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PEDS Advance Access published online on June 21, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm018
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Probing the structural plasticity of an archaeal primordial cobaltochelatase CbiXS

Alexander Pisarchik, Ralf Petri and Claudia Schmidt-Dannert1

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St Paul, MN 55108, USA

1 To whom correspondence should be addressed. E-mail: schmi232{at}umn.edu

Insertion of metal ions into tetrapyrrole macrocycles is catalyzed by a diverse group of enzymes called chelatases. Structures are known for several chelatases catalyzing metal insertion into protoporphyrin IX or sirohydrochlorin. Despite a lack of significant amino acid sequence similarity, these ferro- and cobaltochelatases share a high degree of structural similarity. Cobaltochelatase CbiK and ferrochelatase HemH are bilobial enzymes with two {alpha}/ß domains, which were suggested to origin from a common ancestral protein via gene duplication. Small, single-domain chelatases (CbiXS) were recently described in archaea and are believed to represent primordial chelatases. Here, we tested the structural plasticity of an archaeal cobaltochelatase CbiXS by rearranging its structure with a novel method producing random in-frame deletions, duplications and insertions. A number of functional chelatase variants with insertion of duplicated sequence stretches, encompassing from one to nine secondary structural elements, were obtained. CbiXS was found to tolerate large sequence rearrangements in four out of the nine loop regions of the protein, indicating a high degree of structural plasticity. The predicted topologies of two variants (M51 and M518) are strikingly similar to CbiK and HemH, suggesting that we recreated duplication events that are believed to have created the bilobial chelatases.

Keywords: CbiK/CbiX/chelatase/directed evolution/HemH

Received January 11, 2007; revised March 21, 2007; accepted March 30, 2007.


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