Mechanisms of interaction of Escherichia coli threonine synthase with substrates and inhibitors

Laber, Bernd; Gerbling, Klaus-Peter; Harde, Christoph; Neff, Karl-Heinz; Nordhoff, Erhard; Pohlenz, Hans-Dieter

doi:10.1021/bi00177a035

Cited by 62 publications

(76 citation statements)

References 25 publications

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“…The extreme survival defects of S. cerevisiae thr1⌬ and thr4⌬ mutants in vivo, the inhibition of hyphal formation, the attenuated survival of C. albicans thr1⌬ mutants, and the essentiality of these genes in C. neoformans (34) all validate the potential of Thr1p and Thr4p as antifungal drug targets. Several inhibitors, including phosphohomoserine analogs (17) and the phosphohomoserine analog-containing peptides rhizocticin and plumbemycin (38,40), have been identified that target bacterial threonine synthase; however, the therapeutic efficacy of each is limited since many peptides are subject to serum protease digestion (30,47), various phosphohomoserine analogs are also N-methyl-D-aspartate (NMDA) receptor antagonists (57), and we found no inhibition of yeast by D,L-threo-␤-hydroxyaspartic acid or D,L-2-amino-5-phosphonovaleric acid (data not shown). Nonetheless, the rapidity and profound lethality observed upon exposure of thr1⌬ and thr4⌬ mutants to serum and threonine starvation conditions indicate that Thr1p and Thr4p inhibitors would be fungicidal in vivo.…”

Section: Discussionmentioning

confidence: 99%

Fungal Homoserine Kinase ( thr1 Δ) Mutants Are Attenuated in Virulence and Die Rapidly upon Threonine Starvation and Serum Incubation

Kingsbury

McCusker

2010

Eukaryot Cell

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The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neoformans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae homoserine kinase (thr1⌬) and threonine synthase (thr4⌬) mutants. In striking contrast to aspartate kinase (hom3⌬) mutants, S. cerevisiae thr1⌬ and thr4⌬ mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1⌬ mutants, but not hom3⌬ mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1⌬ and thr4⌬ mutants as well as C. albicans thr1⌬ mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1⌬ mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets.Fungal infections are an increasingly significant cause of human disease and morbidity due to an expanding immunocompromised population. However, only four main classes of broad-spectrum antifungal drugs are currently available (polyenes, azoles, echinocandins, and 5-fluorocytosine), which target only three cellular components: the cell membrane, cell wall, and nucleotide biosynthesis (55). Compared with the identification of antibacterial drug targets, an obstacle to antifungal drug target identification is the eukaryotic nature of both the fungal pathogen and the host, ensuring a considerably higher degree of conserved genes and pathways. Since a subset of amino acid biosynthetic pathways are not present in humans (46), yet are conserved in fungi, and many are required for survival in vivo and/or virulence (22,31,35,36,45,58), various amino acid biosynthetic enzymes are an attractive, unexploited class of antifungal drug targets.The threonine biosynthetic pathway is of particular interest for antifungal drug targets. Threonine is produced from aspartate, via the intermediate homoserine, in a series of five enzymatic steps, initiated by aspartate kinase (Hom3p). Homoserine is converted to threonine by the sequential actions of homoserine kinase (Thr1p) and threonine synthase (Thr4p). Threonine synthesis is regulated by induction of pathway genes via the general control pathway in response to amino acid starvation (26,43) and by feedback regulation of aspartate kinase when threonine is abundant (41, 48). Homoserine and threonine are interm...

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Section: Discussionmentioning

confidence: 99%

Fungal Homoserine Kinase ( thr1 Δ) Mutants Are Attenuated in Virulence and Die Rapidly upon Threonine Starvation and Serum Incubation

Kingsbury

McCusker

2010

Eukaryot Cell

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“…Quantitative determination of individual 2-oxoacids was performed as described previously (Laber et al, 1994). Plant material was frozen in liquid nitrogen and homogenized in 25 mM Mops-NaOH buffer, pH 7.1 (300 pL/g fresh weight).…”

Section: Analysis Of 2-oxoacidsmentioning

confidence: 99%

Repression of Acetolactate Synthase Activity through Antisense Inhibition (Molecular and Biochemical Analysis of Transgenic Potato (Solanum tuberosum L. cv Desiree) Plants)

Höfgen¹,

Laber²,

Schüttke³

et al. 1995

Plant Physiol.

