A metal bridge between two enzyme families - 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Aquifex aeolicus requires a divalent metal for activity
Hs. Duewel et Rw. Woodard, A metal bridge between two enzyme families - 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Aquifex aeolicus requires a divalent metal for activity, J BIOL CHEM, 275(30), 2000, pp. 22824-22831
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.