Enzyme In Lysine Biosynthesis Research Articles

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Homocitrate synthase (HCS) catalyzes the first and committed step in the α-aminoadipate (AAA) pathway of lysine biosynthesis, which occurs in many fungi and certain archaea, and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is comprised of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed α/β-topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain covers the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex, the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Steady state kinetic assays and in vivo yeast growth assays on wild-type enzyme and active site mutants allow us to elucidate its catalytic mechanism, including the residues implicated in catalysis. Together these results yield new insights into the mechanism and regulation of HCS, which provide a platform to identify small molecule inhibitors of HCS that may be optimized and used as anti-fungal agents.

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed alpha/beta topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.

Conformationally constrained diketopimelic acid analogues as inhibitors of dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis and a potential antibiotic target. The enzyme catalyses the condensation of ( S)-aspartate semi-aldehyde (ASA) and pyruvate to form dihydrodipicolinate. Constrained diketopimelic acid derivatives have been designed as mimics of the acyclic enzyme-bound condensation product of ASA and pyruvate. Several of the compounds are shown to be active, slow-binding inhibitors with improved inhibition of DHDPS.

Inhibiting dihydrodipicolinate synthase across species: Towards specificity for pathogens?

Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis and an important antibiotic target. The specificity of a range of heterocyclic product analogues against DHDPS from three pathogenic species, Bacillus anthracis, Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus, and the evolutionarily related N-acetylneuraminate lyase, has been determined. The results suggest that the development of species-specific inhibitors of DHDPS as potential antibacterials is achievable.

Two New Irreversible Inhibitors of Dihydrodipicolinate Synthase: Diethyl (E,E)‐4‐Oxo‐2,5‐heptadienedioate and Diethyl (E)‐4‐Oxo‐2‐heptenedioate.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Two new irreversible inhibitors of dihydrodipicolinate synthase: diethyl ( E, E)-4-oxo-2,5-heptadienedioate and diethyl ( E)-4-oxo-2-heptenedioate

Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis and an important antibiotic target. The enzyme catalyses the condensation of ( S)-aspartate semialdehyde (ASA) and pyruvate to form dihydrodipicolinate. Two new irreversible inhibitors of dihydrodipicolinate synthase are reported, designed to mimic the acyclic enzyme-bound condensation product of ASA and pyruvate. These compounds represent an important new lead in the design of potent inhibitors for this enzyme.

Molecular genetic dissection and potential manipulation of lysine metabolism in seeds

Summary Lysine is among the most important essential amino acids in the diet of human and livestock because it is presented in severely limiting amounts in cereals and other important crops. Attempts to increase lysine levels in plants were made by reducing the sensitivity of the key enzyme in lysine biosynthesis, namely dihydrodipicolinate synthase, to feedback inhibition by lysine. However, these studies showed that in plant seeds, lysine accumulation is determined not only by the rate of its synthesis, but also by the rate of its catabolism via the α-amino adipic acid pathway. Our laboratory is currently studying the regulation of lysine catabolism in plants in order to explore potentials of reducing lysine catabolic fluxes in transgenic plants. In plants, like animals, lysine is catabolized via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. Yet SDH activity of the LKR/SDH polypeptide may be limiting in vivo because of its non-physiological pH optimum of activity. In some plants, like Arabidopsis and canola, this is overcome by the presence of an additional monofunctional SDH enzyme, which is encoded by the same locus that encodes the bifunctional LKR/SDH enzyme. Results from our, and other laboratories imply that attempts to generate high-lysine crop plants should take into account a seed-specific reduction of lysine catabolism.

The Arabidopsis thaliana dhdps gene encoding dihydrodipicolinate synthase, key enzyme of lysine biosynthesis, is expressed in a cell-specific manner.

