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J. Biol. Chem., Vol. 278, Issue 51, 50880-50886, December 19, 2003

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Overexpression of L-Isoaspartate O-Methyltransferase in Escherichia coli Increases Heat Shock Survival by a Mechanism Independent of Methyltransferase Activity^*

Jason Kindrachuk, Jennifer Parent, Gerald F. Davies, Michael Dinsmore, Samuel Attah-Poku, and Scott Napper¶

From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada and the Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E3, Canada

Over time and under stressing conditions proteins are susceptible to a variety of spontaneous covalent modifications. One of the more commonly occurring types of protein damage is deamidation; the conversion of asparagines into aspartyls and isoaspartyls. The physiological significance of isoaspartyl formation is emphasized by the presence of the conserved enzyme L-isoaspartyl O-methyltransferase (PIMT), whose physiological function appears to be in preventing the accumulation of deamidated proteins. Seemingly consistent with a repair function, overexpression of PIMT in Drosophila melanogaster extends lifespan under conditions expected to contribute to protein damage. Based on structural information and sequence homology we have created mutants of residues proposed to be involved in co-factor binding in Escherichia coli PIMT. Both mutants retain S-adenosyl L-methionine binding capabilities but demonstrate dramatically reduced kinetic capabilities, perhaps suggestive of catalytic roles beyond co-factor binding. As anticipated, overexpression of the wild type enzyme in E. coli results in bacteria with increased tolerance to thermal stress. Surprisingly, even greater levels of heat tolerance were observed with overexpression of the inactive PIMT mutants. The increased survival capabilities observed with overexpression of PIMT in E. coli, and possibly in Drosophila, are not due to increased isoaspartyl repair capabilities but rather a temperature-independent induction of the heat shock system as a result of overexpression of a misfolding-prone protein. An alternate hypothesis as to the physiological substrate and function of L-isoaspartyl methyltransferase is proposed.

Received for publication, July 31, 2003 , and in revised form, September 9, 2003.

^* This work was supported by a grant from the Natural Science and Engineering Research Council (to S. N.) and through graduate support from the Saskatchewan Synchrotron Institute (to J. K.). Equipment support was provided by The Saskatchewan Structural Sciences Center. 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 should be addressed. Tel.: 306-966-1546; Fax: 306-966-7478; E-mail: napper{at}sask.usask.ca.

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