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.     

A study of the metabolism of l-αγ-diaminobutyric acid in a Xanthomonas species

1. l-αγ-Diaminobutyric acid is metabolized in Xanthomonas sp. to aspartic β-semialdehyde, aspartic acid and oxaloacetic acid. 2. Aspartic β-semialdehyde is formed from diaminobutyric acid by a pyruvate-dependent γ-transamination. 3. The transaminase has a pH optimum of 9 and exhibits a high degree of substrate specificity, as analogues of diaminobutyric acid and pyruvate are inert in the system. The transaminase is inhibited by carbonyl-binding agents such as hydroxylamine. 4. Aspartic acid is formed from aspartic β-semialdehyde by an NAD⁺-dependent dehydrogenation. 5. The dehydrogenase has a pH optimum of 8·5 and is a thiol enzyme. It is specific for aspartic β-semialdehyde but analogues of NAD⁺ such as 3-acetylpyridine–adenine dinucleotide and deamino-NAD are partly active in the system. 6. The significance of these reactions is discussed in relation to diaminobutyric acid metabolism in plants and mammalian systems.     

A Mouse Model of L-2-Hydroxyglutaric Aciduria,a Disorder of Metabolite Repair

The purpose of the present work was to progress in our understanding of the pathophysiology of L-2-hydroxyglutaric aciduria, due to a defect in L-2-hydroxyglutarate dehydrogenase, by creating and studying a mouse model of this disease. L-2-hydroxyglutarate dehydrogenase-deficient mice (l2hgdh ^-/-) accumulated L-2-hydroxyglutarate in tissues, most particularly in brain and testis, where the concentration reached ≈ 3.5 μmol/g. Male mice showed a 30% higher excretion of L-2-hydroxyglutarate compared to female mice, supporting that this dicarboxylic acid is partially made in males by lactate dehydrogenase C, a poorly specific form of this enzyme exclusively expressed in testes. Involvement of mitochondrial malate dehydrogenase in the formation of L-2-hydroxyglutarate was supported by the commensurate decrease in the formation of this dicarboxylic acid when down-regulating this enzyme in mouse l2hgdh ^-/- embryonic fibroblasts. The concentration of lysine and arginine was markedly increased in the brain of l2hgdh ^-/- adult mice. Saccharopine was depleted and glutamine was decreased by ≈ 40%. Lysine-α-ketoglutarate reductase, which converts lysine to saccharopine, was inhibited by L-2-hydroxyglutarate with a Ki of ≈ 0.8 mM. As low but significant activities of the bifunctional enzyme lysine-α-ketoglutarate reductase/saccharopine dehydrogenase were found in brain, these findings suggest that the classical lysine degradation pathway also operates in brain and is inhibited by the high concentrations of L-2-hydroxyglutarate found in l2hgdh ^-/- mice. Pathological analysis of the brain showed significant spongiosis. The vacuolar lesions mostly affected oligodendrocytes and myelin sheats, as in other dicarboxylic acidurias, suggesting that the pathophysiology of this model of leukodystrophy may involve irreversible pumping of a dicarboxylate in oligodendrocytes. Neurobehavioral testing indicated that the mice mostly suffered from a deficit in learning capacity. In conclusion, the findings support the concept that L-2-hydroxyglutaric aciduria is a disorder of metabolite repair. The accumulation of L-2-hydroxyglutarate exerts toxic effects through various means including enzyme inhibition and glial cell swelling.     

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.     

Efficient biosynthesis of α-aminoadipic acid via lysine catabolism in Escherichia coli

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.     

Lysine-ketoglutarate reductase activity in developing maize endosperm

Lysine-ketoglutarate reductase activity was detected and characterized in the developing endosperm of maize (Zea mays L.). The enzyme showed specificity for its substrates: lysine, α-ketoglutarate, and NADPH. Formation of the reaction product saccharopine was demonstrated. The pH optimum of the enzyme was close to 7, and the K_m for lysine and α-ketoglutarate were 5.2 and 1.8 millimolar, respectively.     

Properties of partially purified saccharopine dehydrogenase from human placenta.

Saccharopine dehydrogenase was previously purified 380-fold from human placenta. The enzyme was shown to catalyze the formation of α-aminoadipic-δ-semialdehyde and glutamate from saccharopine, to have a molecular weight of 480,000 on gel filtration, and not to be separable from l-lysine-α-ketoglutarate reductase. Additional properties of the saccharopine dehydrogenase are now described. The pH optimum for the conversion of saccharopine to glutamate and α-aminoadipic-δ-semialdehyde is 8.5 in Tris-HCl buffer and 8.9 in 2-amino-2-methyl-1,3-propanediol buffer. The specificity of the enzyme for Saccharopine and NAD and the inhibition by glutamate and product analogs were tested. It was found the NADP was the only cofactor that could replace NAD in the enzyme reaction and that several NAD analogs were reaction inhibitors. Glutamate was found to be only moderately effective as an inhibitor. Initial velocity studies revealed that the enzyme has an ordered reaction mechanism. The true K_m values for saccharopine and NAD are 1.15 mm and 0.0645 mm, respectively.     

