Alanine racemase (Alr) is a pyridoxal 5′-phosphate-dependent
enzyme that catalyzes the racemization ofl-alanine tod-alanine. Alr is one of the two targets of the broad-spectrum
antibioticd-cycloserine (DCS), a structural analogue ofd-alanine. Despite being an essential component of regimens
used to treat multi- and extensively drug-resistant tuberculosis for
almost seven decades, resistance to DCS has not been observed in patients.
We previously demonstrated that DCS evades resistance due to an ultralow
rate of emergence of mutations. Yet, we identified a single polymorphism
(converting Asp322 to Asn) in thealrgene, which
arose in 8 out of 11 independent variants identified and that confers
resistance. Here, we present the crystal structure of the Alr variant
D322N in both the free and DCS-inactivated forms and the characterization
of its DCS inactivation mechanism by UV–visible and fluorescence
spectroscopy. Comparison of these results with those obtained with
wild-type Alr reveals the structural basis of the 240-fold reduced
inhibition observed in Alr D322N.