Lysine synthesis in prokaryotes, some phycomycetes and higher plants starts with the condensation of L-aspartate-beta-semialdehyde (L-ASA) and pyruvate into dihydrodipicolinic acid. The enzyme that catalyses this step, dihydrodipicolinate synthase (DHDPS), is inhibited by the end-product lysine and is therefore thought to have a regulatory control on lysine synthesis. We have cloned and sequenced an Arabidopsis thaliana DNA fragment containing 900 bases upstream of the dhdps coding sequence. A transcriptional fusion of this fragment with the beta-glucuronidase reporter gene (uidA. Gus) was used to study the transcription properties of this promoter fragment (DS). No lysine-induced repression on transcription could be detected. Expression of DS-Gus activity in transformed Arabidopsis thaliana and Nicotiana tabacum was found to be cell type-specific. In the vegetative parts of the plant, GUS activity was located in meristems and young vasculature of roots, in vasculature of stem and leaves and in the meristems of young shoots. In flowers, high expression was found in the carpels, style, stigma, developing embryos, tapetum of young anthers and pollen. We demonstrated that the Arabidopsis DS promoter can direct its cell type-specific expression in a heterologous plant, Nicotiana tuabacum. The importance of transcriptional regulation of the dhdps gene, and in more general genes involved in amino acid biosynthesis, is discussed.

Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts

SummaryA major nutritional drawback of many crop plants is their low content of several essential amino acids, particularly lysine. The biosynthesis of lysine in plants is regulated by several feedback loops. Dihydrodipicolinate synthase (DHPS) from Escherichia coli, a key enzyme in lysine biosynthesis, which is considerably less sensitive to lysine accumulation than the endogenous plant enzyme has been expressed in chloroplasts of tobacco leaves. Expression of the bacterial enzyme was accompanied by a significant increase in the level of free lysine. No increase in protein‐bound lysine was evident. Free lysine accumulation was positively correlated with the level of DHPS activity in various transgenic plants. Compartmentalization of DHPS in the chloroplast was essential for its participation in lysine biosynthesis as no lysine overproduction was obtained in transgenic plants that expressed the bacterial enzyme in the cytoplasm. The elevated level of free lysine in the transgenic plants was sufficient to inhibit, in vivo, a second key enzyme in lysine biosynthesis, namely, aspartate kinase, with no apparent influence on lysine accumulation. The present report not only provides a better understanding of the regulation of lysine biosynthesis in higher plants but also offers a new strategy to improve the production of this essential amino acid.

Inhibition of α-aminoadipate-semialdehyde dehydrogenase from Trichosporon adeninovorans bvy lysine and lysine analogues

The α-aminoadipate-semialdehyde dehydrogenase (EC 1.2.1.31) of Trichosporon adeninovorans, an enzyme of lysine biosynthesis, was partially purified, some properties of the enzyme were studied and a novel regulatory pattern was found. The K m values of the enzyme were estimated to be 0.78 mM for α-aminoadipate, 1.0 mM for ATP, 0.23 mM for NADPH and 0.77 mM for MgCl 2. It is demonstrated that the enzyme can be regulated by lysine and lysine analogues. l-Lysine ( K i of 0.09 mM), S-(B- aminoethyl)- l-cysteine ( K i of 0.007 mM and δ-hydroxylysine ( K i of 1.65 mM) inhibited the enzyme activity. The inhibition was competitive with respect to α-aminodipate and non-competitive with respect to both ATP and NADPH.

Localization of the Enzymes of Quinolizidine Alkaloid Biosynthesis in Leaf Chloroplasts of Lupinus polyphyllus

Studies with purified chloroplasts of Lupinus polyphyllus LINDL. leaflets indicate that the first two enzymes of quinolizidine alkaloid biosynthesis, lysine decarboxylase and 17-oxosparteine synthase, are localized in the chloroplast stroma. Thus, both enzymes share the same subcellular compartment as the biosynthetic pathway of lysine, the precursor of quinolizidine alkaloids. The activity of diaminopimelate decarboxylase, the final enzyme in lysine biosynthesis, is about two to three orders of magnitude higher than that of the enzymes of alkaloid formation.