Depletion of Glycolytic Intermediates Plays a Key Role in Glucose-Phosphate Stress in Escherichia coli

In bacteria like Escherichia coli, the accumulation of glucose-6-phosphate (G6P) or its analogs such as α-methyl glucoside-6-phosphate (αMG6P) results in stress that appears in the form of growth inhibition. The small RNA SgrS is an essential part of the response that helps E. coli combat glucose-phosphate stress; the growth of sgrS mutants during stress caused by αMG is significantly impaired. The cause of this stress is not currently known but may be due to either toxicity of accumulated sugar-phosphates or to depletion of metabolic intermediates. Here, we present evidence that glucose-phosphate stress results from depletion of glycolytic intermediates. Addition of glycolytic compounds like G6P and fructose-6-phosphate rescues the αMG growth defect of an sgrS mutant. These intermediates also markedly decrease induction of the stress response in both wild-type and sgrS strains grown with αMG, implying that cells grown with these intermediates experience less stress. Moreover, αMG transport assays confirm that G6P relieves stress even when αMG is taken up by the cell, strongly suggesting that accumulated αMG6P per se does not cause stress. We also report that addition of pyruvate during stress has a novel lethal effect on the sgrS mutant, resulting in cell lysis. The phosphoenolpyruvate (PEP) synthetase PpsA, which converts pyruvate to PEP, can confer resistance to pyruvate-induced lysis when ppsA is ectopically expressed in the sgrS mutant. Taken as a whole, these results provide the strongest evidence thus far that depletion of glycolytic intermediates is at the metabolic root of glucose-phosphate stress.     

Cnr interferes with dimerization of the replication protein alpha in phage-plasmid P4

DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein α. In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation). Mutations in α (α^cr) that prevent regulation by Cnr cause P4 over-replication and cell death. Using the two-hybrid system in Saccharomyces cerevisiae and a system based on λ immunity in E.coli for in vivo detection of protein–protein interactions, we found that: (i) α protein interacts with Cnr, whereas α^cr proteins do not; (ii) both α–α and α^cr–α^cr interactions occur and the interaction domain is located within the C-terminal of α; (iii) Cnr–Cnr interaction also occurs. Using an in vivo competition assay, we found that Cnr interferes with both α–α and α^cr–α^cr dimerization. Our data suggest that Cnr and α interact in at least two ways, which may have different functional roles in P4 replication control.     

Lysine-2,3-Aminomutase and β-Lysine Acetyltransferase Genes of Methanogenic Archaea Are Salt Induced and Are Essential for the Biosynthesis of Nɛ-Acetyl-β-Lysine and Growth at High Salinity

The compatible solute N-acetyl-β-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of N-acetyl-β-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and β-lysine acetyltransferase (ablB), which are assumed to catalyze N-acetyl-β-lysine formation from α-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei Gö1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei Gö1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Δabl mutants of M. maripaludis no longer produced N-acetyl-β-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for N-acetyl-β-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.

     

Mutation of OsALDH7 causes a yellow-colored endosperm associated with accumulation of oryzamutaic acid A in rice

Aldehyde dehydrogenase proteins consist of a superfamily and the family 7 (ALDH7) is a typical group with highly conserved proteins across species. It catalyzes oxidation of α-aminoadipic semialdehyde (AASA) in lysine degradation, participates in protection against hyperosmotic stress, and detoxifies aldehydes in human; however, its function in plants has been much less documented. Here we reported a mutant with yellow-colored endosperm in rice, and showed that the yellow endosperm was caused by mutation of OsALDH7. OsALDH7 is expressed in all tissues detected, with the highest level in mature seeds. We found that oryzamutaic acid A accumulated during late seed development and after a year-long storage in the colored endosperm, whereas it was undetectable in the wild type endosperm. Moreover, lysine degradation was enhanced in yeast over-expressing OsALDH7 and as a result, content of lysine, glutamate and saccharopine was changed, suggesting a role of OsALDH7 in lysine catabolism.     

The regulation of propionate oxidation in Prototheca zopfii

1. Whole cell suspensions of Prototheca zopfii grown on propionate oxidize propionate, acrylate, malonic semialdehyde and acetate immediately, whereas acetate-grown cells only oxidize acrylate or propionate rapidly after a lag of 20–30min. This adaptation to propionate is slowed down by 8-azaguanine or p-fluorophenylalanine, and is not influenced by adding an ammonium salt or an amino acid mixture. 2. The adaptation involves induction of the enzymes of β-oxidation of propionate. 3. A small proportion (5–8%) of the activities of propionyl-CoA dehydrogenase, β-hydroxypropionate dehydrogenase and malonic semialdehyde dehydrogenase are consistently associated with mitochondria isolated from propionate-grown cells. 4. Such mitochondria will oxidize propionyl-CoA, β-hydroxypropionate and malonic semialdehyde, and the respiration rates with these substrates in the presence of inorganic phosphate are ADP-dependent. 5. Mitochondria from acetate-grown cells do not contain detectable activities of the enzymes of propionate oxidation.     

