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Originally published In Press as doi:10.1074/jbc.M000133200 on May 15, 2000

J. Biol. Chem., Vol. 275, Issue 30, 22824-22831, July 28, 2000
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A Metal Bridge between Two Enzyme Families
3-DEOXY-D-MANNO-OCTULOSONATE-8-PHOSPHATE SYNTHASE FROM AQUIFEX AEOLICUS REQUIRES A DIVALENT METAL FOR ACTIVITY*

Henry S. Duewel and Ronald W. WoodardDagger

From the Interdepartmental Program in Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1065

The enzymes 3-deoxy-D-manno-octulosonic acid-8-phosphate synthase (KDO8PS) and 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase (DAHPS) catalyze analogous condensation reactions between phosphoenolpyruvate and D-arabinose 5-phosphate or D-erythrose 4-phosphate, respectively. While several similarities exist between the two enzymatic reactions, classic studies on the Escherichia coli enzymes have established that DAHPS is a metalloenzyme, whereas KDO8PS has no metal requirement. Here, we demonstrate that KDO8PS from Aquifex aeolicus, representing only the second member of the KDO8PS family to be characterized in detail, is a metalloenzyme. The recombinant KDO8PS, as isolated, displays an absorption band at 505 nm and contains approximately 0.4 and 0.2-0.3 eq of zinc and iron, respectively, per enzyme subunit. EDTA inactivates the enzyme in a time- and concentration-dependent manner and eliminates the absorption at 505 nm. The addition of Cu2+ to KDO8PS produces an intense absorption at 375 nm, while neither Co2+ nor Ni2+ produce 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 mass spectrometry suggests that the enzyme preferentially binds one, two, or four metal ions per tetramer. These observations strongly suggest that A. aeolicus KDO8PS is a metalloenzyme in vivo and point to a previously unrecognized relationship between the KDO8PS and DAHPS families.

* This work was supported by National Institutes of Health Grant GM 53069 (to R. W. W) and a Natural Sciences and Engineering Research Council of Canada postdoctoral fellowship (to H. S. D).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: College of Pharmacy, 428 Church St., Ann Arbor, MI 48109-1065. Tel.: 734-764-7366; Fax: 734-763-5633; E-mail: rww@umich.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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