, suggesting that this residue is not involved in the formation of the hemiacetal intermediate, but might be involved indirectly in the abstraction of the proton and in stabilizing the quinonoid intermediate.
Serine hydroxymethyltransferase (SHMT), a pyridoxal-5′-phosphate (pyridoxalP)-containing enzyme catalyzes a physiologically important reaction, namely the transfer of the hydroxymethyl group of serine to 5,6,7,8-tetrahydrofolate (H4-folate) to yield glycine and N5,N10-methylene H4-folate, a key intermediate in the biosynthesis of amino acids and nucleic acids [1,2]. For these reasons, it has been suggested that SHMT is a potential target in cancer chemotherapy [3–5]. The enzyme from prokaryotic sources is a dimer , whereas that from eukaryotic sources is a tetramer [4,7]. It contains 1 mol of pyridoxalP per mol of subunit with an absorbance maximum at 425 nm. Spectrally distinct intermediates are generated during the reaction of the enzyme with its substrates .
The availability of the crystal structures of human liver cytosolic recombinant SHMT (hcSHMT) , rabbit liver cytosolic recombinant SHMT (rcSHMT)  and Escherichia coli SHMT (eSHMT)  have enabled a more critical examination of the role of specific amino acid residues in the different steps of catalysis. The first step in catalysis is a nucleophilic attack by the amino group of l-serine on the internal aldimine (Scheme 1, structure I) to form the geminal diamine (Scheme 1, structure II). R363 in eSHMT and the corresponding residue R401 in sheep liver cytosolic recombinant SHMT (scSHMT) were identified previously as essential for binding of the carboxy group of the amino acid substrate [10,11]. The X-ray structure confirmed that this residue is at hydrogen bonding distance from the carboxy group of the substrate and is well positioned to carry out this function [5,8,9]. The geminal diamine structure derived by analogy with the X-ray structure of the reduced internal aldimine showed that the position of the pyridoxalP ring was altered to facilitate the reaction following interaction with the substrate carboxy residue. It is postulated that conversion from the geminal diamine to external aldimine (Scheme 1, structure III) occurs via proton transfer from the substrate amino group to K256 and that the orientation of the pyridoxalP ring is perpendicular to the bond that is being cleaved. Mutation of the K256 in scSHMT led to the loss of pyridoxalP and disruption of the oligomeric structure , as this Lys residue is located at the interface of the tight dimers [5,8,9]. From the three-dimensional structure of hcSHMT and rcSHMT, it is apparent that the side chains of E74 and Y82 are oriented towards the -OH group of the serine [5,8]. Abstraction of the proton from the hydroxy group of serine followed by Cα-Cβ bond cleavage leads to the formation of the quinonoid intermediate with an absorbance maximum at 492.5 nm (Scheme 1, structure IV). The formaldehyde formed during this process is transferred to the N5,N10 position of the H4-folate, which enhances the rate of proton abstraction. Subsequently, the external aldimine is formed with the product, glycine (Scheme 1, structure V), which is converted to the geminal diamine (Scheme 1, structure VI). The catalytic cycle is completed by the release of glycine and formation of internal aldimine with the ɛ-amino group of K256 (Scheme 1, structure I).
Figure Scheme 1. . Schematic representation of pyridoxalP and active site residues participating in the different steps of the reaction catalyzed by scSHMT. (I) PyridoxalP is linked in scSHMT to the ɛ-amino group of K256 to form an internal aldimine, absorbing at 425 nm (I). The carboxy group of substrate, l-serine binds to R401. E74 and Y82 are positioned to facilitate further reaction. Geminal diamine (II) in which both the amino group of the substrate and ɛ-amino group of K256 are linked to pyridoxalP. This intermediate absorbs at 343 nm. The external aldimine, which absorbs at 425 nm (III) of pyridoxalP and the substrate, is formed by breaking the bond between ɛ-amino group of K256 and pyridoxalP. The abstraction of the proton from the hydroxy group of l-serine possibly by E74 and/or Y82 results in the formation of the quinonoid intermediate (IV), absorbing at 492.5 nm. H4-Folate enhances the formation of the quinonoid intermediate and N5,N10-CH2-H4-folate. PyridoxalP-glycine external aldimine (V) absorbing at 425 nm is converted to the corresponding geminal diamine (VI) (absorbing at 343 nm) by reaction with ɛ-amino group of K256. Release of the product glycine results in the formation of internal aldimine (I) for the next round of catalysis. Adapted from Schirch .
