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PubMed2006

The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography.

Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.

Binding Sites↗
PubMed1999

Enzymic evaluation of thiamin, riboflavin and pyridoxine status of parturient mothers and their newborn infants in a Mediterranean area of Spain.

OBJECTIVE: To determine the biochemical status of thiamin, riboflavin and pyridoxine in parturient mothers and their newborn infants in a Mediterranean region. DESIGN: Transveral study. SETTING: St Joan University Hospital and Faculty of Medicine & Health Sciences, Universitat Rovira i Virgili, Reus, Spain. SUBJECTS: 131 healthy parturient mothers, with normal pregnancies and deliveries in St Joan University Hospital, and their newborn infants. INTERVENTIONS: Erythrocyte haemolysates were prepared from maternal blood at delivery and infants' umbilical cord blood and used to measure micronutrient status using the transketolase, glutathione reductase and aspartate aminotransferase coenzyme stimulation tests. RESULTS: Maternal and infant coenzyme activities were significantly correlated, but infant coenzyme status was better than maternal, with significantly higher basal and stimulated activity (P < 0.001) and significantly lower activation coefficients (P < 0.001). Inadequate thiamin, riboflavin or pyridoxine status occured in 38.2 62.6% (50-82) of the mothers and 3.1-37.4% (4 49) of the infants; 85.2% (46/54), 12.9% (4/31) and 24.1% (12/54) of infants born to mothers with biochemical deficiency of either thiamin, riboflavin or pyridoxine, respectively also had inadequate status. Maternal deficiencies in more than one vitamin further increased the risk of infant thiamin and pyridoxine deficiency. Maternal and infant riboflavin status were significantly correlated with fetal development (e.g. length at birth, P < 0.001). The incidence of thiamin deficiency in paturient mothers in Spain was the highest out of a 12-country comparison. CONCLUSIONS: Inadequate status for each vitamin was evident in mothers and infants. Maternal status of each individual vitamin, but especially riboflavin, was affected by maternal status of the other vitamins. Infant thiamin status was the most adversely affected by maternal deficiencies in more than one vitamin. Infant riboflavin status, however, was apparently protected from adverse maternal status.

Adolescent↗
PubMed2003

Current mechanistic understanding of thiamin diphosphate-dependent enzymatic reactions.

The mechanism of thiamin diphosphate-dependent enzymatic reactions is discussed, concentrating on two enzymes involved in decarboxylating pyruvic acid, the yeast pyruvate decarboxylase and the pyruvate dehydrogenase multienzyme complex from Escherichia coli. The availability of high-resolution X-ray structures for several thiamin diphosphate-dependent enzymes, the use of site-specifically substituted protein variants (resulting from site-directed mutagenesis), the development of model reactions for the various putative intermediates, and the application of new mechanistic tools in solution have all contributed to a much better understanding of the role of the protein component in catalysis. Perhaps the most important advance in our understanding of these mechanisms concerns the role of the 4'-aminopyrimidine component of the coenzyme, widely ignored prior to the publication of the X-ray results. The current view is that the two aromatic rings both contribute to catalysis, perhaps carrying out an intramolecular proton transfer to initiate the various reactions, an ability that makes this coenzyme virtually unique among coenzymes.

Catalysis↗
PubMed2004

Inhibition of thiamin diphosphate dependent enzymes by 3-deazathiamin diphosphate.

3-Deazathiamin diphosphate (deazaTPP) and a second thiamin diphosphate (TPP) analogue having a benzene ring in place of the thiazolium ring have been synthesised. These compounds are both extremely potent inhibitors of pyruvate decarboxylase from Zymomonas mobilis; binding is competitive with TPP and is essentially irreversible even though no covalent linkage is formed. DeazaTPP binds approximately seven-fold faster than TPP and at least 25,000-fold more tightly (K(i) less than 14 pM). DeazaTPP is also a potent inhibitor of the E1 subunit of alpha-ketoglutarate dehydrogenase from E. coli and binds more than 70-fold faster than TPP. In this case slow reversal of the inhibition could be observed and a K(i) value of about 5 nM was calculated (ca. 500-fold tighter binding than TPP).

