Primary structure and tetrahydropteroylglutamate binding site of rabbit liver cytosolic 5,10-methenyltetrahydrofolate synthetase.

The primary sequence of 5,10-methenyltetrahydrofolate synthetase from rabbit liver was determined by amino acid sequencing of the purified enzyme. The enzyme contains 201 amino acid residues with a predicted mass of 22,779 Da. The enzyme is located in the cytosolic fraction of liver homogenates. Carbodiimide-activated 5-formyltetrahydropteroylmonoglutamate and the pentaglutamate form of the substrate both irreversibly inactivate the enzyme by forming a covalent bond to Lys-18. Non-activated 5-formyltetrahydropteroylpentaglutamate protected against this inactivation. Substrate specificity studies showed that increasing the number of glutamate residues from zero to five on 5-formyltetrahydropteroate results in a 2 order of magnitude increase in the affinity of the substrate for the enzyme but only a 3-fold increase in the value of Vmax

Serine hydroxymethyltransferase: origin of substrate specificity

All forms of serine hydroxymethyltransferase, for which a primary structure is known, have five threonine residues near the active-site lysyl residue (K229) that forms the internal aldimine with pyridoxal phosphate. For Escherichia coli serine hydroxymethyltransferase each of these threonine residues has been changed to an alanine residue. The resulting five mutant enzymes were purified and characterized with respect to kinetic and spectral properties. The mutant enzymes T224A and T227A showed no significant changes in kinetic and spectral properties compared to the wild-type enzyme. The T225A and T230A enzymes exhibited differences in K(m) and k(cat) values but exhibited the same spectral properties as the wild-type enzyme. The four threonine residues at positions 224, 225, 227, and 230 do not play a critical role in the mechanism of the enzyme. The T226A enzyme had nearly normal affinity for substrates and coenzymes but had only 3% of the catalytic activity of the wild-type enzyme. The spectrum of the T226A enzyme in the presence of amino acid substrates showed a large absorption maximum at 343 nm with only a small absorption band at 425 nm, unlike the wild-type enzyme whose enzyme-substrate complexes absorb at 425 nm. Rapid reaction studies showed that when amino acid substrates and substrate analogues were added to the T226A enzyme, the internal aldimine absorbing at 422 nm was rapidly converted to a complex absorbing at 343 nm in a second-order process. This was followed by a very slow first-order formation of a complex absorbing at 425 nm. Variation of the initial rapid second-order process as a function of pH suggested that the anionic form of the amino acid forms the first complex with the enzyme. The results are interpreted as being due to the rapid formation of a gem-diamine complex between amino acids and T226A enzyme with a rate-determining formation of the external aldimine. This suggests that Thr-226 plays an important role in converting the gem-diamine complex to the external aldimine complex. Variation of the kinetic constants with amino acid structure suggests that the T226A enzyme distinguishes between substrates and substrate analogues in the formation of the gem-diamine complex

The primary structure of rabbit liver mitochondrial serine hydroxymethyltransferase.

The complete amino acid sequence of mitochondrial serine hydroxymethyltransferase from rabbit liver was determined. The sequence was obtained from analysis of peptides isolated from chymotryptic, cyanogen bromide, and limited acid cleavages of the protein. The enzyme consists of four identical subunits, each of 475 residues, i.e. 8 residues shorter than the subunit of the corresponding cytosolic isoenzyme. The sequences of the two rabbit proteins are easily aligned, provided a gap of 5 residues near the amino terminus and a gap of 3 residues near the carboxyl terminus are included in the mitochondrial sequence. The overall degree of identity between the two isoenzymes is 61.9%, whereas the structural identity of each eukaryotic isoenzyme with the corresponding Escherichia coli enzyme is about 40%. The rabbit isoenzymes are about 70 residues longer than the E. coli enzyme, with one-half of these residues accounted for by insertions in both isoenzymes near their carboxyl terminus. Predictions of secondary structure and calculations of hydropathy profiles are also presented, suggesting an even more extensive degree of identity in the three-dimensional folding of the three proteins, in accord with the known similarity of their catalytic properties. Evidence was obtained for the existence of additional molecular forms of the mitochondrial protein, differing in the absence of some amino acid residues at the amino terminus of the polypeptide chain

Crystal Structures of Human Pyridoxal Kinase in Complex with the Neurotoxins, Ginkgotoxin and Theophylline: Insights into Pyridoxal Kinase Inhibition

Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B6, pyridoxal 5′-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B6 is converted to PLP by PL kinase. PLP is the B6 vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B6, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects

Nuclear localised more sulphur accumulation1 epigenetically regulates sulphur homeostasis in Arabidopsis thaliana

Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the more sulphur accumulation1 (msa1-1) mutant. The MSA1 protein is localized to the nucleus and is required for both S adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over- accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation

Mice have a transcribed L-threonine aldolase/GLY1 gene, but the human GLY1 gene is a non-processed pseudogene

BACKGROUND: There are three pathways of L-threonine catabolism. The enzyme L-threonine aldolase (TA) has been shown to catalyse the conversion of L-threonine to yield glycine and acetaldehyde in bacteria, fungi and plants. Low levels of TA enzymatic activity have been found in vertebrates. It has been suggested that any detectable activity is due to serine hydroxymethyltransferase and that mammals lack a genuine threonine aldolase. RESULTS: The 7-exon murine L-threonine aldolase gene (GLY1) is located on chromosome 11, spanning 5.6 kb. The cDNA encodes a 400-residue protein. The protein has 81% similarity with the bacterium Thermotoga maritima TA. Almost all known functional residues are conserved between the two proteins including Lys242 that forms a Schiff-base with the cofactor, pyridoxal-5'-phosphate. The human TA gene is located at 17q25. It contains two single nucleotide deletions, in exons 4 and 7, which cause frame-shifts and a premature in-frame stop codon towards the carboxy-terminal. Expression of human TA mRNA was undetectable by RT-PCR. In mice, TA mRNA was found at low levels in a range of adult tissues, being highest in prostate, heart and liver. In contrast, serine/threonine dehydratase, another enzyme that catabolises L-threonine, is expressed very highly only in the liver. Serine dehydratase-like 1, also was most abundant in the liver. In whole mouse embryos TA mRNA expression was low prior to E-15 increasing more than four-fold by E-17. CONCLUSION: Mice, the western-clawed frog and the zebrafish have transcribed threonine aldolase/GLY1 genes, but the human homolog is a non-transcribed pseudogene. Serine dehydratase-like 1 is a putative L-threonine catabolising enzyme

Domain structure and function of 10-formyltetrahydrofolate dehydrogenase.

10-Formyltetrahydrofolate dehydrogenase catalyzes the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate. Previous studies have shown that the enzyme binds the physiological pentaglutamate form of tetrahydrofolate product so tightly that it remains bound during size exclusion chromatography (Cook, R. J., and Wagner, C. (1982) Biochemistry 21, 4427-4434). In addition to the dehydrogenase activity, the enzyme from rat liver has been reported to exhibit both 10-formyltetrahydrofolate hydrolase and aldehyde dehydrogenase activities (Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Chem. 266, 4965-4973). We have purified the enzyme from rabbit liver and found that it catalyzes the same three reactions with similar kinetic constants and that it is a 99-kDa homotetramer, as reported previously for the rat and pig enzymes. Previous studies have suggested that the enzyme is composed of three domains and has separate folate binding sites for the dehydrogenase and hydrolase activities. We have investigated the domain structure of the rabbit enzyme. Differential scanning calorimetry reveals two thermal transitions, indicating the presence of two independently folded domains. The pentaglutamate form of tetrahydrofolate and NADP+ each stabilize one of the thermal transitions, showing that these ligands bind to separate domains. Limited proteolytic digestions by several proteases cleave the enzyme in a linker region between the two domains. After proteolytic cleavage, the domains no longer remain associated and do not catalyze the 10-formyltetrahydrofolate dehydrogenase reaction. Isolation and characterization of the intact domains revealed that the N-terminal domain only catalyzes the NADP(+)-independent 10-formyltetrahydrofolate hydrolase activity and the C-terminal domain only catalyzes the NADP(+)-dependent aldehyde dehydrogenase activity. The kinetic constants of these isolated domains are similar to those of the intact enzyme. Binding studies on the native enzyme using fluorescence and isothermal titration calorimetry indicated that the enzyme binds one molecule of tetrahydrofolate and two molecules of NADP+ per tetramer. Dissociation constants for both ligands were also determined by these methods

Similarity between serine hydroxymethyltransferase and other pyridoxal phosphate-dependent enzymes.

A structural homology of the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine hydroxymethyltransferase (SHMT) with aspartate aminotransferase (AAT) is proposed. Although the two sequences are very dissimilar, a reasonable alignment was obtained using the profile analysis method. Sequences of AAT and dialkylglycine decarboxylase (DGD), for which crystal structure data are available, have been aligned on the basis of their structure superposition. A profile was then calculated and SHMT sequence aligned to it. Three of the four residues conserved in all aminotransferases (including the PLP-binding lysine) are matched. A profile search with DGD-AAT-SHMT profile is more selective and sensitive than individual sequence profiles for PLP-dependent enzyme detection. Potential homologies with the eryC1 gene product involved in erythromycin biosynthesis and with amino acid decarboxylases were observed. Homology with AAT will be used as a guideline for planning site-directed mutagenesis experiments on SHMT