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

• Published in: The Journal of biological chemistry Vol. 279; no. 43; pp. 45110 - 45120
• Main Authors: Shulami, Smadar, Furdui, Cristina, Adir, Noam, Shoham, Yuval, Anderson, Karen S, Baasov, Timor
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 22.10.2004
• Subjects: Aldehyde-Lyases - biosynthesis; Aquifex pyrophilus; Bacteria - enzymology; Binding Sites; Cadmium - chemistry; Cadmium - pharmacology; Catalysis; Chelating Agents - pharmacology; Cloning, Molecular; Cysteine - chemistry; Dose-Response Relationship, Drug; Edetic Acid - pharmacology; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - metabolism; Ions; Kinetics; Magnesium - chemistry; Magnesium - pharmacology; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Plasmids - metabolism; Spectrophotometry; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M404561200

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, Cys 11 was replaced by Asn ( Ap C11N), and in the metal-independent Escherichia coli KDO8P synthase a reciprocal mutation, Asn 26 to Cys, was prepared ( Ec N26C). The Ap C11N 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 Ap C11N KDO8P synthase mutant has become a bone fide metal-independent enzyme. The isolated Ec N26C mutant had similar metal content and spectral properties as the metal-dependent wild-type A. pyrophilus KDO8P synthase. EDTA-treated Ec N26C retained about 6% of the wild-type activity, and the addition of Mn 2+ or Cd 2+ stimulated its activity to ∼30% of the wild-type maximal activity. This suggests that Ec N26C 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.