Lysine biosynthesis in selected pathogenic fungi: characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans.

The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. Until now, this unique metabolic pathway has never been investigated in the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. Five of the eight enzymes (homocitrate synthase, homoisocitrate dehydrogenase, alpha-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase) of the alpha-aminoadipate pathway and glucose-6-phosphate dehydrogenase, a glycolytic enzyme used as a control, were demonstrated in wild-type cells of these organisms. All enzymes were present in Saccharomyces cerevisiae and the pathogenic organisms except C. neoformans 32608 serotype C, which exhibited no saccharopine reductase activity. The levels of enzyme activity varied considerably from strain to strain. Variation among organisms was also observed for the control enzyme. Among the pathogens, C. albicans exhibited much higher homocitrate synthase, homoisocitrate dehydrogenase, and alpha-aminoadipate reductase activities. Seven lysine auxotrophs of C. albicans and one of Candida tropicalis were characterized biochemically to determine the biochemical blocks and gene-enzyme relationships. Growth responses to alpha-aminoadipate- and lysine-supplemented media, accumulation of alpha-aminoadipate semialdehyde, and the lack of enzyme activity revealed that five of the mutants (WA104, WA153, WC7-1-3, WD1-31-2, and A5155) were blocked at the alpha-aminoadipate reductase step, two (STN57 and WD1-3-6) were blocked at the saccharopine dehydrogenase step, and the C. tropicalis mutant (X-16) was blocked at the saccharopine reductase step. The cloned LYS1 gene of C. albicans in the recombinant plasmid YpB1078 complemented saccharopine dehydrogenase (lys1) mutants of S. cerevisiae and C. albicans. The Lys1+ transformed strains exhibited significant saccharopine dehydrogenase activity in comparison with untransformed mutants. The cloned LYS1 gene has been localized on a 1.8-kb HindIII DNA insert of the recombinant plasmid YpB1041RG1. These results established the gene-enzyme relationship in the second half of the alpha-aminoadipate pathway. The presence of this unique pathway in the pathogenic fungi could be useful for their rapid detection and control.     

Conversion of Pipecolic Acid into Lysine in Penicillium chrysogenum Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the lys7 Gene Encoding Saccharopine Reductase

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.     

Effects of Supersuppressor Genes on Enzymes Controlling Lysine Biosynthesis in Saccharomyces

Yeast supersuppressor genes capable of masking the effects of several lysine mutant genes (ly(1-1), ly(9-1), ly(2-1)) were studied with respect to their effects on the respective enzymes (saccharopine dehydrogenase, saccharopine reductase, and alpha-amino-adipic acid reductase). In all strains tested, the supersuppressors functioned by allowing enzyme synthesis not found in the unsuppressed mutant. Studies by optical methods of saccharopine dehydrogenase and saccharopine reductase extracted from suppressed ly(1-1) and ly(9-1) cells, respectively, revealed that the K(m) values for these enzymes were significantly greater than those found in wild type. Saccharopine dehydrogenase from suppressed ly(9-1) cells was found to have K(m) values similar to wild type. These findings are consistent with the inference that a supersuppressor may act by enabling nonsense codons to be read, producing altered enzyme protein. Recent findings that lysine degradation in mammals may involve saccharopine and that the human diseases, hyperlysinemia and saccharopinuria, may be due to metabolic blocks in this route of lysine degradation suggest the ly(1-1) and ly(9-1) yeast mutants as models for the human condition and its possible euphenic treatment.     

Enzymatic measurement of saccharopine with saccharopine dehydrogenase

We have developed an enzymatic method for measuring saccharopine, a key intermediate in lysine metabolism. With the enzyme saccharopine dehydrogenase, saccharopine can be oxidized to lysine and alpha-ketoglutarate with the corresponding conversion of NAD to NADH. The natural equilibrium favors saccharopine formation, but using hydrazine to trap one of the products, alpha-ketoglutarate, shifts the reaction toward quantitative oxidation of saccharopine. A stable endpoint is reached in 15-20 min, and although high concentrations of alpha-ketoglutarate slow the reaction, the end product is fully recovered. Unlike previous assays this technique is specific, convenient, and capable of measuring saccharopine directly in protein-free biological fluids or extracts.     

