Degradation of lipoxygenase-derived oxylipins by glyoxysomes from sunflower and cucumber cotyledons.

Background: Oilseed germination is characterized by the degradation of storage lipids. It may proceed either via the direct action of a triacylglycerol lipase, or in certain plant species via a specific lipid body 13-lipoxygenase. For the involvement of a lipoxygenase previous results suggested that the hydroxy- or oxo-group that is being introduced into the fatty acid backbone by this lipoxygenase forms a barrier to continuous β-oxidation.

Observation of a stable carbene at the active site of a thiamin enzyme.

Carbenes are highly reactive chemical compounds that are exploited as ligands in organometallic chemistry and are powerful organic catalysts. They were postulated to occur as transient intermediates in enzymes, yet their existence in a biological system could never be demonstrated directly. We present spectroscopic and structural data of a thiamin enzyme in a noncovalent complex with substrate, which implicate accumulation of a stable carbene as a major resonance contributor to deprotonated thiamin.

Alternating sites reactivity is a common feature of thiamin diphosphate-dependent enzymes as evidenced by isothermal titration calorimetry studies of substrate binding.

Thiamin diphosphate (ThDP)-dependent enzymes play vital roles in cellular metabolism in all kingdoms of life. In previous kinetic and structural studies, a communication between the active centers in terms of a negative cooperativity had been suggested for some but not all ThDP enzymes, which typically operate as functional dimers. To further underline this hypothesis and to test its universality, we investigated the binding of substrate analogue methyl acetylphosphonate (MAP) to three different ThDP-dependent enzymes acting on substrate pyruvate, namely, the Escherichia coli E1 component of the pyruvate dehydrogenase complex, E.

A δ38 deletion variant of human transketolase as a model of transketolase-like protein 1 exhibits no enzymatic activity.

Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy.

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin.

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.

Significant catalytic roles for Glu47 and Gln 110 in all four of the C-C bond-making and -breaking steps of the reactions of acetohydroxyacid synthase II.

Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the first common step in the biosynthesis of branched-chain amino acids, condensation of pyruvate with a second 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme II from Escherichia coli is specific for pyruvate as the first donor substrate but exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. In previous studies relying on steady state and transient kinetics, substrate competition and detailed analysis of the distribution of intermediates in the steady-state, we have identified several residues which confer specificity for the donor and acceptor substrates, respectively.

Conversion of pyruvate decarboxylase into an enantioselective carboligase with biosynthetic potential.

Pyruvate decarboxylase (PDC) catalyzes the decarboxylation of pyruvate into acetaldehyde and CO(2) and requires the cofactors thiamin diphosphate and Mg(2+) for activity. Owing to its catalytic promiscuity and relaxed substrate specificity, PDC catalyzes carboligation side reactions and is exploited for the asymmetric synthesis of 2-hydroxy ketones such as (R)-phenylacetyl carbinol, the precursor of (-)-ephedrine. Although PDC variants with enhanced carboligation efficiency were generated in the past, the native reaction, i.

Double duty for a conserved glutamate in pyruvate decarboxylase: evidence of the participation in stereoelectronically controlled decarboxylation and in protonation of the nascent carbanion/enamine intermediate .

Pyruvate decarboxylase (PDC) catalyzes the nonoxidative decarboxylation of pyruvate into acetaldehyde and carbon dioxide and requires thiamin diphosphate (ThDP) and a divalent cation as cofactors. Recent studies have permitted the assignment of functional roles of active site residues; however, the underlying reaction mechanisms of elementary steps have remained hypothetical. Here, a kinetic and thermodynamic single-step analysis in conjunction with X-ray crystallographic studies of PDC from Zymomonas mobilis implicates active site residue Glu473 (located on the re-face of the ThDP thiazolium nucleus) in facilitating both decarboxylation of 2-lactyl-ThDP and protonation of the 2-hydroxyethyl-ThDP carbanion/enamine intermediate.

Valine 375 and phenylalanine 109 confer affinity and specificity for pyruvate as donor substrate in acetohydroxy acid synthase isozyme II from Escherichia coli.

Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate-dependent enzyme that catalyzes the condensation of pyruvate with either another pyruvate molecule (product acetolactate) or 2-ketobutyrate (product acetohydroxybutyrate) as the first common step in the biosynthesis of branched-chain amino acids in plants, bacteria, algae, and fungi. AHAS isozyme II from Escherichia coli exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as acceptor, which was shown to result from a stronger hydrophobic interaction of the ethyl substituent of 2-KB with the side chain of Trp464 in multiple, apparently committed steps of catalysis. Here, we have elucidated the molecular determinants conferring specificity for pyruvate as the sole physiological donor substrate.

Structural and kinetic studies on native intermediates and an intermediate analogue in benzoylformate decarboxylase reveal a least motion mechanism with an unprecedented short-lived predecarboxylation intermediate.

The thiamin diphosphate- (ThDP-) dependent enzyme benzoylformate decarboxylase (BFDC) catalyzes the nonoxidative decarboxylation of benzoylformic acid to benzaldehyde and carbon dioxide. To date, no structural information for a cofactor-bound reaction intermediate in BFDC is available. For kinetic analysis, a chromophoric substrate analogue was employed that produces various absorbing intermediates during turnover but is a poor substrate with a 10(4)-fold compromised kcat.

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.