Download figure to PowerPoint
It was suggested from analysis of modeled ternary complex of serine and H4-folate of the rcSHMT that the pteridine ring of H4-folate is too far displaced (≈ 8 Å) from the active site to directly accept the C3-hydroxy group of l-serine to its N5,N10 positions. The phenolic group of Y82, which interposes between the hydroxymethyl group of the l-serine and the N5,N10 positions of H4-folate, could form a hemiacetal with the formyl group produced by the deprotonation of the hydroxy group of serine by E74 . It was also proposed that the short hydrogen bond between Y82 and exocyclic O4 of the pteridine ring could activate the Y82 hydroxy group for the formation of such an intermediate. This would place the hemiacetal group ≈ 5.2 Å from N5 of the folate to which it is to be transferred. It was therefore suggested that Y82 could serve as a shuttle for the hydroxymethyl group between l-serine-external aldimine and the folate cofactor or form the hemiacetal directly by a SN2 displacement at the Cα-bond. It was postulated that if this hypothesis was true, the Y82F mutant should cleave serine rapidly and release formaldehyde .
In order to examine the functions of Y82 and E74 as postulated above [5,8], Y82 was mutated to F and E74 to Q and K in scSHMT. Our results show that E74 is not involved in abstracting the proton from hydroxymethyl group of l-serine but participates in converting the enzyme from an open to closed form upon the addition of l-serine. It is also clear that Y82 is not involved in the formation of a hemiacetal intermediate but has a role in stabilizing the quinonoid intermediate. The mutation of this residue may indirectly affect the formation of the quinonoid intermediate, thereby decreasing the catalytic efficiency.
- Top of page
- Materials and methods
A major objective in the study of any enzyme-catalyzed reaction is the elucidation of the role of specific amino acids in the proposed catalytic mechanism. Analysis of the structural features of hcSHMT and rcSHMT led to the proposition that E75 and E57 (equivalent residues) are involved in abstraction of the proton from the hydroxy group of l-serine [5,8]. Critical examination of the recently determined structure of eSHMT-5-formyl H4-folate-glycine ternary complex suggested that this residue (E57) is in a protonated form and that the carbonyl oxygen is hydrogen bonded to a water molecule from which it must be accepting a proton. It is close to the protonated N10 atom of folate carrying the methylene group . This analysis raises some doubts about the role of this residue as a primary proton acceptor from the hydroxy group of l-serine.
The sequence identity (94%) of scSHMT with hcSHMT suggested that it may have a similar three-dimensional structure. E74 corresponds to E75 and E57 in hcSHMT and rcSHMT, respectively. It can be seen from Table 1 that the mutation of E74 to Q led to a marked decrease in hydroxymethyltransferase activity (0.012 U·mg−1). The residual activity could be monitored conveniently (Table 1). The similar Km value for l-serine suggested that the interaction of the enzyme with l-serine was probably not seriously affected by mutation (Table 1). The characteristic CD intensity at 425 nm of scSHMT was decreased by ≈ 50% upon addition of 100 mm l-serine. A similar decrease in CD intensity was observed with E74Q, suggesting that the mutant was capable of binding serine as efficiently as scSHMT (data not shown). These results suggest that a step in catalysis beyond substrate binding may be affected. The observation that comparable amounts of the quinonoid intermediate (0.35 and 0.39 µmol per µmol of subunit of scSHMT and E74Q, respectively) were seen in the presence of glycine and H4-folate with both E74Q and scSHMT suggested that the proton abstraction step was probably unaffected by the mutation. It was interesting to observe the peak corresponding to the quinonoid intermediate upon addition of glycine to E74Q, suggesting that a significant amount of proton abstraction from the Cα-carbon occurs even in the absence of H4-folate. The small amount of H4-folate independent hydroxymethyltransfer could be due to the ability of the mutant to abstract the equivalent proton from the hydroxymethyl group of serine. The step following formation of the quinonoid intermediate is the removal of HCHO generated at the active site, when l-serine is used as a substrate. l-Serine and HCHO decreased the concentration of quinonoid intermediate in a similar manner (Fig. 3), indicating that the interactions of H4-folate and removal of HCHO from the active site are not probably affected by the mutation.
