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J. Biol. Chem., Vol. 283, Issue 30, 20797-20804, July 25, 2008

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Domain Characterization of a 4--Glucanotransferase Essential for Maltose Metabolism in Photosynthetic Leaves^*

Jon M. Steichen¹, Ryan V. Petty, and Thomas D. Sharkey^¶2

From the Departments of Botany and Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706 and the ^¶Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824

Maltose metabolism during the conversion of transitory (leaf) starch to sucrose requires a 4--glucanotransferase (EC 2.4.1.2 [EC] 5) in the cytosol of leaf cells. This enzyme is called DPE2 because of its similarity to the disproportionating enzyme in plastids (DPE1). DPE1 does not use maltose; it primarily transfers a maltosyl unit from one maltotriose to a second maltotriose to make glucose and maltopentaose. DPE2 is a modular protein consisting of a family 77 glycosyl hydrolase domain, similar to DPE1, but unlike DPE1 the domain is interrupted by an insertion of 150 amino acids as well as an N-terminal extension that consists of two carbohydrate binding modules. Phylogenetic analysis shows that the DPE2-type enzyme is present in a limited but highly diverse group of organisms. Here we show that DPE2 transfers the non-reducing glucosyl unit from maltose to glycogen by a ping-pong mechanism. The forward reaction (consumption of maltose) is specific for the β-anomer of maltose, while the reverse reaction (production of maltose) is not stereospecific for the acceptor glucose. Additionally, through deletion mutants we show that the glycosyl hydrolase domain alone provides disproportionating activity with a much higher affinity for short maltodextrins than the complete wild-type enzyme, while absence of the carbohydrate binding modules completely abolishes activity with large complex carbohydrates, reflecting the presumed function of DPE2 in vivo.

Received for publication, April 21, 2008 , and in revised form, May 22, 2008.

^* This work was supported by United States Dept. of Energy Grant DE-FG02-04ER15565. 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. S1 and S2.

¹ Present address: Dept. of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA 92093.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, MI State University, East Lansing, MI 48824. Tel.: 608-345-8167; E-mail: tsharkey{at}msu.edu.

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