苏氨酸醛缩酶的催化机理、分子改造及合成应用

苏氨酸醛缩酶催化醛和甘氨酸羟醛缩合,一步反应即可构建产物β-羟基-α-氨基酸的两个手性中心,从原子经济性和环境影响角度,是非常具有潜力的绿色合成光学纯β-羟基-α-氨基酸的方式之一.多个不同生物来源的苏氨酸醛缩酶得到分离和表征,较低的β-碳立体选择性以及反应过程中动力学和热力学控制难题,使其在合成应用中面临很大挑战.文...     

Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool

Amino acids are not only fundamental protein constituents but also serve as precursors for many essential plant metabolites. Although amino acid biosynthetic pathways in plants have been identified, pathway regulation, catabolism, and downstream metabolite partitioning remain relatively uninvestigated. Conversion of Thr to Gly and acetaldehyde by Thr aldolase (EC 4.1.2.5) was only recently shown to play a role in plant amino acid metabolism. Whereas one Arabidopsis thaliana Thr aldolase (THA1) is expressed primarily in seeds and seedlings, the other (THA2) is expressed in vascular tissue throughout the plant. Metabolite profiling of tha1 mutants identified a >50-fold increase in the seed Thr content, a 50% decrease in seedling Gly content, and few other significant metabolic changes. By contrast, homozygous tha2 mutations cause a lethal albino phenotype. Rescue of tha2 mutants and tha1 tha2 double mutants by overproduction of feedback-insensitive Thr deaminase (OMR1) shows that Gly formation by THA1 and THA2 is not essential in Arabidopsis. Seed-specific expression of feedback-insensitive Thr deaminase in both tha1 and tha2 Thr aldolase mutants greatly increases seed Ile content, suggesting that these two Thr catabolic enzymes compete for a common substrate pool.     

Inducibility and some properties of the threonine dehydratase of sheep liver

The threonine dehydratase extracted from sheep liver is in an essentially inactive form, referred to here as the precursor, but can be activated by incubation at high temperature in alkaline solution. A method for purification of the activated enzyme to a state approaching homogeneity has been devised. The activated enzyme catalyses the deamination of serine but rapidly loses activity during the process. The enzyme inactivated by incubation with serine can be reactivated under the same conditions as those described for the precursor, the kinetic parameters being the same in both cases. Individual sheep livers differ widely in their content of threonine dehydratase, and the possible role of dietary factors in this variability has been examined. The average amount of the enzyme in the livers of sheep fed on high-plant-protein diets did not differ significantly from that in livers obtained from slaughterhouses, but was higher than that in livers from animals fed only on grass or normal concentrates. Large increases in enzyme concentration were, however, obtained when sheep were injected intraperitoneally with a protein hydrolysate. The enzyme from the livers of these sheep could be purified by the same technique and to the same specific activity as the enzyme from the livers of control sheep.     

Comparison of conformational properties of proline and threonine residues

The conformational role of Thr was investigated by 13C-n.m.r. and CD methods using a following series of tetrapeptides: Thr-Ala-Ala-Ala, Ala-Thr-Ala-Ala, Ala-Ala-Thr-Ala and Ala-Ala-Ala-Thr. It was found that introduction of Thr in every position of the tetraalanine peptide chain distinctly influences conformational equilibria of the peptides. An increase of beta-turn forms in conformational equilibria is induced by ionization of the terminal carboxyl group, independent of threonine position in the peptide chain. Threonine in position 1 or 3 of the peptide chain seems to have some importance for beta-turn formation in acid solution.     

Purification and properties of threonine aldolase from Clostridium pasterianum

1. Threonine aldolase was purified about 200-fold in 10% yield from Clostridium pasteurianum and its properties were examined. The final preparation gave three bands after ionophoresis on polyacrylamide gel. 2. The purified enzyme was shown to produce glycine and acetaldehyde in stoicheiometric amounts from threonine. The reverse reaction was demonstrated qualitatively. 3. The enzyme has a broad pH optimum at 6.5–7.0. 4. The enzyme is highly specific for l-threonine. 5. The enzyme is completely inhibited by 1mm concentrations of hydroxylamine and semicarbazide. Activity is decreased to 20% of the original by treatment with cysteine plus mercaptoethanol; most of the loss is regained on incubation with pyridoxal phosphate. It is concluded that pyridoxal phosphate is a prosthetic group. 6. The relationship between velocity and substrate concentration is atypical but indicates a K_m value of 0.42mm. 7. The enzyme was demonstrated in several other strictly anaerobic bacteria.     

Influence of threonine exporters on threonine production in Escherichia coli

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.     

Threonine aldolases—screening, properties and applications in the synthesis of non-proteinogenic β-hydroxy-α-amino acids

Threonine aldolases (TAs) constitute a powerful tool for catalyzing carbon–carbon bond formations in synthetic organic chemistry, thus enabling an enantio- and diastereoselective synthesis of β-hydroxy-α-amino acids. Starting from the achiral precursors glycine and an aldehyde, two new stereogenic centres are formed in this catalytic step. The resulting chiral β-hydroxy-α-amino acid products are important precursors for pharmaceuticals such as thiamphenicol, a l-threo-phenylserine derivative or l-threo-3,4-dihydroxyphenylserine. TAs are pyridoxal-5-phosphate-dependent enzymes, which, in nature, catalyze the cleavage of l-threonine or l-allo-threonine to glycine and acetaldehyde in a glycine biosynthetic pathway. TAs from a broad number of species of bacteria and fungi have been isolated and characterised as biocatalysts for the synthesis of β-hydroxy-α-amino acids. In this review, screening methods to obtain novel TAs, their biological function, biochemical characterisation and preparative biotransformations with TAs are described.     

Studies on the neurochemical properties of cystathionine

Following the intracerebral administration of [³⁵S]cystathionine, the synaptosome fraction of rat brain was labelled, the greatest uptake of amino acid being associated with hypothalamus.The uptake of [³⁵S]cystathionine by synaptosome preparations isolated from different regions of brain, was typical of that exhibited by amino acids which are not neurotransmitters.Depolarization of the synaptic membrane had no effect on the efflux of [³⁵S]cystathionine from preloaded synaptosomes.The intracerebral administration of cystathionine resulted in an elevation of the levels of brain cyclic AMP, the effect being particularly evident in the cerebellum. Attempts to reproduce this effect in vitro were unsuccessful.