Regulation of lysine catabolism through lysine-ketoglutarate reductase and saccharopine dehydrogenase in Arabidopsis.

In plant and mammalian cells, excess lysine is catabolized by a pathway that is initiated by two enzymes, namely, lysine-ketoglutarate reductase and saccharopine dehydrogenase. In this study, we report the cloning of an Arabidopsis cDNA encoding a bifunctional polypeptide that contains both of these enzyme activities linked to each other. RNA gel blot analysis identified two mRNA bands-a large mRNA containing both lysine-ketoglutarate reductase and saccharopine dehydrogenase sequences and a smaller mRNA containing only the saccharopine dehydrogenase sequence. However, DNA gel blot hybridization using either the lysine-ketoglutarate reductase or the saccharopine dehydrogenase cDNA sequence as a probe suggested that the two mRNA populations apparently are encoded by the same gene. To test whether these two mRNAs are functional, protein extracts from Arabidopsis cells were fractionated by anion exchange chromatography. This fractionation revealed two separate peaks-one containing both coeluted lysine-ketoglutarate reductase and saccharopine dehydrogenase activities and the second containing only saccharopine dehydrogenase activity. RNA gel blot analysis and in situ hybridization showed that the gene encoding lysine-ketoglutarate reductase and saccharopine dehydrogenase is significantly upregulated in floral organs and in embryonic tissues of developing seeds. Our results suggest that lysine catabolism is subject to complex developmental and physiological regulation, which may operate at gene expression as well as post-translational levels.     

真菌中赖氨酸生物合成途径研究进展

本文综述了真菌中赖氨酸的生物合成途径及途径中的关键基因——酵母氨酸脱氢酶基因,详细介绍了赖氨酸从头合成途径,阐述了酵母氨酸脱氢酶的作用机理及理化性质,以期为探索赖氨酸合成途径及途径中的基因提供资料和思路。     

Lysine catabolism in Haemonchus contortus and Teladorsagia circumcincta

Catabolism of lysine through the pipecolate, saccharopine and cadaverine pathways has been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Both enzymes of the saccharopine pathway (lysine ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH)) were active in L3 and adult worms of both species. All three enzymes which catabolise lysine to α-amino adipic semialdehyde via pipecolate (lysine oxidase (LO), Δ(1)-piperideine-2-carboxylate reductase (Pip2CR) and pipecolate oxidase (PipO)) were present in adult worms, whereas the pathway was incomplete in L3 of both species; Pip2CR activity was not detected in the L3 of either parasite species. In adult worms, the saccharopine pathway would probably be favoured over the pipecolate pathway as the K(m) for lysine was lower for LKR than for LO. Neither lysine dehydrogenase nor lysine decarboxylase activity was detected in the two parasite species. Enzyme activities and substrate affinities were higher for all five enzymes in adult worms than in L3. An unexpected finding was that both LKR and SDH were dual co-factor enzymes and not specific for either NAD(+) or NADP(+), as is the case in other organisms. This novel property of LKR/SDH suggests it could be a good candidate for anthelmintic targeting.     

Genetic manipulation of lysine catabolism in maize kernels

In plants, lysine catabolism is thought to be controlled by a bifunctional enzyme, lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). Lysine is converted to saccharopine, through condensation with alpha-ketoglutarate, by LKR, and subsequently to glutamate and alpha-aminoadipate-delta-semialdehyde by SDH. To investigate lysine catabolism in maize kernels, we generated transgenic plants with suppressed LKR/SDH activity in either endosperm or embryo. We found that the suppression of LKR/SDH in endosperm induced an increase in free lysine in developing endosperm, which peaked at 32 days after pollination. At later stages of kernel development, most of the free lysine was found in the embryo along with an elevated level of saccharopine. By combining endosperm LKR/SDH suppression with embryo LKR/SDH suppression through crosses, the saccharopine level in embryo was reduced and resulted in higher lysine accumulation in mature kernels. These results reveal new insights into how free lysine level is regulated and distributed in developing maize kernels and demonstrate the possibility of engineering high lysine corn via the suppression of lysine catabolism.     

