Enzymes of lysine metabolism from Coix lacryma-jobi seeds

Lysine, threonine, methionine and isoleucine are synthesized through the aspartate metabolic pathway. The concentrations of soluble lysine and threonine in cereal seeds are very low. Coix lacryma-jobi (coix) is a maize-related grass and the enzymological aspects of the aspartate metabolic pathway are completely unknown. In order to obtain information on lysine metabolism in this plant species, two enzymes involved in the biosynthesis of these amino acids (aspartate kinase 〚AK, EC 2.7.2.4〛 and homoserine dehydrogenase 〚HSDH, EC 1.1.1.3〛) and two enzymes involved in lysine degradation (lysine 2-oxoglutarate reductase 〚LOR, EC 1.5.1.8〛 and saccharopine dehydrogenase 〚SDH, EC 1.5.1.9〛) were isolated and partially characterized in coix seeds. AK activity was inhibited by threonine and lysine separately, suggesting the presence of two isoenzymes, one sensitive to lysine and the other sensitive to threonine, with the latter corresponding to approximately 60% of the total AK activity. In contrast to previous results from other plant species, the threonine-sensitive AK eluted from an ion exchange chromatography column at higher KCl concentration than the lysine-sensitive form. The HSDH activity extracted from the seeds was partially inhibited by threonine, indicating the presence of threonine-sensitive and threonine-resistant isoenzymes. LOR and SDH activities were detected only in the endosperm tissue and co-purified on an anion exchange chromatography column, suggesting that the two activities may be linked on a single bifunctional polypeptide, as observed for other plant species. One single SDH activity band was observed on non-denaturing PAGE gels. The K_m for saccharopine of SDH was determined as 0.143 mM and the K_m for NAD as 0.531 mM. Although SDH activity was shown to be stable, LOR, AK and HSDH were extremely unstable, under all buffer systems tested.     

Intestinal lysine metabolism is driven by the enteral availability of dietary lysine in piglets fed a bolus meal

Previous steady-state continuous-feeding studies have shown that the gut mucosa removes substantial amounts of both dietary and systemic amino acids. However, enteral nutrition is often given under non-steady-state conditions as a bolus meal, and this has been shown to influence systemic metabolism. Therefore, our aim was to quantify the relative metabolism of dietary and systemic lysine by the portal-drained viscera (PDV) under non-steady-state conditions after a single bolus meal. Five 28-day-old piglets implanted with arterial, venous, and portal catheters and with an ultrasonic portal flow probe were given an oral bolus feeding of a milk formula containing a trace quantity of intrinsically 15N-labeled soy protein and a continuous intravenous infusion of [U-13C]lysine for 8 h. Total lysine use by the PDV was maximal 1 h after the meal (891 micromol x kg(-1) x h(-1)) and was predominantly of dietary origin (89%), paralleling the enteral delivery of dietary lysine. Intestinal lysine use returned to a low level after 4 h postprandially and was derived exclusively from the arterial supply until 8 h. Cumulative systemic appearance of dietary lysine reached 44 and 80% of the ingested amount 4 and 8 h after the meal, respectively, whereas the PDV first-pass use of dietary lysine was 55 and 32% of the intake for these two periods, respectively. We conclude that the first-pass utilization rate of dietary lysine by the PDV is directly increased by the enteral lysine availability and that it is higher with a bolus than with continuous oral feeding.     

Biosynthesis and degradation of saccharopine, an intermediate of lysine metabolism

Lysine-2-oxoglutarate reductase was prepared from ox liver and its characteristics were examined. Its activity was totally inhibited in the presence of NH(4)Cl. Under conditions that inhibit saccharopine formation, and in the presence of NADP(+), ox liver mitochondria were found to catalyse the hydrolysis of saccharopine to lysine and alpha-oxoglutarate. The enzyme involved was named saccharopine oxidoreductase. It was partially purified and separated from lysine-oxoglutarate reductase. Comparison of the properties of these two enzymes showed that saccharopine degradation was stimulated under conditions that inhibit its formation. The effect of pH, various cofactors and stability during incubation confirm that saccharopine biosynthesis from, and degradation to, lysine are catalysed by two distinct enzymes.     

Coordination of energy and carbon metabolism in lysine producing Brevibacterium strains

The present paper deals with the coordination of energy metabolism, glucose consumption rate, glycolytic and TCA cycle enzyme activities in the lysine-producing bacterium Brevibacterium flavum. It is shown, that inhibition of the elctron transport chain causes changes of the following sequence:

at first, TCA cycle enzymes are activated;

secondly, TCA cycle enzyme activity decreases, and glycolytic enzyme activities as well as glucose transport rate increase; there is a slight increase in Q_o2 and a considerable one of O₂ consumption in cyanide-resistant respiration pathway;

thirdly, TCA cycle enzyme activities and glucose transport rate decrease.

It is supposed, that coordination of carbon and energy metabolism in B. flavum depends on intracellular ATP concentration or energy charge value.     

Protein lysine acylation and cysteine succination by intermediates of energy metabolism

In the past few years, several new protein post-translational modifications that use intermediates in metabolism have been discovered. These include various acyl lysine modifications (formylation, propionylation, butyrylation, crotonylation, malonylation, succinylation, myristoylation) and cysteine succination. Here, we review the discovery and the current understanding of these modifications. Several of these modifications are regulated by the deacylases, sirtuins, which use nicotinamide adenine dinucleotide (NAD), an important metabolic small molecule. Interestingly, several of these modifications in turn regulate the activity of metabolic enzymes. These new modifications reveal interesting connections between metabolism and protein post-translational modifications and raise many questions for future investigations.     

