Inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine

Batson, Sarah; Chiara, Cesira de; Majce, Vita; Lloyd, Adrian J.; Gobec, Stanislav; Rea, Dean; Fülöp, Vilmos; Thoroughgood, Christopher W.; Simmons, Katie J.; Dowson, Christopher G.; Fishwick, Colin W. G.; Carvalho, Luiz Pedro S. de; Roper, David I.

doi:10.1038/s41467-017-02118-7

Cited by 65 publications

(72 citation statements)

References 31 publications

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“…A carbonate anion (CO3 2-) was modelled to clearly planar electron density at the expected position of the γ-Pi of bound ATP. This was previously observed in structures E. coli Ddl (33). The unexpected presence of products in several of these structures suggests that the TtDdl used for crystallography copurified with substrates/products.…”

Section: Determination Of Kinetic Parameterssupporting

confidence: 68%

d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

Pederick

Thompson

Bell

et al. 2020

Journal of Biological Chemistry

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The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine–d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl–d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+. Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

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Section: Determination Of Kinetic Parameterssupporting

confidence: 68%

d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

Pederick

Thompson

Bell

et al. 2020

Journal of Biological Chemistry

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“…DCS could be binding both d ‐Ala pockets and so display different binding behaviour. For Mt Ddl ( K i,DSC of 25 μ m ) and Ec Ddl ( K i,CDS of 185 μ m ), it was reported that slow binding of DCS occurs but not at a high concentration (1000 μ m ). This DCS concentration dependent behaviour appears to also apply to Bp Ddl.…”

Section: Resultsmentioning

confidence: 99%

Burkholderia pseudomallei d‐alanine‐d‐alanine ligase; detailed characterisation and assessment of a potential antibiotic drug target

et al. 2019

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Burkholderia pseudomallei is a serious, difficult to treat Gram‐negative pathogen and an increase in the occurrence of drug‐resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We have selected and characterised the essential enzyme d‐alanine‐d‐alanine ligase (BpDdl), required for the ATP‐assisted biosynthesis of a peptidoglycan precursor. A recombinant supply of protein supported high‐resolution crystallographic and biophysical studies with ligands (AMP and AMP+d‐Ala‐d‐Ala), and comparisons with orthologues enzymes suggest a ligand‐induced conformational change occurring that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow‐up and then in combination with a computational analysis of the active site, we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure‐based approaches.

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“…At this dosage, the DCS concentration in blood serum is generally 25-30 μg/ml, which is similar to the concentrations used in our in vitro and in vivo experiments. The known neurological side effects associated with DCS therapy [24, 25] mean that this antibiotic is unlikely to be considered for the treatment of MRSA infections unless alternative therapeutic approaches have been exhausted. Oxacillin/DCS combination therapy was significantly more effective than DCS or oxacillin alone over a 5-day therapeutic window suggesting that further studies on using DCS to augment β -lactams as a treatment option for recalcitrant staphylococcal infections are merited.…”

Section: Discussionmentioning

confidence: 99%

Impaired alanine transport or exposure to D-cycloserine increases the susceptibility of MRSA to β-lactam antibiotics

Gallagher

Shears

Fingleton

et al. 2019

Preprint

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Running title: Re-sensitization of MRSA to b-lactams 23 33 DCS re-sensitised MRSA to β-lactams in vitro and significantly enhanced MRSA eradication by 34 oxacillin in a mouse bacteraemia model. These findings reveal alanine transport as a new 35 therapeutic target to enhance the susceptibility of MRSA to β-lactam antibiotics. 36 37 Abstract word count: 161 38 3 Manuscript word count: 3,494 39 40 58 resistance (HoR) [5-7]. Exposure of HeR isolates to b-lactam antibiotics induces expression of 59 mecA, which encodes the alternative penicillin binding protein 2a (PBP2a) and can select for 60 mutations in accessory genes resulting in a HoR phenotype, including mutations that affect 61 the stringent response and c-di-AMP signalling [8-12]. Because accessory genes can influence 62 the expression of methicillin resistance in MRSA, targeting the pathways associated with such 63 genes may identify new ways to increase the susceptibility of MRSA to β-lactams. To pursue 64 this, we performed a forward genetic screen to identify loci that impact the expression of 65 resistance to β-lactam antibiotics in MRSA. Using the Nebraska Transposon Mutant Library, 66 which comprises 1,952 sequence-defined transposon insertion mutants [13], inactivation of 67 a putative amino acid permease gene, cycA, was found to reduce resistance to cefoxitin, the 68 β-lactam drug recommended by the Clinical and Laboratory Standards Institute for measuring 69 mecA-mediated methicillin resistance in MRSA isolates. Amino acid transport and 70

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Inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine

Cited by 65 publications

References 31 publications

d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

Burkholderia pseudomallei d‐alanine‐d‐alanine ligase; detailed characterisation and assessment of a potential antibiotic drug target

Impaired alanine transport or exposure to D-cycloserine increases the susceptibility of MRSA to β-lactam antibiotics

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