Purification and Characterization of the Bifunctional Enzyme Lysine-Ketoglutarate Reductase-Saccharopine Dehydrogenase from Maize

The first enzyme of the lysine degradation pathway in maize (Zea mays L.), lysine-ketoglutarate reductase, condenses lysine and [alpha]-ketoglutarate into saccharopine using NADPH as a cofactor, whereas the second, saccharopine dehydrogenase, converts saccharopine to [alpha]-aminoadipic-[delta]-semialdehyde and glutamic acid using NAD+ or NADP+ as a cofactor. The reductase and dehydrogenase activities are optimal at pH 7.0 and 9.0, respectively. Both enzyme activities, co-purified on diethylaminoethyl-cellulose and gel filtration columns, were detected on nondenaturing polyacrylamide gels as single bands with identical electrophoretic mobilities and share tissue specificity for the endosperm. The highly purified preparation containing the reductase and dehydrogenase activities showed a single polypeptide band of 125 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native form of the enzyme is a dimer of 260 kD. Limited proteolysis with elastase indicated that lysine-ketoglutarate reductase and saccharopine dehydrogenase from maize endosperm are located in two functionally independent domains of a bifunctional polypeptide.     

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.     

Isolation and characterization of α-aminoadipate-δ-semialdehyde overproducing mutants from yeasts

Abstract We isolated from Candida maltosa mutants lacking saccharopine reductase ( lys9 ) and saccharopine dehydrogenase ( lys1 ). They accumulated α-aminoadipate-δ-semialdehyde (AASA) in the cell and excreted it into the culture medium. In the presence of 15 g glucose/l, 1.25 g NH₄H₂PO₄/l and 50 mg l -lysine/l in a minimal salt medium C. maltosa G285 ( lys1 ) produced about 80–90 mg AASA/l within 48 h. It is the first report of lysine-requiring yeast mutants that accumulate and excrete AASA. In contrast, Pichia guilliermondii lys9 mutants lacked this AASA overproduction. The AASA accumulation by C. maltosa mutants may be explained by the low feedback regulation of their homocitrate synthase and the equilibrium of the enzyme reactions involved in the lysine biosynthesis.     

In vitro dephosphorylation inhibits the activity of soybean lysine-ketoglutarate reductase in a lysine-regulated manner

In plant seeds, the essential amino acid lysine auto-regulates its own level by modulating the activity of its catabolic enzyme lysine-ketoglutarate reductase via an intracellular signaling cascade, mediated by Ca²⁺ and protein phosphorylation/dephosphorylation. In the present report, it has been further tested whether the activity of soybean lysine-ketoglutarate reductase, as well as that of saccharopine dehydrogenase, the second enzyme in the pathway of lysine catabolism, are modulated by direct phosphorylation of the bifunctional polypeptide containing both of these linked activities. Incubation of purified lysine-ketoglutarate reductase/ saccharopine dehydrogenase with casein kinase II resulted in a significant phosphorylation of the bifunctional enzyme. Moreover, in vitro dephosphorylation of the bifunctional polypeptide with alkaline phosphatase significantly inhibited the activity of lysine-ketoglutarate reductase, but not of its linked enzyme saccharopine dehydrogenase. The inhibitory effect of alkaline phosphatase on lysine-ketoglutarate reductase activity was dramatically stimulated by binding of lysine to the enzyme. Our results suggest that in plant seeds, active lysine-ketoglutarate reductase is a phospho-protein, and that its activity is modulated by opposing actions of protein kinases and phosphatases. Moreover, this modulation is subject to a compound regulation by lysine.     

