There is a growing need for the cost-effective synthesis of less resistant prone antibiotics, such as texiobactin, which contains D-threonine. Here, the previously uncharacterised crystal structure of a Pseudomonas putida pyridoxal-5’-phosphate (PLP)-dependent broad specificity racemase (PpBarT), which had been determined to reversibly catalyse the enantiomeric conversion of L- to D-threonine, has been determined at 1.23 Å resolution. This novel crystal structure was solved by molecular replacement, with a 72.3 % sequence identity search model, and refined (Rfree, 0.1979; FOM, 0.7644) using the CCP4 suite. DALI structural alignments of PpBarT with other PLP-dependent racemases, revealed the conservation of a homodimeric, N-terminal (α/β)8 barrel and C-terminal β sheet structure (Cα-RMSD values between, 0.5-2.2 Å). Conserved catalytic residues, lysine74 and tyrosine319’ were observed to lay within a relatively positively charged active site cavity, at each end of the dimeric interface. Autodock Vina molecular docking experiments of D-threonine and its PLP-bound intermediate, revealed that threonine’s α-carboxyl and α-amino groups are involved in substrate recognition and stabilisation within the PpBarT active site (putatively co-ordinated by arginine173). Thus, PpBarT-threonine docking experiments, revealed limitations for the in situ enantiomeric conversion of L- to D-threonine within texiobactin, although allowing potential use for cost effective D-threonine stock production. Molecular docking experiments of a known racemase inhibitor, D-cycloserine, and several of its PLP-bound intermediates, revealed that a co-ordinated water molecule, within the PpBarT active site, resides in a putative position such that it could facilitate the formation of a PLP-bound oxime derivative; supporting a recently published, irreversible racemase inhibition mechanism.
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