The Crystal Structure of a Homodimeric Pseudomonas Glyoxalase I Enzyme Reveals Asymmetric Metallation Commensurate with Half‐of‐Sites Activity

The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal‐dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X‐ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal‐binding arrangement consistent with half‐of‐sites activity: one active site contains a single activating Ni²⁺ ion, whereas the other contains two inactivating Zn²⁺ ions. Enzymological experiments prompted by the binuclear Zn²⁺ site identified a novel catalytic property of GloA2. The enzyme can function as a Zn²⁺/Co²⁺‐dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal‐binding arrangements simultaneously, each of which catalyzes a different reaction.