Familial hyperlysinemia: enzyme studies, diagnostic methods, comments on terminology.

Enzyme assays of skin fibroblasts from five children with familial hyperlysinemia from unrelated families are added to the previous report of three children from two unrelated families. In all instances there was a deficiency in lysine-ketoglutarate reductase, saccharopine dehydrogenase, and saccharopine oxidoreductase activities. To complete the studies on the enzymes associated with familial hyperlysinemia, saccharopine oxidoreductase was partially purified from human liver and characterized. The activity did not separate from that of lysine-ketoglutarate reductase or saccharopine dehydrogenase. A simple screening test for familial hyperlysinemia is described based on the evolution of 14CO2 from lysine-14C by skin fibroblasts. The test differentiated, without overlap, seven patients with familial hyperlysinemia from control subjects. The relation of the two genetic entities involving lysine degradation, familial hyperlysinemia and saccharopinuria, is discussed. It is suggested that familial hyperlysinemia, type I, be applied to patients with major defects in lysine-ketoglutarate reductase and saccharopine dehydrogenase, and that familial hyperlysinemia, type II, to be used to designate patients in whom significant amounts of lysine-ketoglutarate reductase are retained. The nomenclature would be consistent with that of an analogous disease, orotic aciduria.     

Lysine metabolism in higher plants

Summary. The essential amino acid lysine is synthesised in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine. Enzyme kinetic studies and the analysis of mutants and transgenic plants that overaccumulate lysine, have indicated that the major site of the regulation of lysine synthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, there is strong evidence that lysine is also subject to catabolism in plants, specifically in the seed. The two enzymes involved in lysine breakdown, lysine 2-oxoglutarate reductase (also known as lysine α-ketoglutarate reductase) and saccharopine dehydrogenase exist as a single bifunctional protein, with the former activity being regulated by lysine availability, calcium and phosphorylation/dephosphorylation. Received December 21, 1999 Accepted February 7, 2000     

Two unlinked lysine genes (LYS9 and LYS14) are required for the synthesis of saccharopine reductase in Saccharomyces cerevisiae.

Three lysine auxotrophs, strains AU363, 7305d, and 8201-7A, were investigated genetically and biochemically to determine their gene loci, biochemical lesions, and roles in the lysine biosynthesis of Saccharomyces cerevisiae. These mutants were leaky and blocked after the alpha-aminoadipate step. Complementation studies placed these three mutations into a single, new complementation group, lys14. Tetrad analysis from appropriate crosses provided evidence that the lys14 locus represented a single nuclear gene and that lys14 mutants were genetically distinct from the other mutants (lys1, lys2, lys5, and lys9) blocked after the alpha-aminoadipate step. The lys14 strains, like lys9 mutants, accumulated alpha-aminoadipate-semialdehyde and lacked significant amounts of saccharopine reductase activity. On the bases of these results, it was concluded, therefore, that LYS9 and LYS14, two distinct genes, were required for the biosynthesis of saccharopine reductase in wild-type S. cerevisiae.     

A spectrophotometric assay for meso-diaminopimelate decarboxylase and L-alpha-amino-epsilon-caprolactam hydrolase

A spectrophotometric assay for the activities of mesodiaminopimelate decarboxylase and L-alpha-amino-epsilon-caprolactam hydrolase is described. With the commercially available enzyme saccharopine dehydrogenase lysine formed either by decarboxylation of meso-diaminopimelate or by hydrolysis of L-alpha-amino-epsilon-caprolactam is converted to saccharopine with the concomitant oxidation of NADH, which is monitored by the decrease in absorbance at 340 nm. For meso-diaminopimelate decarboxylase this assay can be performed either as an endpoint determination, when working with crude extracts, or as a continuous spectrophotometric assay of partially purified enzyme preparations. The activity of L-alpha-amino-epsilon-caprolactam hydrolase can only be assayed by the endpoint method because of the great differences in the pH optima of the hydrolase and the saccharopine dehydrogenase.