A metal bridge between two enzyme families - 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Aquifex aeolicus requires a divalent metal for activity

The enzymes 3-deoxy-D-manno-octulosonic acid-8-phosphate synthase (KDO8PS) and 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase (DAHPS) cataly ze analogous condensation reactions between phosphoenolpyruvate and D-arabi nose 5-phosphate or D-erythrose 4-phosphate, respectively. While several si milarities exist between the two enzymatic reactions, classic studies on th e Escherichia coli enzymes have established that DAMPS is a metalloenzyme, whereas KDO8PS has no metal requirement. Here, we demonstrate that KDO8PS f rom Aquifex aeolicus, representing only the second member of the KDO8PS fam ily to be characterized in detail, is a metalloenzyme, The recombinant KDO8 PS, as isolated, displays an absorption band at 505 nm and contains approxi mately 0.4 and 0.2-0.3 eq of zinc and iron, respectively, per enzyme subuni t. EDTA inactivates the enzyme in a time- and concentration-dependent manne r and eliminates the absorption at 505 nm, The addition of Cu2+ to RDO8PS p roduces an intense absorption at 375 nm, while neither Co2+ nor Ni2+ produc e such an effect, The EDTA-treated enzyme is reactivated by a wide range of divalent metal ions including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+ Ni2 + and Zn2+ and is reversibly inhibited by higher concentrations (>1 mM) of certain metals. Analysis of several metal forms of the enzyme by plasma mas s spectrometry suggests that the enzyme preferentially binds one, two, or f our metal ions per tetramer, These observations strongly suggest that A. ae olicus KDO8PS is a metalloenzyme in vivo and point to a previously unrecogn ized relationship between the KDO8PS and DAMPS families.