No abstract

Serine hydroxymethyltransferase (SHMT), a member of the ␣-class of pyridoxal phosphate-dependent enzymes, catalyzes the reversible conversion of serine to glycine and tetrahydrofolate to 5,10-methylene tetrahydrofolate. We present here the crystal structures of the native enzyme and its complexes with serine, glycine, glycine, and 5-formyl tetrahydrofolate (FTHF) from Bacillus stearothermophilus. The first structure of the serine-bound form of SHMT allows identification of residues involved in serine binding and catalysis. The SHMT-serine complex does not show any significant conformational change compared with the native enzyme, contrary to that expected for a conversion from an "open" to "closed" form of the enzyme. However, the ternary complex with FTHF and glycine shows the reported conformational changes. In contrast to the Escherichia coli enzyme, this complex shows asymmetric binding of the FTHF to the two monomers within the dimer in a way similar to the murine SHMT. Comparison of the ternary complex with the native enzyme reveals the structural basis for the conformational change and asymmetric binding of FTHF. The four structures presented here correspond to the various reaction intermediates of the catalytic pathway and provide evidence for a direct displacement mechanism for the hydroxymethyl transfer rather than a retroaldol cleavage.Serine hydroxymethyltransferase (SHMT; 1 EC 2.1.2.1) is a PLP-dependent enzyme that plays a central role in the onecarbon metabolism. It catalyzes the reversible inter-conversion of serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate, a key intermediate in the biosynthesis of purine, thymidine, choline, and methionine (1, 2). In addition to this physiological reaction, SHMT has also been shown to catalyze THF-independent aldolytic cleavage, decarboxylation, racemization, and transamination reactions (3). The importance of SHMT in DNA synthesis coupled with the observed high level of enzyme activity in rapidly proliferating cells has focused attention on SHMT as a potential target for the development of anticancer and antimicrobial agents (4 -6).Several mechanisms have been proposed for the hydroxymethyl transfer, the most favored being the retroaldol cleavage (7,8). The crystal structures of human liver SHMT (hcSHMT) and rabbit liver SHMT (rcSHMT) and Escherichia coli SHMT (eSHMT) as well as murine cytoplasmic SHMT (mcSHMT) have been reported (9 -12). The structure of a reduced form of rcSHMT representing a gem diamine equivalent has also been reported (10). Although these structures have provided a wealth of information regarding the architecture of the enzyme, active site, and residues involved in substrate binding and catalysis, several aspects of SHMT catalytic mechanism remain uncertain (7, 13). A detailed comparison and analysis of several structures of the enzyme corresponding to different intermediate steps and in complex with various substrates, substrate analogs, and product analogs are required to unravel the finer molecular details of ...

The O-acetylserine sulfhydrylase (OASS) reaction has been studied using a number of spectral probes including UV--visible, fluorescence, circular dichroism, and 31P NMR spectroscopy. The addition of L-cysteine, L-alanine, and glycine to OASS results in a shift in lambda max of 412 nm for the internal Schiff base to 418 nm resulting from the formation of the external Schiff base. The addition of L-serine or O-methyl-D,L-serine gives decreases of the absorbance of unliganded enzyme at 412 nm of about 50% and 20%, respectively, concomitant with an increase in the absorbance at 320 nm and a shift in the lambda max of the remaining visible absorbance to 418 nm. The spectral shifts observed in the presence of L-serine are suggestive of establishing an equilibrium between different forms of external Schiff base. The concentration dependence of the changes at 440 (L-cysteine) and 320 nm (L-serine) provides an estimate of the dissociation constant for the external aldimine. The pH dependence of the dissociation constant suggests the alpha-amine of the amino acid must be unprotonated for nucleophilic attack at C4' of PLP, and an enzyme side chain must be unprotonated to hydrogen-bond the thiol or hydroxyl side chain of the amino acid. When L-cysteine is the amino acid, the thiol side chain must be protonated to hydrogen-bond to the unprotonated enzyme side chain. The 31P NMR chemical shift is increased from 5.2 ppm for unliganded enzyme to 5.3 ppm in the presence of L-cysteine, signaling a tighter interaction at the 5'-phosphate upon formation of the external Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of aging and dietary restriction on the expression of several enzymes (superoxide dismutase, catalase and glutathione peroxidase) that are involved in free radical detoxification were studied in liver tissue from male Fischer F344 rats. The expression (i.e., activities and mRNA levels) of superoxide dismutase (Cu-Zn) and catalase decreased with age in liver. Dietary restriction (40% restriction of energy intake) increased the activities of superoxide dismutase (24 to 38%) and catalase (64 to 75%) in liver at 21 and 28 mo of age. Glutathione peroxidase activity in liver of diet-restricted rats was significantly higher (37%) at 28 mo of age than that of rats fed ad libitum. The age-related changes in the relative levels of mRNA for superoxide dismutase, catalase and glutathione peroxidase paralleled the changes in the activities of these enzymes in rats fed ad libitum or rats fed the restricted diet. Thus, the changes in the activities of superoxide dismutase, catalase and glutathione peroxidase with age and dietary restriction appear to arise from changes in the levels of mRNAs coding for these enzymes. Free radical damage, as measured by thiobarbituric acid-reactive material and lipofuscin accumulation, was lower in diet-restricted rats than in rats fed ad libitum.

The structure of the Ascaris suum mitochondrial NAD-malic enzyme in binary complex with NAD has been solved to a resolution of 2.3 A by X-ray crystallography. The structure resembles that of the human mitochondrial enzyme determined in complex with NAD [Xu, Y., Bhargava, G., Wu, H., Loeber, G., and Tong, L. (1999) Structure 7, 877-889]. The enzyme is a tetramer comprised of subunits possessing four domains organized in an "open" structure typical of the NAD-bound form. The subunit organization, as in the human enzyme, is a dimer of dimers. The Ascaris enzyme contains 30 additional residues at its amino terminus relative to the human enzyme. These residues significantly increase the interactions that promote tetramer formation and give rise to different subunit-subunit interactions. Unlike the mammalian enzyme, the Ascaris malic enzyme is not regulated by ATP, and no ATP binding site is observed in this structure. Although the active sites of the two enzymes are similar, residues interacting with NAD differ between the two. The structure is discussed in terms of the mechanism and particularly with respect to previously obtained kinetic and site-directed mutagenesis experiments.