Benzene Derivatives↗
PubMed2006

Thiamine models and perspectives on the mechanism of action of thiamine-dependent enzymes.

Thiamine dependent enzymes catalyze ligase and lyase reactions near a carbonyl moiety. Chemical models for these reactions serve as useful tools to substantiate a detailed mechanism of action. This tutorial review covers all such studies performed thus far, emphasizing the role of each part around the active site and the conformation of the cofactor during catalysis.

Binding Sites↗
PubMed1972

Deoxyribonucleic acid synthesis in mammalian systems. Permealysed Ehrlich ascites cells in vitro first label Okazaki-type low-molecular-weight deoxyribonucleic acid and then high-molecular-weight deoxyribonucleic acid.

During the evaluation of a method of preparing permealysed Ehrlich ascites cells, shortterm labelling experiments were carried out with d[(3)H]TTP. In the first minute the bulk of the label appeared as low-molecular-weight pieces of DNA. Subsequently the label appeared in DNA of much higher molecular weight. A brief description of the preparation procedure and the properties of the product is provided. Evidence is presented to show that the nucleotide was incorporated directly without intermediate conversion into dTMP or thymidine.

Animals↗
PubMed1974

Oxidative decarboxylation of glycollate and glyoxylate by leaf peroxisomes.

1. The conditions under which peroxisomal preparations from leaves of spinach beet and spinach catalyse the release of (14)CO(2) from [1-(14)C]glycollate and [1-(14)C]glyoxylate were investigated. 2. At pH8, (14)CO(2) production from [1-(14)C]glyoxylate was accompanied by equivalent quantities of formate. The accumulation of oxalate and the effects of various reagents, especially catalase inhibitors, show that glyoxylate is non-enzymically oxidized by H(2)O(2), which is generated by the oxidation of glyoxylate to oxalate by the action of glycollate oxidase. 3. (14)CO(2) is shown to be generated from [1-(14)C]glycollate at pH8 by a similar reaction, but the H(2)O(2) is generated mainly by the oxidation of glycollate to glyoxylate. 4. The physiological significance of these reactions is discussed, with special reference to their role in photorespiration.

Animals↗
PubMed2004

Effects of deletions at the C-terminus of tobacco acetohydroxyacid synthase on the enzyme activity and cofactor binding.

AHAS (acetohydroxyacid synthase) catalyses the first committed step in the biosynthesis of branched-chain amino acids, such as valine, leucine and isoleucine. Owing to the unique presence of these biosynthetic pathways in plants and micro-organisms, AHAS has been widely investigated as an attractive target of several classes of herbicides. Recently, the crystal structure of the catalytic subunit of yeast AHAS has been resolved at 2.8 A (1 A=0.1 nm), showing that the active site is located at the dimer interface and is near the herbicide-binding site. In this structure, the existence of two disordered regions, a 'mobile loop' and a C-terminal 'lid', is worth notice. Although these regions contain the residues that are known to be important in substrate specificity and in herbicide resistance, they are poorly folded into any distinct secondary structure and are not within contact distance of the cofactors. In the present study, we have tried to demonstrate the role of these regions of tobacco AHAS by constructing variants with serial deletions, based on the structure of yeast AHAS. In contrast with the wild-type AHAS, the truncated mutant which removes the C-terminal lid, Delta630, and the internal deletion mutant without the mobile loop, Delta567-582, impaired the binding affinity for ThDP (thiamine diphosphate), and showed different elution profiles representing a monomeric form in gel-filtration chromatography. Our results suggest that these regions are involved in the binding/stabilization of the active dimer and ThDP binding.

Acetolactate Synthase↗