PEDS Advance Access originally published online on May 27, 2004
Protein Engineering Design and Selection 2004 17(4):383-390; doi:10.1093/protein/gzh046
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Designing a metal-binding site in the scaffold of Escherichia coli KDO8PS
Centre for Protein Engineering, MRC, Hills Road, Cambridge CB2 2QH, UK
1 To whom correspondence should be addressed. E-mail: arf25{at}cam.ac.uk
KDO8PS (3-deoxy-D-manno-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase) enzymes catalyse analogous condensation reactions between phosphoenolpyruvate and arabinose 5-phosphate or erythrose 4-phosphate, respectively. All known DAH7PS and some of KDO8PS enzymes (Aquifex aeolicus KDO8PS) require a metal ion for activity whereas another class of KDO8PS (including Escherichia coli KDO8PS) does not. Based on sequence alignment of all known KDO8PS and DAH7PS enzymes, we identified a single amino acid residue that might define the metal dependence of KDO8PS activity. One of the four metal-binding residues, a cysteine, is conserved only among metal-binding KDO8PS and DAH7PS enzymes and is replaced by an asparagine residue in other KDO8PS enzymes. We introduced a metal binding site into E.coli KDO8PS by a single N26C and a double M25P N26C mutation, which led to an increased kcat of the enzymes in the presence of activating Mn2+ ions. The M25P N26C mutant of E.coli KDO8PS had a value of kcat/KM in the presence of Mn2+ ions four times higher than A.aeolicus KDO8PS. KDO8PS and DAH7PS may have evolved from a common ancestor protein that required a divalent metal ion for activity. A non-metal-binding KDO8PSs may have evolved from an ancestor protein that was able to bind Mn2+ but no longer required Mn2+ to function and eventually lost one of metal-binding residues.
Received April 29, 2004; accepted May 14, 2004.
Edited by Greg Winter