Structural Basis for Catalytic Racemization and Substrate Specificity of an N-Acylamino Acid Racemase Homologue from Deinococcus radiodurans

N-Acylamino acid racemase (NAAAR) catalyzes the racemization of N-acylamino acids and can be used in concert with an aminoacylase to produce enantiopure α-amino acids, a process that has potential industrial applications. Here we have cloned and characterized an NAAAR homologue from a radiation-resistant ancient bacterium, Deinococcus radiodurans. The expressed NAAAR racemized various substrates at an optimal temperature of 60 °C and had Km values of 24.8 mM and 12.3 mM for N-acetyl-d-methionine and N-acetyl-l-methionine, respectively. The crystal structure of NAAAR was solved to 1.3 Å resolution using multiwavelength anomalous dispersion (MAD) methods. The structure consists of a homooctamer in which each subunit has an architecture characteristic of enolases with a capping domain and a (β/α)7 β barrel domain. The NAAAR·Mg2+ and NAAAR·N-acetyl-l-glutamine·Mg2+ structures were also determined, allowing us to define the Lys170-Asp195-Glu220-Asp245-Lys269 framework for catalyzing 1,1-proton exchange of N-acylamino acids. Four subsites enclosing the substrate are identified: catalytic site, metal-binding site, side-chain-binding region, and a flexible lid region. The high conservation of catalytic and metal-binding sites in different enolases reflects the essentiality of a common catalytic platform, allowing these enzymes to robustly abstract α-protons of various carboxylate substrates efficiently. The other subsites involved in substrate recognition are less conserved, suggesting that divergent evolution has led to functionally distinct enzymes.