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

Abstract

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.

Unlocking the PIP-box: A peptide library reveals interactions that drive high-affinity binding to human PCNA

“…The supernatant containing peptide-bound protein was stored at −80 °C. Crystals were grown by hanging-drop vapor-diffusion method in 24-well linbro plates containing 500 μl well solution, by mixing 1 μl protein and peptide with an equal volume of the well solution ( 29 , 30 , 31 ). Initial cocrystallization screens with all p21 μ -modified peptides and hPCNA were attempted.…”

“…Diffracting crystals of hPCNA bound to p21 μ –F150Y were formed in 0.18 M magnesium acetate and 20% PEG at room temperature after 8 weeks. Crystals were mounted on cryoloops, cryoprotected using paratone-N, and flash-cooled in liquid nitrogen ( 29 , 30 , 31 ). Data were collected at 100 K using the MX1 beamline at the Australian Synchrotron ( 32 ).…”

“…Crystals were mounted on cryoloops, cryoprotected using paratone-N, and flash cooled in liquid nitrogen (29)(30)(31). Data was collected at 100 K using the MX1 beamline at the Australian Synchrotron (Clayton, Vic).…”

Identification of Potential Inhibitors From Urginea indica Metabolites Against Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae Receptors

“…In current study, D-alanine-D-alanine ligase A (DdlA) and MAPK1 of Xoo and M. oryzae, respectively, were selected for molecular docking studies with bioactive compounds acquired from gas chromatography-mass spectrometry (GC-MS). DdlA catalyzes the biosynthesis of dalanyl-d-alanine, an essential precursor of peptidoglycan (Pederick et al, 2020). On the other side, MAPK1 regulates cell division through phosphorylation of protein residues (Xu et al, 2017).…”

Discovery of novel antibacterial agents: Recent developments in D‐alanyl‐D‐alanine ligase inhibitors

“…D‐alanyl‐D‐alanine ligase (Ddl, EC 6.3.2.4) is an indispensable adenosine triphosphate (ATP)‐dependent bacterial enzyme involved in the biosynthesis of peptidoglycan, which catalyzes the ligation of two D‐alanine molecules into one D‐alanyl‐D‐alanine dipeptide (D‐Ala‐D‐Ala; Pederick et al., 2020; Tytgat, Colacino, et al., 2009; Tytgat, Vandevuer, et al., 2009; Walsh, 1989). This dipeptide is an essential component of the intracellular peptidoglycan precursor, uridine diphospho‐ N ‐acetylmuramic acid (UDP‐MurNAc)‐pentapeptide (Boudrioua et al., 2020; Van Heijenoort, 2001), which maintains the integrity of the bacterial cell wall by cross‐linking the peptidoglycan chain (Hrast et al., 2012), and is crucial for the survival of pathogens (Fakhar et al., 2016; Gholizadeh et al., 2001; Halouska et al., 2014; Mullins et al., 1990; Zhang et al., 2018).…”