The structure of eSHMT in the presence of N5-formyl H4-folate and glycine has revealed that the carbonyl oxygen atom of E57 could form an H-bonding interaction with a water molecule. It is possible that the carbonyl oxygen atom in Q could carry out a similar function and enable proton abstraction by the E74Q mutant. However, the E74K mutant SHMT, which cannot participate in such interactions, had similar activity (0.1% of scSHMT). This mutant also generated the quinonoid intermediate upon addition of glycine. Furthermore, addition of H4-folate increased the concentration of the quinonoid intermediate (Fig. 2C). These results suggest that the carboxy group of E74 residue may not function as a proton acceptor. Additional support for this suggestion is that H4-folate independent reactions, such as transamination and retroaldol cleavage of lallo threonine (Table 2), were unaffected, strongly suggesting that the mutation has probably incapacitated the enzyme to undergo the conformational change consequent to l-serine binding. Catalytic efficiency with l-serine as the substrate, however, decreased by 410-fold (Table 1). It is therefore possible that E74 is involved in the conversion of the enzyme from an open to a closed conformation which is required for physiological reactions to occur. As demonstrated previously with SHMT [18,27], conversion of the open form to the closed form of the enzyme by l-serine is characterized by its increased thermal stabilty. In a similar manner, the thermal stability of scSHMT is enhanced by addition of l-serine but not glycine (Table 3). However, the thermal stability of E74Q was not enhanced by the interaction with l-serine or glycine (Table 3).
This is further confirmed by DSC data which suggest the possibility of a few critical domains or patches in the protein becoming very fragile or very stable upon the interaction of scSHMT and its mutants with l-serine. Addition of l-serine may have modified E74Q in such a way that these domains behave rather independently, as suggested by peak values in which the total enthalpic contribution of the protein molecule is provided by two or more domains. It is likely that under ‘stress conditions’, such as binding of ligand, structural changes or additional patches could be induced in the protein resulting in new transitions. The DSC data of E74Q and scSHMT in the absence of ligand and the presence of glycine were, however, identical, suggesting that thermal unfolding of these enzymes was identical and no conformational change occurs in the presence of glycine.
The altered conformation upon binding of l-serine in the case of E74Q was also monitored by differential susceptibility to tryptic digestion. SDS/PAGE patterns of tryptic digests of scSHMT and E74Q in the presence or absence of glycine were very similar, as were the tryptic digestion patterns of scSHMT either alone or in the presence of l-serine were similar (Fig. 5, lanes 3 and 5). However, the appearance of an additional ≈ 35 kDa band in the SDS/PAGE of E74Q in the presence of l-serine (Fig. 5, lane 9) suggested that this mutant was converted to a new conformation different to the closed form generated by the interaction of l-serine to scSHMT, as well as to the open form.
The following observations support the contention that E74 may be involved in conversion of the enzyme from an open to a closed form and that the mutation to Q or K hampers this conversion. E74Q was characterized by limited physiological reaction in the absence of H4-folate, a prominent quinonoid intermediate peak upon the addition of glycine alone, inability to enhance thermal stabilty in the presence of l-serine, multimodal thermograms of DSC in the presence of l-serine and altered susceptibility to tryptic digestion in the presence of l-serine.
In addition to the role of E74 in the catalysis, it was postulated that Y82 was involved in the formation of hemiacetal intermediate with the formyl group produced by Cα–Cβ bond cleavage [5,8]. Our results clearly show that mutation of this residue did not cause a marked decrease in activity either with serine or alternate substrates thereby implying that Y82 may not have a crucial role in catalysis. It was also postulated by Scarsdale et al.  that if hemiacetal hypothesis was correct then the Y82F mutant should cleave serine and rapidly release formaldehyde. A rapid release of HCHO after the addition of l-serine to Y82F was not observed. The absence of a quinonoid intermediate in the presence of glycine and H4-folate may be due to the effect of this residue on the stability of the quinonoid intermediate.
The results presented here show that E74 is not involved in abstracting the proton from hydroxymethyl group of l-serine, but in the conversion of the enzyme from an open to a closed form. Y82, is not involved directly in proton abstraction or Cα-Cβ bond cleavage, but may have a role in stabilizing the quinoniod intermediate.