Enzymatic breakdown of threonine by threonine aldolase

1. The enzyme which splits threonine to acetaldehyde and glycine has been partially purified from rat liver (five- to sixfold purification) and the name threonine aldolase proposed for it. 2. The general properties of threonine aldolase have been studied. The enzyme is unstable to a pH below 5. The pH optimum of the enzyme reaction is at 7.5-7.7. The initial rate of production of acetaldehyde is proportional to the enzyme concentration, and when the enzyme concentration is constant, the production of acetaldehyde is proportional to the time, provided that the substrate is in excess. The enzyme is inhibited by the carbonyl group reagent, hydroxylamine. Attempts to demonstrate that pyridoxal phosphate is a cofactor were unsuccessful. 3. The enzyme splits only L-allothreonine and L-threonine and is inactive against the D-forms of these amino acids. 4. The enzyme reaction on DL-allothreonine follows first order kinetics. From the first order velocity constants and the initial rates of the rates of the reaction at various substrate concentrations the Michaelis constant, Ks, for this substrate has been evaluated. Michaelis constants have also been determined for threonine. 5. The optimum temperature for the enzymatic breakdown of DL-allothreonine at pH 7.65 was found to be 50 degrees C. in phosphate buffer and 48 degrees C. in tris-maleate buffer. The rate of thermal inactivation of the enzyme threonine aldolase obeys a first order reaction. The heat of thermal inactivation was calculated by the aid of the van't Hoff-Arrhenius equation to be 43,000 cal. per mole for the temperature range 41.2-46.6 degrees C. 6. Equivalent amounts of acetaldehyde and glycine were formed from DL-allothreonine and the enzymatic breakdown of DL-allothreonine was found to be irreversible.     

Role of threonine dehydrogenase in Escherichia coli threonine degradation.

Threonine was used as nitrogen source by Escherichia coli K-12 through a pathway beginning with the enzyme threonine dehydrogenase. The 2-amino-3-ketobutyrate formed was converted to glycine, and the glycine was converted to serine, which acted as the actual nitrogen donor. The enzyme formed under anaerobic conditions and known as threonine deaminase (biodegradative) is less widespread than threonine dehydrogenase and may be involved in energy metabolism rather than in threonine degradation per se.     

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.     

Analysis of threonine uptake in Escherichia coli threonine production strains

Threonine uptake in Escherichia coli wild-type and in threonine-producing strains decreased throughout threonine production. In contrast to previously published results, the SstT uptake system is not the sole serine/threonine permease in E. coli, since a novel transport system was detected in an sstT deletion strain.     

Production of threonine byBrevibacterium flavum containing threonine biosynthesis genes fromEscherichia coli

Genes of the threonine operon ofEscherichia coli were used for the construction of aBrevibacterium flavum strain excreting threonine. Using the shuttle vector pCEM300 and a newly constructed shuttle vector pEC71 (7.1 kb, Km^r/Nm^r), various plasmids carryingE. coli thr genes were prepared. Mutants resistant to the threonine analog 2-amino-3-hydroxyvaleric acid (AHV) were isolated after the ethyl methanesulfonate treatment ofB. flavum carrying these recombinant plasmids. A mutant ofB. flavum CCM 351 carrying the cloned genesthrA andthrB accumulated 12 g/L of threonine after 48 h of cultivation.     

Minor threonine dehydratase encoded within the threonine synthetic region of Bacillus subtilis

Challenging auxotrophs on metabolites that are precursors of a biosynthetic step involving a mutated enzyme has revealed a new class of suppressor mutations which act by derepressing a minor enzyme activity not normally detected in the wild-type strain. These indirect, partial suppressor mutations which allow isoleucine auxotrophs to grow on homoserine or threonine have been analyzed to determine their effect on enzymes involved in the biosynthesis of these amino acids. It has been found that one class of these suppressor mutations (sprA) leads to the derepression of homoserine kinase, homoserine dehydrogenase, and a minor threonine dehydratase that is not sufficiently active to be detected in the wild-type strain. The gene encoding this second threonine dehydratase activity has been found to be located between the structural genes for homoserine kinase and homoserine dehydrogenase. The results of these experiments indicate that plating of auxotrophs on precursors of a biosynthetic step involving mutated enzymes could prove to be a valuable method for the detection of regulatory mutants as well as a possible tool in studying the evolution of biochemical pathways.     