Catabolite repression of the lac operon. Separate repression of two enzymes

1. Catabolite repression of β-galactosidase and of thiogalactoside transacetylase was studied in several strains of Escherichia coli K 12, in an attempt to show whether a single site within the structural genes of the lac operon co-ordinately controls translational repression for the two enzymes. In all experiments the rate of synthesis of the enzymes was compared in glycerol–minimal medium and in glucose–minimal medium. 2. In a wild-type strain, glucose repressed the synthesis of the two enzymes equally. 3. The possibility that repression was co-ordinate was investigated by studies of mutant strains that carry deletions in the genes for β-galactosidase or galactoside permease or both. In all of the strains with deletions, the repression of thiogalactoside transacetylase persisted, and it is concluded that there is no part of the structural gene for β-galactosidase that is essential for catabolite repression of thiogalactoside transacetylase. 4. Subculture of one strain through several transfers in rich medium greatly increased its susceptibility to catabolite repression by glucose. It is concluded that unknown features of the genotype can markedly affect sensitivity to catabolite repression. 5. These results make it clear that one cannot draw valid conclusions about the effect of known genotypic differences on catabolite repression from a comparison of two separate strains; to study the effect of a particular genetic change in a lac operon it is necessary to construct a partially diploid strain so that catabolite repression suffered by one lac operon can be compared with that suffered by another. 6. Four such partial diploids were constructed. In all of them catabolite repression of β-galactosidase synthesized by one operon was equal in extent to catabolite repression of thiogalactoside transacetylase synthesized by the other. 7. Taken together, these results suggest that catabolite repression of β-galactosidase and thiogalactoside transacetylase is separate but equal.     

Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli

Summary In bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast. In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate. The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate. Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced. The popC gene of E. coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase. Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E. coli glutamate 1-semialdehyde aminotransferases. The cyanobacterial and barley enzymes share 72% identical residues. The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.     

Cross Dimerization of Amyloid-β and αSynuclein Proteins in Aqueous Environment: A Molecular Dynamics Simulations Study

Self-assembly of the intrinsically unstructured proteins, amyloid beta (Aβ) and alpha synclein (αSyn), are associated with Alzheimer’s Disease, and Parkinson’s and Lewy Body Diseases, respectively. Importantly, pathological overlaps between these neurodegenerative diseases, and the possibilities of interactions between Aβ and αSyn in biological milieu emerge from several recent clinical reports and in vitro studies. Nevertheless, there are very few molecular level studies that have probed the nature of spontaneous interactions between these two sequentially dissimilar proteins and key characteristics of the resulting cross complexes. In this study, we have used atomistic molecular dynamics simulations to probe the possibility of cross dimerization between αSyn_1–95 and Aβ _1–42, and thereby gain insights into their plausible early assembly pathways in aqueous environment. Our analyses indicate a strong probability of association between the two sequences, with inter-protein attractive electrostatic interactions playing dominant roles. Principal component analysis revealed significant heterogeneity in the strength and nature of the associations in the key interaction modes. In most, the interactions of repeating Lys residues, mainly in the imperfect repeats ‘KTKEGV’ present in αSyn_1–95 were found to be essential for cross interactions and formation of inter-protein salt bridges. Additionally, a hydrophobicity driven interaction mode devoid of salt bridges, where the non-amyloid component (NAC) region of αSyn_1–95 came in contact with the hydrophobic core of Aβ _1–42 was observed. The existence of such hetero complexes, and therefore hetero assembly pathways may lead to polymorphic aggregates with variations in pathological attributes. Our results provide a perspective on development of therapeutic strategies for preventing pathogenic interactions between these proteins.     

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP⁺-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.     

Cloning and Sequencing of a 2,5-Dichlorohydroquinone Reductive Dehalogenase Gene Whose Product Is Involved in Degradation of γ-Hexachlorocyclohexane by Sphingomonas paucimobilis

Sphingomonas (formerly Pseudomonas) paucimobilis UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a halogenated organic insecticide, as a sole carbon and energy source. In a previous study, we showed that γ-HCH is degraded to 2,5-dichlorohydroquinone (2,5-DCHQ) (Y. Nagata, R. Ohtomo, K. Miyauchi, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 176:3117–3125, 1994). In the present study, we cloned and characterized a gene, designated linD, directly involved in the degradation of 2,5-DCHQ. The linD gene encodes a peptide of 343 amino acids and has a low level of similarity to proteins which belong to the glutathione S-transferase family. When LinD was overproduced in Escherichia coli, a 40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis revealed that expression of the linD gene was induced by 2,5-DCHQ in S. paucimobilis UT26. Thin-layer chromatography and gas chromatography-mass spectrometry analyses with the LinD-overexpressing E. coli cells revealed that LinD converts 2,5-DCHQ rapidly to chlorohydroquinone (CHQ) and also converts CHQ slowly to hydroquinone. LinD activity in crude cell extracts was increased 3.7-fold by the addition of glutathione. All three of the Tn5-induced mutants of UT26, which lack 2,5-DCHQ dehalogenase activity, had rearrangements or a deletion in the linD region. These results indicate that LinD is a glutathione-dependent reductive dehalogenase involved in the degradation of γ-HCH by S. paucimobilis UT26.