Mis‐annotations of a promising antibiotic target in high‐priority gram‐negative pathogens

Abstract

The rise of antibiotic resistance combined with the lack of new products entering the market has led to bacterial infections becoming one of the biggest threats to global health. Therefore, there is an urgent need to identify novel antibiotic targets, such as dihydrodipicolinate synthase (DHDPS), an enzyme involved in the production of essential metabolites in cell wall and protein synthesis. Here, we utilised a 7‐residue sequence motif to identify mis‐annotation of multiple DHDPS genes in the high‐priority Gram‐negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae. We subsequently confirmed these mis‐annotations using a combination of enzyme kinetics and X‐ray crystallography. Thus, this study highlights the need to ensure genes encoding promising drug targets, like DHDPS, are annotated correctly, especially for clinically important pathogens.PDB ID6UE0.

The First Inhibitor of Meso-Diaminopimelate Biosynthesis with Antibacterial Activity Against Multi-Drug Resistant Bacteria

“…Subsequently, the inhibitory activity of PMSH against recombinant AbDHDPS was quantitated using a DHDPS-DHDPR coupled assay (37). Different concentrations of PMSH were titrated to the assay with substrates fixed at previously determined K M values (34), resulting in an IC 50 value of 35 µM (Figure 3B).…”

“…The DHDPS-DHDPR coupled assay was used to quantify DHDPS activity as previously described (40, 43) by measuring the oxidation of NADPH at 340 nm. Assays were conducted in a Cary 4000 UV/Vis spectrophotometer (Varian, Mulgrave, Victoria, Australia) with substrates fixed at the previously determined K M values (34). AbDHDPS enzyme and reactions were incubated at 37 °C for 12 mins before initiation with ASA.…”

“…The active site is located within the (β/α) 8 -barrel, which is highly conserved across DHDPS enzymes, and the allosteric cleft is found in the dimer interface (Figure 2C) (8, 30–33). We have recently characterised the kinetic parameters of the recombinant AbDHDPS enzyme, including affinities for the substrates, pyruvate and ASA, to allow for screening of inhibitors in vitro (34). The structural and functional characterisation of this enzyme offers an excellent platform for inhibitor identification in the pursuit of antibacterial agents against A. baumannii .…”

“…We have recently characterised the kinetic parameters of the recombinant AbDHDPS enzyme, including affinities for the substrates, pyruvate and ASA, to allow for screening of inhibitors in vitro (34). The structural and functional characterisation of this enzyme offers an excellent platform for inhibitor identification in the pursuit of antibacterial agents against A. baumannii.…”

Overcoming Intrinsic and Acquired Resistance Mechanisms Associated with the Cell Wall of Gram-Negative Bacteria

“…One approach to negate such resistance mechanisms is to move upstream of the peptidoglycan layer and look at the synthesis of its components. A promising antibiotic target is the diaminopimelate (DAP) pathway [ 70 , 71 , 72 , 73 , 74 ]. The DAP pathway is responsible for the production of meso -diaminopimelate ( meso -DAP) and l -lysine, which are critical building blocks for cell wall and protein synthesis [ 75 , 76 , 77 ].…”