RT Journal Article
SR Electronic
T1 Computational and experimental analyses of alanine racemase suggest new avenues for developing allosteric small-molecule antibiotics
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.10.29.514358
DO 10.1101/2022.10.29.514358
A1 Arie Van Wieren
A1 Jacob D Durant
A1 Sudipta Majumdar
YR 2022
UL http://biorxiv.org/content/early/2022/10/30/2022.10.29.514358.abstract
AB Given the ever-present threat of antibacterial resistance, there is an urgent need to identify new antibacterial drugs and targets. One such target is alanine racemase (Alr), an enzyme required for bacterial cell-wall biosynthesis. Alr is an attractive drug target because it is essential for bacterial survival but is absent in humans. Here, we investigate the Alr from M. tuberculosis (MT), the pathogen responsible for human tuberculosis, as a model Alr enzyme. MT-Alr functions exclusively as an obligate homodimer formed by two identical monomers. Both monomers contribute to the overall composition of their active sites. Therefore, disrupting the dimer interface could inhibit MT-Alr activity. Using computational methods, we identified seven interfacial residues predicted to be responsible for MT-Alr dimerization. Mutating one of the seven residues, Lys261, to alanine resulted in a completely inactive enzyme. Further investigation suggested a potential drug-binding site near Lys261 that might be useful for allosteric drug discovery.Summary The bacterial protein alanine racemase (Alr) converts L-alanine to D-alanine, a critical component of the bacterial cell wall. Cycloserine, a known antibiotic, inhibits Alr by binding to the same pocket that alanine binds. Several human proteins have similar pockets, so cycloserine has severe side effects. We identified additional Alr pockets and discovered that altering one of them abolishes Alr activity. Molecules that bind this pocket may similarly impact Alr activity, helping to address the ongoing antibiotic resistance crisis.Competing Interest StatementThe authors have declared no competing interest.AlrAlanine racemaseMTMycobacterium tuberculosisPLPPyridoxal 5-phosphatePCRPolymerase chain reactionSDS-PAGESodium dodecyl sulfate-polyacrylamide gel electrophoresisIMACImmobilized metal affinity chromatographyKLDKinase ligase DpnI enzymesODOptical density