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Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, valine, and isoleucine, is the biochemical target of different herbicides. To investigate the effects of repression of ALS activity through antisense gene expression we cloned an ALS gene from potato (Solanum fuberosum L. cv Désirée), constructed a chimeric antisense gene under control of the cauliflower mosaic virus 35s promoter, and created transgenic potato plants through Agrobacterium fumefaciens-mediated gene transfer. Two regenerants revealed severe growth retardation and strong phenotypical effects resembling those caused by ALS-inhibiting herbicides. Antisense gene expression decreased the steady-state leve1 of ALS mRNA in these plants and induced a corresponding decrease in ALS activity of up to 85%. This reduction was sufficient to generate plants almost inviable without amino acid supplementation. In both ALS antisense and herbicide-treated plants, we could exclude accumulation of 2-oxobutyrate and/or 2-aminobutyrate as the reason for the observed deleterious effects, but we detected elevated levels of free amino acids and imbalances in their relative proportions. Thus, antisense inhibition of ALS generated an in vivo model of herbicide action. Furthermore, expression of antisense RNA to the enzyme of interest provides a general method for validation of potential herbicide targets.

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“…1B). The bacterial reaction was studied by following the absorbence of PLP and was shown to present at least seven steps (Laber et al 1994).In plants, OPH is a branch point intermediate between the methionine/S-adenosyl-methionine (SAM) and threonine/ isoleucine pathways (Giovanelli et al 1984). Thus TS competes for OPH with cystathionine-␥-synthase (CGS), the first committed enzyme in the methionine pathway (Fig.…”

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confidence: 99%

Crystal structure of threonine synthase from Arabidopsis thaliana

Thomazeau

2001

Protein Science

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Threonine synthase (TS) is a PLP-dependent enzyme that catalyzes the last reaction in the synthesis of threonine from aspartate. In plants, the methionine pathway shares the same substrate, O-phospho-Lhomoserine (OPH), and TS is activated by S-adenosyl-methionine (SAM), a downstream product of methionine synthesis. This positive allosteric effect triggered by the product of another pathway is specific to plants. The crystal structure of Arabidopsis thaliana apo threonine synthase was solved at 2.25 Å resolution from triclinic crystals using MAD data from the selenomethionated protein. The structure reveals a fourdomain dimer with a two-stranded ␤-sheet arm protruding from one monomer onto the other. This domain swap could form a lever through which the allosteric effect is transmitted. The N-terminal domain (domain 1) has a unique fold and is partially disordered, whereas the structural core (domains 2 and 3) shares the functional domain of PLP enzymes of the same family. It also has similarities with SAM-dependent methyltransferases. Structure comparisons allowed us to propose potential sites for pyridoxal-phosphate and SAM binding on TS; they are close to regions that are disordered in the absence of these molecules.Keywords: Allostery; MAD; pyridoxal phosphate; S-adenosyl-methionine; threonine synthesis Aspartate is a precursor for the synthesis of several other amino acids, including lysine, threonine, isoleucine, and methionine. Threonine synthase (TS) (EC 4.2.99.2) is the last enzyme of the threonine synthesis pathway (Fig. 1A). It is a PLP-dependent enzyme and a 110-kD homodimer in solution. It catalyzes the conversion of O-phospho-L-homoserine (OPH) into threonine and inorganic phosphate (Fig. 1B). The bacterial reaction was studied by following the absorbence of PLP and was shown to present at least seven steps (Laber et al. 1994).In plants, OPH is a branch point intermediate between the methionine/S-adenosyl-methionine (SAM) and threonine/ isoleucine pathways (Giovanelli et al. 1984). Thus TS competes for OPH with cystathionine-␥-synthase (CGS), the first committed enzyme in the methionine pathway (Fig. 1A). Flux regulation between the threonine and methionine pathways does not result from end-product inhibition but, rather, via a strong stimulation of TS by SAM (Giovanelli et al. 1984). Indeed, previous results showed that the allosteric binding of SAM leads to an eightfold increase in the rate of catalysis and to a 25-fold decrease in the K M value for OPH (Curien et al. 1998). Saturation isotherms of TS by SAM disclosed positive cooperativity and indicated that at least two SAM molecules can bind on each dimeric enzyme (Curien et al. 1998). From these results, it was concluded that the dramatic modification in kinetic properties of plant TS originates from an allosteric and cooperative transition that is induced by SAM. Furthermore, this transition occurs at a

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Mechanisms of interaction of Escherichia coli threonine synthase with substrates and inhibitors

Cited by 62 publications

References 25 publications

Fungal Homoserine Kinase ( thr1 Δ) Mutants Are Attenuated in Virulence and Die Rapidly upon Threonine Starvation and Serum Incubation

Fungal Homoserine Kinase ( thr1 Δ) Mutants Are Attenuated in Virulence and Die Rapidly upon Threonine Starvation and Serum Incubation

Repression of Acetolactate Synthase Activity through Antisense Inhibition (Molecular and Biochemical Analysis of Transgenic Potato (Solanum tuberosum L. cv Desiree) Plants)

Crystal structure of threonine synthase from Arabidopsis thaliana

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