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PEDS Advance Access published online on May 27, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh046
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Received April 29, 2004
Accepted May 14, 2004

Article

Designing a metal-binding site in the scaffold of E. coli KDO8PS

Z. Oliynyk 1, L. Briseño-Roa 1, T. Janowitz 1, P. Sondergeld 1, A. R. Fersht 1*

1 Centre for Protein Engineering, MRC, Hills Rd., Cambridge, CB2 2QH, UK

* To whom correspondence should be addressed. E-mail: arf25{at}cam.ac.uk.


  Abstract

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 (A. aeolicus KDO8PS) require a metal ion for activity while another class of KDO8PS (including E. 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 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.

Keywords: KDO8PS, DAH7PS, metal-dependent, evolution, metal-binding


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