Regulation of monkey liver serine hydroxymethyltransferase by nicotinamide nucleotide.

The positive homotropic binding of tetrahydrofolate to monkey liver serine hydroxymethyltransferase was abolished on preincubating the enzyme with NADH and NADPH. NAD+ was a negative heterotropic effector, whereas NADP+ was without effect. The allosteric effects of nicotinamide nucleotides on the serine hydroxymethyltransferase, reported for the first time, lead to a better understanding of the regulation of the metabolic interconversion of folate coenzymes.     

The serine hydroxymethyltransferase of Plasmodium lophurae.

Plasmodium lophurae serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified and characterized by (NH4)2SO4 fractionation and chromatography on Sephadex G-100. The enzyme, precipitated by 3.0.3.3 M (NH4)2SO4, had a molecular weight of 68,300 as estimated by exclusion chromatography on G-100. The pH optimum of the enzyme was 6.8-7.6 in sodium phosphate-citrate buffer. Citrate stabilized the enzyme during storage in phosphate buffer at 4 C. The Km was 4.3 X 10(-3) M for L-serine and 2.5 X 10(-4) M for tetrahydrofolate.     

Mechanism for folate-independent aldolase reaction catalyzed by serine hydroxymethyltransferase

Serine hydroxymethyltransferase catalyzes the cleavage of β-hydroxyamino acids into glycine and aldehydes in the absence of tetrahydrofolate. The enzyme accepts various β-hydroxyamino acids as the substrate of this reaction. The reaction rate varies depending on the substituent and stereochemistry at the Cβ atom: the erythro forms and the β-phenyl substituent are preferred over the threo forms and the β-methyl substituent, respectively. Although several mechanisms have been proposed, what determines the substrate preference remains unclear. We first performed quantum mechanical calculations to assess the validity of the reaction mechanisms. The results indicate that the retro-aldol mechanism starting with abstraction of the proton from the β-hydroxyl group is plausible. This also suggests that Cα-Cβ bond cleavage is the rate-limiting step. We next measured the dependence of the rate constants on temperature with four representative substrates and calculated the activation energies and pre-exponential factors from the Arrhenius plots. The activation energies of the erythro forms were lower than those of the threo forms. The β-phenyl substituent lowered the activation energy in the threo form, whereas it did not alter the activation energy but increased the pre-exponential factor in the erythro form. We present a unified model to explain the origin of the substituent and stereochemical preferences by combining the theoretical and experimental results. A possible biological role of the tetrahydrofolate-independent activity in thermophiles is also discussed.     

Structural basis for inhibition of DNA replication by aphidicolin

Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.     

Inhibition of monkey liver serine hydroxymethyltransferase by Cibacron Blue 3G-A.

Cibacron Blue 3G-A inhibited monkey liver serine hydroxymethyltransferase competitively with respect to tetrahydrofolate and non-competitively with respect to L-serine. NADH, a positive heterotropic effector, failed to protect the enzymes against inhibition by the dye and was unable to desorb the enzyme from Blue Sepharose CL-6B gel matrix. The binding of the dye to the free enzyme was confirmed by changes in the dye absorption spectrum. The results indicate that the dye probably binds at the tetrahydrofolate-binding domain of the enzyme, rather than at the 'dinucleotide fold'.     

Subcellular distribution of serine hydroxymethyltransferase in Plasmodium lophurae.

Subcellular fractions of the bird malaria, $\text{Plasmodium lophurae}$ were prepared by differential centrifugation. Cytochrome oxidase activity was located in the mitochondrial fraction. A major portion of glutamate dehydrogenase activity was found in the mitochondrial fraction with the remainder in the ribosomal and cytosolic fractions. Malate dehydrogenase and serine hydroxymethyltransferase activities were located primarily in the cytosolic fraction.     

Enzymatic mechanism for the hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate by serine hydroxymethyltransferase.

Serine hydroxymethyltransferase in the presence of glycine catalyzes the hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamate to (6S)-5-formyltetrahydropteroylpolyglutamate. The enzyme also catalyzes the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from a compound in equilibrium with (6R)-5,10-methenyltetrahydropteroylpolyglutamate believed to be (6R,11R)-5,10-hydroxymethylenetetrahydropteroylpolyglutamate , a putative intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate [Stover, P., & Schirch, V. (1992) Biochemistry (preceding paper in this issue)]. The enzymatic mechanism for the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from these substrates and the role of glycine in the reaction was addressed. Evidence suggests that (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is a catalytically competent intermediate in the enzyme-catalyzed hydrolysis of (6R)-5,10-methenyltetrahydropteroyltetraglutamate. The enzyme displays a high Km of 40 microM for (6R)-5,10-methenyltetrahydropteroyltetraglutamate, while the Km for (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is below 0.5 microM. The kcat values for both reactions are identical and equal to the rate of formation of an enzyme ternary complex absorbing at 502 nm which is formed from glycine and (6S)-5-formyltetrahydropteroylpolyglutamate. The hydrolysis reaction proceeds with exchange of the C11 formyl proton of (6R)-5,10-methenyltetrahydropteroyltetraglutamate, suggesting that the enzyme-catalyzed reaction occurs by the same C11 carbanion inversion mechanism as the nonenzymatic reaction. Isotope exchange experiments using [2-3H]glycine and differential scanning calorimetry data suggest both a catalytic and a conformational role for glycine in the enzymatic reaction. The results are discussed in terms of the similarity in mechanisms of the SHMT-catalyzed retroaldol cleavage of serine and hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamates.     

Identification of active-site residues of sheep liver serine hydroxymethyltransferase.

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.     

Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.     

Haploinsufficiency of cytosolic serine hydroxymethyltransferase in the Smith-Magenis syndrome.

Folate-dependent one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth, and serine hydroxymethyltransferase (SHMT) is central to this process. Our studies reveal that the gene for cytosolic SHMT (cSHMT) maps to the critical interval for Smith-Magenis syndrome (SMS) on chromosome 17p11.2. The basic organization of the cSHMT locus on chromosome 17 was determined and was found to be deleted in all 26 SMS patients examined by PCR, FISH, and/or Southern analysis. Furthermore, with respect to haploinsufficiency, cSHMT enzyme activity in patient lymphoblasts was determined to be approximately 50% that of unaffected parent lymphoblasts. Serine, glycine, and folate levels were also assessed in three SMS patients and were found to be within normal ranges. The possible effects of cSHMT hemizygosity on the SMS phenotype are discussed.     

Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner

The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.     

The role of serine hydroxymethyltransferase in cell proliferation: DNA synthesis from serine following mitogenic stimulation of lymphocytes

It is shown that the time-course of incorporation of radioactivity from [3-¹⁴C]serine into nucleic acids parallels DNA synthesis following mitogenic stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA). The activity of serine hydroxymethyltransferase was elevated about four-fold in PHA-stimulated lymphocytes compared to that in unstimulated control ceils. It is suggested that lymphocytes, in common with other proliferating cell systems:, may synthesize serine de novo for utilization in pathways of nucleotide biosynthesis following mitogenic stim--ulation.     

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

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 alpha-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.