Threonine diffusion and threonine transport in Corynebacterium glutamicum and their role in threonine production

Transmembrane threonine fluxes (i.e., uptake, diffusion, and carrier-mediated excretion) all contribut-ing to threonine production by a recombinant strain of Corynebacterium glutamicum, were analyzed and quantitated. A threonine-uptake carrier that transports threonine in symport with sodium ions was identified. Under production conditions (i.e., when internal threonine is high), this uptake system catalyzed predominantly threonine/threonine exchange. Threonine export via the uptake system was excluded. Threonine efflux from the cells was shown to comprise both carrier-mediated excretion and passive diffusion. The latter process was analyzed after inhibition of all carrier-mediated fluxes. Threonine diffusion was found to proceed with a first-order rate constant of 0.003 min^–1 or 0.004 μl min^–1 (mg dry wt.)^–1, which corresponds to a permeability of 8 × 10^–10 cm s^–1. According to this permeability, less than 10% of the efflux observed under optimal conditions takes place via diffusion, and more than 90% must result from the activity of the excretion carrier. In addition, the excretion carrier was identified by (1) inhibition of its activity by amino acid modifying reagents and (2) its dependence on metabolic energy in the form of the membrane potential. Activity of the excretion system depended on the membrane potential, but not on the presence of sodium ions. Threonine export in antiport against protons is proposed. Received: 25 August 1995 / Accepted: 18 October 1995     

Metabolism of threonine by Fusaria growth on threonine as the sole carbon and nitrogen source

Three strains ofFusarium supporting aerobic growth onl-threonine as the sole source of energy and carbon and nitrogen, initially metabolised threonine to acetyl-CoA and glycine via induciblel-threonine:NAD⁺ dehydrogenase plus 2-amino-3-oxobutyrate:CoA ligase activities. Comparative enzyme induction patterns after growth of the three strains on a wide range of carbon sources indicated that the glycine produced by the NAD⁺ plus CoASH-dependent cleavage of threonine was subsequently utilised as an energy source and biosynthetic precursor via the glycine-serine pathway, pyruvate carboxylase, and ultimately the TCA cycle. Acetyl-CoA, the second initial C₂ threonine catabolism product, was subsequently assimilated via a combined TCA plus glyoxylate cycle.     

Metabolism of threonine in newborn infants

Threonine kinetics, threonine oxidative pathway, and the relationship between threonine and whole body protein turnover were quantified in 10 healthy term infants during the first 48 h after birth. The kinetic data were obtained 6 h after the last feed (fasting) and in response to formula feeding, using [U-(13)C(4),(15)N]threonine, [(2)H(5)]phenylalanine, and [(15)N]glycine tracers. The rate of carbon dioxide production (Vco(2)) and (13)C enrichment of the expired CO(2) were measured to quantify the rate of oxidation of threonine. The rate of appearance (R(a)) of threonine (136 +/- 37 micromol.kg(-1).h(-1)) was higher in newborn infants than that reported in adults. Formula feeding resulted in a significant decrease in threonine R(a) (P < 0.05). A significant positive correlation was seen between phenylalanine R(a) and threonine R(a), both during fasting and after formula feeding (r(2) = 0.65). In contrast to a 1:1 ratio of threonine and phenylalanine in mixed muscle protein, threonine R(a) relative to phenylalanine R(a) was 2.2 +/- 0.4. The fractional rate of threonine flux oxidized was 20% during fasting and 26% (P < 0.05) in response to nutrient administration. There was a significant correlation between plasma threonine concentration and threonine oxidation (r(2) = 0.75). No measurable incorporation of threonine in plasma glycine was seen. These data suggest that threonine is exclusively degraded by the glycine-independent serine/threonine dehydratase pathway. A higher flux of threonine relative to phenylalanine indicates higher turnover of threonine enriched proteins.     

Viral serine/threonine protein kinases

Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.     

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.