Inhibitors of Lysine Biosynthesis as Antibacterial Agents

Hutton, Craig A.; Southwood, Timothy J; Turner, Jennifer

doi:10.2174/1389557033405359

Cited by 95 publications

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“…This pathway is of special interest for antibacterial drugs given that the absence of DAP in mammalian cells allows for the use of the DAP biosynthetic genes as bacteria-specific drug targets (Hutton et al 2003). The first two stages of the DAP pathway are common for the biosynthesis of amino acids of the aspartate family, namely, lysine, threonine, and methionine, and are catalyzed by aspartokinase and aspartate semialdehyde dehydrogenase.…”

Section: Introductionmentioning

confidence: 99%

A loop–loop interaction and a K-turn motif located in the lysine aptamer domain are important for the riboswitch gene regulation control

Blouin¹,

Lafontaine²

2007

RNA

107

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The lysine riboswitch is associated to the lysC gene in Bacillus subtilis, and the binding of lysine modulates the RNA structure to allow the formation of an intrinsic terminator presumably involved in transcription attenuation. The complex secondary structure of the lysine riboswitch aptamer is organized around a five-way junction that undergoes structural changes upon ligand binding. Using single-round transcription assays, we show that a loop-loop interaction is important for lysine-induced termination of transcription. Moreover, upon close inspection of the secondary structure, we find that an unconventional kinkturn motif is present in one of the stems participating in the loop-loop interaction. We show that the K-turn adopts a pronounced kink and that it binds the K-turn-binding protein L7Ae of Archaeoglobus fulgidus in the low nanomolar range. The functional importance of this K-turn motif is revealed from single-round transcription assays, which show its importance for efficient transcription termination. This motif is essential for the loop-loop interaction, and consequently, for lysine binding. Taken together, our results depict for the first time the importance of a K-turn-dependent loop-loop interaction for the transcription regulation of a lysine riboswitch.

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Section: Introductionmentioning

confidence: 99%

A loop–loop interaction and a K-turn motif located in the lysine aptamer domain are important for the riboswitch gene regulation control

Blouin¹,

Lafontaine²

2007

RNA

107

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“…The product of the reaction, L-lysine, is an important building block for the biosynthesis of the peptidoglycan cell wall, housekeeping proteins, and virulence factors of bacteria. Consequently, DAPDC represents a promising target for the development of novel antibacterial agents (4).…”

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confidence: 99%

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Peverelli

Costa

Kirby

et al. 2016

Journal of Biological Chemistry

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Diaminopimelate decarboxylase (DAPDC) catalyzes the final step in the diaminopimelate biosynthesis pathway of bacteria. The product of the reaction is the essential amino acid L-lysine, which is an important precursor for the synthesis of the peptidoglycan cell wall, housekeeping proteins, and virulence factors of bacteria. Accordingly, the enzyme is a promising antibacterial target. Previous structural studies demonstrate that DAPDC exists as monomers, dimers, and tetramers in the crystal state. However, the active oligomeric form has not yet been determined. We show using analytical ultracentrifugation, small angle x-ray scattering, and enzyme kinetic analyses in solution that the active form of DAPDC from Bacillus anthracis, Escherichia coli, Mycobacterium tuberculosis, and Vibrio cholerae is a dimer. The importance of dimerization was probed further by generating dimerization interface mutants (N381A and R385A) of V. cholerae DAPDC. Our studies indicate that N381A and R385A are significantly attenuated in catalytic activity, thus confirming that dimerization of DAPDC is essential for function. These findings provide scope for the development of new antibacterial agents that prevent DAPDC dimerization. Diaminopimelate decarboxylase (DAPDC)3 (E.C. 4.1.1.20) is a member of the pyridoxal 5Ј-phosphate (PLP)-dependent decarboxylases (1). It catalyzes the irreversible and stereospecific decarboxylation of meso-diaminopimelate (meso-DAP) in the final step of the diaminopimelate (DAP) biosynthesis pathway of bacteria and plants (2, 3). The product of the reaction, L-lysine, is an important building block for the biosynthesis of the peptidoglycan cell wall, housekeeping proteins, and virulence factors of bacteria. Consequently, DAPDC represents a promising target for the development of novel antibacterial agents (4).A DAPDC reaction mechanism has been previously proposed (5, 6). The reaction mechanism is thought to be initiated by the formation of a Schiff base between PLP and a conserved active site lysine from the (Y/F)AXKA motif (7). The substrate, meso-DAP, then binds and forms a subsequent Schiff base with PLP. This is suggested to be coordinated by the highly conserved CE(S/T)XD motif provided by an adjacent subunit (7). Decarboxylation of meso-DAP is then mediated by the sulfhydryl nucleophile provided by the conserved cysteine of the CE(S/T)XD motif thereby leaving the substrate cradled between two monomers (8). This proposed mechanism suggests DAPDC functions as a dimer.To support this assertion, previous structural studies of DAPDC from Methanocaldococcus jannaschii (PDB codes 1TWI and 1TUF) (9), Aquifex aeolicus (PDB code 2P3E), and Brucella melitensis (PDB code 3VAB) show that the enzyme crystallizes as a homodimer, with each monomer comprised of two domains (9). Domain I forms an ␣/␤ barrel, whereas domain II is comprised of a mixed ␤-sheet flanked by ␣-helices (9). The overall fold is similar to the functionally related enzyme ornithine decarboxylase, which also uses PLP as a cofactor in a decarboxylatio...

