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J. Biol. Chem., Vol. 281, Issue 20, 14514-14522, May 19, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/20/14514    most recent M600996200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gonzalez, C. F. Articles by Yakunin, A. F. Search for Related Content PubMed PubMed Citation Articles by Gonzalez, C. F. Articles by Yakunin, A. F. Social Bookmarking                      What's this?

Molecular Basis of Formaldehyde Detoxification

CHARACTERIZATION OF TWO S-FORMYLGLUTATHIONE HYDROLASES FROM ESCHERICHIA COLI, FrmB AND YeiG^*

Claudio F. Gonzalez, Michael Proudfoot, Greg Brown, Yurij Korniyenko, Hirotada Mori, Alexei V. Savchenko, and Alexander F. Yakunin¹

From the Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada and theGraduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan

The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and -naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (K_m = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser¹⁴⁵, Asp²³³, and His²⁵⁶ are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical /-hydrolase fold and a classical serine hydrolase catalytic triad (Ser¹⁶¹, His²⁷⁶, Asp²⁴¹). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.

Received for publication, February 1, 2006 , and in revised form, March 22, 2006.

^* This work was supported by Genome Canada through the Ontario Genomics Institute, National Institutes of Health Grant GM62414-01, CREST (Japan Science and Technology), and the Inamori Foundation (to H. M.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

¹ To whom correspondence should be addressed. Tel.: 416-946-0075; Fax: 416-978-8528; E-mail: a.iakounine{at}utoronto.ca.

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