The primordial metabolism: an ancestral interconnection between leucine,arginine, and lysine biosynthesis

Background

It is generally assumed that primordial cells had small genomes with simple genes coding for enzymes able to react with a wide range of chemically related substrates, interconnecting different metabolic routes. New genes coding for enzymes with a narrowed substrate specificity arose by paralogous duplication(s) of ancestral ones and evolutionary divergence. In this way new metabolic pathways were built up by primordial cells. Useful hints to disclose the origin and evolution of ancestral metabolic routes and their interconnections can be obtained by comparing sequences of enzymes involved in the same or different metabolic routes. From this viewpoint, the lysine, arginine, and leucine biosynthetic routes represent very interesting study-models. Some of the lys, arg and leu genes are paralogs; this led to the suggestion that their ancestor genes might interconnect the three pathways. The aim of this work was to trace the evolutionary pathway leading to the appearance of the extant biosynthetic routes and to try to disclose the interrelationships existing between them and other pathways in the early stages of cellular evolution.

Results

The comparative analysis of the genes involved in the biosynthesis of lysine, leucine, and arginine, their phylogenetic distribution and analysis revealed that the extant metabolic "grids" and their interrelationships might be the outcome of a cascade of duplication of ancestral genes that, according to the patchwork hypothesis, coded for unspecific enzymes able to react with a wide range of substrates. These genes belonged to a single common pathway in which the three biosynthetic routes were highly interconnected between them and also to methionine, threonine, and cell wall biosynthesis. A possible evolutionary model leading to the extant metabolic scenarios was also depicted.

Conclusion

The whole body of data obtained in this work suggests that primordial cells synthesized leucine, lysine, and arginine through a single common metabolic pathway, whose genes underwent a set of duplication events, most of which can have predated the appearance of the last common universal ancestor of the three cell domains (Archaea, Bacteria, and Eucaryotes). The model proposes a relative timing for the appearance of the three routes and also suggests a possible evolutionary pathway for the assembly of bacterial cell-wall.

     

Enzymes of starch metabolism in developing grains of high lysine barley mutant

The absolute activities of ADPG(UDPG)-pyrophosphorylase, starch phosphorylase, ADPG(UDPG)-starch synthetase, NDP-kinase and inorganic pyrophosphatase have been studied in high lysine mutant barley Notch-2 and its parent NP 113 grains during development. In general, mutant Notch-2 grains had higher average activities of UDPG-pyrophosphorylase and starch phosphorylase and lower activity of ADPG(UDPG)-starch synthetase per grain than the parent NP 113 during grain development. Activities of NDP-kinase, ADPG-pyrophosphorylase and inorganic pyrophosphatase differed only to a small extent between the mutant Notch-2 and NP 113. It is suggested that the lower activity of ADPG(UDPG)-starch synthetase might be responsible for the reduced accumulation of starch in the mutant Notch-2 grain as compared with parent NP 113 during development.     

Enzymes of carbohydrate metabolism in developing grains of high lysine barley mutant

Activities of UDP(ADP)-sucrose synthetase, hexokinase, phosphoglucoisomerase and phosphoglucomutase have been studied in both a high lysine mutant barley, Notch-2 and its parent NP 113 during development. The Notch-2 mutant had higher average activities of UDP(ADP)-sucrose synthetase, hexokinase and phosphoglucomutase and lower activity on a grain basis of phosphoglucoisomerase than NP 113. This reflected the decreased dry matter in the mutant grain. In general, the average activities of hexokinase and phosphoglucomutase per grain did not differ significantly between Notch-2 and NP 113. It is suggested that the lower level of phosphoglucoisomerase in Notch-2 compared with NP 113 would limit the synthesis of glucose 6-phosphate, which in turn would result in reduced starch synthesis.     

Energetic and carbon metabolism regulation in lysine producing brevibacterium flavum strains

Coordinated regulation of carbon and energetic metabolism enzymes is characteristic of lysine producing strains of Brevibacterium flavum. ATP is the regulating effector and changes in the stationary ATP-concentration in cells cause certain alternations of enzyme activities in the basic metabolism pathways. The goal of the experiments was the use of the biochemical regulations of methabolism in order to increase the productivity of lysine biosynthesis. Following results were received:

• Activation of TCA-cycle enzymes is compulsory for intensive lysine biosynthesis.
• The most essential of several parallel electron transport pathways in the ETC of Br. flavum is the NADH dependent, cyanid resistant, hydroxamate sensitive oxydation pathway.
• Calculations have shown, that the most economical variant is the synthesis of oxalacetate (precursor of lysine) by PEP carboxylation. Therefore strains with elevated PEP-carboxylase activity synthesize lysine in a more economical way.

     

The reaction of p-nitrophenyl acetate with lysine and lysine derivatives

The kinetics of the reaction of p-nitrophenyl acetate with lysine and its derivatives has been investigated in water-dioxane solutions over the pH range 8.1–10.1. The reaction was found to be second order, and the unprotonated amino group was shown to be the reactive species. Intrinsic second-order rate constants were calculated.