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“…design (Coulter et al 1999;Hutton et al 2003Hutton et al , 2007. However, a potent inhibitor of DHDPS has yet to be characterized.…”

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confidence: 99%

Conserved main‐chain peptide distortions: A proposed role for Ile203 in catalysis by dihydrodipicolinate synthase

et al. 2008

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In recent years, dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) has received considerable attention from a mechanistic and structural viewpoint. DHDPS catalyzes the reaction of (S)-aspartate-b-semialdehyde with pyruvate, which is bound via a Schiff base to a conserved active-site lysine (Lys161 in the enzyme from Escherichia coli). To probe the mechanism of DHDPS, we have studied the inhibition of E. coli DHDPS by the substrate analog, b-hydroxypyruvate. The K i was determined to be 0.21 (60.02) mM, similar to that of the allosteric inhibitor, (S)-lysine, and b-hydroxypyruvate was observed to cause timedependent inhibition. The inhibitory reaction with b-hydroxypyruvate could be qualitatively followed by mass spectrometry, which showed initial noncovalent adduct formation, followed by the slow formation of the covalent adduct. It is unclear whether b-hydroxypyruvate plays a role in regulating the biosynthesis of meso-diaminopimelate and (S)-lysine in E. coli, although we note that it is present in vivo. The crystal structure of DHDPS complexed with b-hydroxypyruvate was solved. The active site clearly showed the presence of the inhibitor covalently bound to the Lys161. Interestingly, the hydroxyl group of bhydroxypyruvate was hydrogen-bonded to the main-chain carbonyl of Ile203. This provides insight into the possible catalytic role played by this peptide unit, which has a highly strained torsion angle (v ;201°). A survey of the known DHDPS structures from other organisms shows this distortion to be a highly conserved feature of the DHDPS active site, and we propose that this peptide unit plays a critical role in catalysis.Keywords: dihydrodipicolinate synthase; lysine biosynthesis; conserved peptide distortion; X-ray crystallography Abbreviations: HTPA, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid; DHDPS, dihydrodipicolinate synthase; DHDPR, dihydrodipicolinate reductase; (S)-ASA, (S)-aspartate-bsemialdehyde; DAP, meso-diaminopimelate; MS, mass spectrometry.Article and publication are at

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Inhibitors of Lysine Biosynthesis as Antibacterial Agents

Cited by 95 publications

References 0 publications

A loop–loop interaction and a K-turn motif located in the lysine aptamer domain are important for the riboswitch gene regulation control

A loop–loop interaction and a K-turn motif located in the lysine aptamer domain are important for the riboswitch gene regulation control

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Conserved main‐chain peptide distortions: A proposed role for Ile203 in catalysis by dihydrodipicolinate synthase

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