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J. Biol. Chem., Vol. 279, Issue 43, 45110-45120, October 22, 2004

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A Reciprocal Single Mutation Affects the Metal Requirement of 3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) Synthases from Aquifex pyrophilus and Escherichia coli^*

Smadar Shulami, Cristina Furdui¶, Noam Adir||, Yuval Shoham||**, Karen S. Anderson¶, and Timor Baasov||

From the Department of Biotechnology and Food Engineering, the Department of Chemistry, and the ^||Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000, Israel and the ^¶Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase is metal-dependent in one class of organisms and metal-independent in another. We have used a rapid transient kinetic approach combined with site-directed mutagenesis to characterize the role of the metal ion as well as to explore the catalytic mechanisms of the two classes of enzymes. In the metal-dependent Aquifex pyrophilus KDO8P synthase, Cys¹¹ was replaced by Asn (ApC11N), and in the metal-independent Escherichia coli KDO8P synthase a reciprocal mutation, Asn²⁶ to Cys, was prepared (EcN26C). The ApC11N mutant retained about 10% of the wild-type maximal activity in the absence of metal ions. Addition of divalent metal ions did not affect the catalytic activity of the mutant enzyme and its catalytic efficiency (k_cat/K_m) was reduced by only 12-fold, implying that the ApC11N KDO8P synthase mutant has become a bone fide metal-independent enzyme. The isolated EcN26C mutant had similar metal content and spectral properties as the metal-dependent wild-type A. pyrophilus KDO8P synthase. EDTA-treated EcN26C retained about 6% of the wild-type activity, and the addition of Mn²⁺ or Cd²⁺ stimulated its activity to 30% of the wild-type maximal activity. This suggests that EcN26C KDO8P synthase mutant has properties similar to that of metal-dependent KDO8P synthases. The combined data indicate that the metal ion is not directly involved in the chemistry of the KDO8P synthase catalyzed reaction, but has an important structural role in metal-dependent enzymes in maintaining the correct orientation of the substrates and/or reaction intermediate(s) in the enzyme active site.

Received for publication, April 26, 2004 , and in revised form, August 11, 2004.

^* This work was supported by U.S.-Israel Binational Science Foundation (Grant 2002-126) (to T. B. and K. S. A.), by Rubin Scientific and Medical Fund for promotion of research at the Technion (Grant 060-624), and by National Institutes of Health Grants GM61413 and GM71805, (to K. S. A.). Additional support was provided by the Fund for the Promotion of Research at the Technion, and by the Otto Meyerhof Center for Biotechnology, Technion, established by the Minerva Foundation (Munich, Germany). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence may be addressed: Dept. of Biotechnology and Food Engineering, Technion, Haifa 32000, Israel. Tel.: 972-4-8293072; Fax: 972-4-8320742; E-mail: yshoham{at}techunix.technion.ac.il. To whom correspondence may be addressed: Dept. of Chemistry, Technion, Haifa 32000, Israel. Tel.: 972-4-8292590; Fax: 972-4-8295703; E-mail: chtimor{at}techunix.technion.ac.il.

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