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J. Biol. Chem., Vol. 281, Issue 47, 36149-36161, November 24, 2006

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Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family^*

Ekaterina Kuznetsova^¶, Michael Proudfoot, Claudio F. Gonzalez, Greg Brown, Marina V. Omelchenko^||, Ivan Borozan, Liran Carmel^||, Yuri I. Wolf^||, Hirotada Mori^**, Alexei V. Savchenko^¶, Cheryl H. Arrowsmith¹, Eugene V. Koonin^||, Aled M. Edwards, The Banbury Chair of Medical Research at the University of Toronto^¶2, and Alexander F. Yakunin^¶3

From the Banting and Best Department of Medical Research and the Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L6, Canada, the ^¶Ontario Center for Structural Proteomics, Ontario Cancer Institute, Toronto, Ontario M5G 2C4, Canada, the ^||National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, the ^**Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan, and Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L6, Canada

Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, -phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and -phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical "one enzyme-one substrate" model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases.

Received for publication, June 7, 2006 , and in revised form, September 21, 2006.

^* This work was supported by Genome Canada through the Ontario Genomics Institute, the National Institutes of Health (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 Figs. 1 and 2 and Tables 1-3.

¹ A Scientist of the Canadian Institutes of Health Research.

²To whom correspondence may be addressed: Banting and Best Dept. of Medical Research, University of Toronto, 112 College St., Rm. 117, Toronto, Ontario M5G 1L6, Canada. Tel.: 416-946-3436; Fax: 416-978-8528; E-mail: aled.edwards{at}utoronto.ca. ³To whom correspondence may be addressed: Banting and Best Dept. of Medical Research, University of Toronto, 112 College St., Rm. 024, Toronto, Ontario M5G 1L6, Canada. Tel.: 416-946-0075; Fax: 416-946-0078; E-mail: a.iakounine{at}utoronto.ca.

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