Charakterisierung der Saccharopin-Dehydrogenase aus Phycomyces blakesleeanus

Saccharopine-dehydrogenase, an enzyme of lysine biosynthesis via amino-adipate pathway could be demonstrated in Phycomyces blakesleeanus . The functionality of enzyme activity and volume of extracts is linear up to 0.5 ml extract. Between 20° and 40 °C the curve of enzyme activity is also linear. In in vitro tests the pH optimum of enzyme activity could be measured at pH 6.8. The Michaelis-Menten-constants were determined in extracts purified by fractionated ammonium sulfate precipitation (50-70%). The Km constant of saccharopine-dehydrogenase from Phycomyces blakesleeanus was for lysine 1.34 mM, a-ketoglutarate 0,23 mM, and for the coenzyme NADH 120µM. The significance of these enzyme data for the biosynthesis of lysine in the fungus was discussed.

A kinetic study of homocitrate synthetase activity in the yeast Saccharomycopsis lipolytica

1. 1.|A rapid method for estimating the activity of the first enzyme of lysine biosynthesis in yeasts (acetyl-coenzyme A: 2-ketoglutarate C-acetyl transferase, EC 4.1.3.21) is described. 2. 2.|In the wild type strain, the fixation of one substrate, S-acetyl coenzyme A, shows sigmoidal saturation kinetics. The initial rate experiments indicate that the reaction obeys an ordered mechanism, 2-ketoglutaric acid binding before S-acetyl coenzyme A. 3. 3.|The activity is completely inhibited in vitro by lysine and by some lysine analogs, which all show cooperative binding and have an heterotrophic effect on 2-ketoglutaric binding sites. A second class of effectors is found, including 2-aminoadipic acid, pipecolid acid and dipicolinic acid, which all affect the cooperativity of S-acetyl coenzyme A binding sites. 4. 4.|Two types of mutations which modify these inhibition patterns without affecting the catalytic activity are described. One results in a desensitization towards lysine and lysine analogs only. The other entirely abolishes the susceptibility towards the second type of inhibitors, without affecting the susceptibility to lysine. 5. 5.|No variations of the specific activity could be detected in the wild type strain at all; mutants showing an increased or a reduced activity were isolated. 6. 6.|Our results do not support the existence of isoenzymes at the level of homocitrate synthetase in this yeast.

Reversible, coenzyme-A-mediated inactivation of biosynthetic condensing enzymes in yeast: a possible regulatory mechanism.

alpha-Isopropylmalate synthase [3-hydroxy-4-methyl-3-carboxyvalerate 2-oxo-3-methylbutyrate-lyase (CoA-acetylating); EC 4.1.3.12], the enzyme catalyzing the first committed step in leucine biosynthesis, and homocitrate synthase [3-hydroxy-3-carboxyadipate 2-oxoglutarate-lyase (CoA-acetylating); EC 4.1.3.21], the first enzyme in lysine biosynthesis in yeast, are rapidly inactivated in the presence of low concentrations of coenzyme A, a product of both reactions. Closely related compounds like 3-dephospho-coenzyme A or oxidized coenzyme A are almost without effect, as are other sulfhydryl compounds. Citrate (si)-synthase [citrate oxaloacetate-lyase (pro-3S-CH2-COO-minus leads to acetyl-CoA); EC 4.1.3.7] appears to be completely resistant against inactivation by coenzyme A. Inactivated alpha-isopropylmalate and homocitrate synthases can be reactivated by dialysis, but not by adding excess substrate. Protection against coenzyme-A-mediated inactivation is provided by relatively high concentrations of the alpha-ketoacid substrate or the specific end product inhibitor of each of the two enzymes. The coenzyme-A-mediated inactivation of alpha-isopropylmalate synthase has been more closely investigated. It requires the presence of divalent metal ions, with Zn++being most effective. The inactivation does not require molecular oxygen. It occurs in the presence of low concentrations of substrates and is observed in toluene-treated cells. These results, together with evidence that alpha-isopropylmalate synthase and homocitrate synthase are located in the mitochondria, suggest a mechanism by which increasing intra-mitochondrial coenzyme A concentrations might serve as a signal of decreasing acetyl-coenzyme A levels, triggering a temporary inactivation of biosynthetic acetyl-coenzyme A-consuming reactions in order to channel the available acetyl-coenzyme A into the citrate cycle.