Cofactor-Independent Amino Acid Epimerases with Catalytic Serines Instead of Cysteines

D-amino acids play important roles in nature and are often produced from their L-stereoisomers by racemase or epimerase enzymes. One interesting class of amino acid racemases and epimerases are the cofactor-independent enzymes, which rely on a pair of active site cysteine residues for catalysis in an unusual chemical mechanism with seemingly mismatched acidity values. One classic example of these enzymes is diaminopimelic acid epimerase (DapF-CC), which produces D,L-diaminopimelic acid (DAP) as the penultimate step in lysine biosynthesis in most bacteria and photosynthetic organisms, and for Gram-negative bacterial peptidoglycan. In this work, we characterized for the first time enzymes of the cofactor-independent racemase and epimerase class that use paired catalytic serines (DapF-SS) instead of cysteines. DapF-SS enzymes catalyze reversible epimerization of DAP with similar kinetic parameters to that of DapF-CC enzymes. Sequence alignment and structural models suggest DapF-SS to have high homology to DapF-CC, and biochemical characterization provides evidence for a similar two-base mechanism. However, mutation of catalytic serine(s) to cysteine(s) nearly abolished activity, suggesting that these enzymes are not the result of simple mutations. A sequence similarity network identified thousands of other predicted DapF-SS enzymes from diverse bacterial phyla. Expression and isolation of several of these other enzymes found two with so far unidentified substrate(s), suggesting the Ser-Ser active site architecture may not be limited to just DAP epimerases. DapF-SS is active under oxidative conditions, while DapF-CC enzymes are inactivated by disulfide bond formation, providing a possible explanation as to why this second type of cofactor-independent epimerase evolved.