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J. Biol. Chem., Vol. 278, Issue 16, 14203-14210, April 18, 2003
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From the The first step of proline biosynthesis is
catalyzed by
Identification of Regions of the Tomato
-Glutamyl Kinase That
Are Involved in Allosteric Regulation by Proline*
§,
,**,,
Department of Biological Sciences and
¶ Department of Horticulture and Landscape Architecture,
Purdue University, West Lafayette, Indiana 47906-1392
-glutamyl kinase (GK). To better understand the
feedback inhibition properties of GK, we randomly mutagenized a plasmid
carrying tomato tomPRO1 cDNA, which encodes
proline-sensitive GK. A pool of mutagenized plasmids was transformed
into an Escherichia coli GK mutant, and proline-overproducing derivatives were selected on minimal medium containing the toxic proline analog 3,4-dehydro-DL-proline.
Thirty-two mutations that conferred 3,4-dehydro-DL-proline
resistance were obtained. Thirteen different single amino acid
substitutions were identified at nine different residues. The residues
were distributed throughout the N-terminal two-thirds of the
polypeptide, but 9 mutations affecting 6 residues were in a cluster of
16 residues. GK assays revealed that these amino acid substitutions
caused varying degrees of diminished sensitivity to proline feedback inhibition and also resulted in a range of increased proline
accumulation in vivo. GK belongs to a family of amino acid
kinases, and a predicted three-dimensional model of this enzyme was
constructed on the basis of the crystal structures of three related
kinases. In the model, residues that were identified as important for
allosteric control were located close to each other, suggesting that
they may contribute to the structure of a proline binding site. The putative allosteric binding site partially overlaps the dimerization and substrate binding domains, suggesting that the allosteric regulation of GK may involve a direct structural interaction between the proline binding site and the dimerization and catalytic domains.
*
This work was supported by the United States Department of
Agriculture Grant 93-37100-8871.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present Address: National Center for Alternative Energy,
Environment, and Innovative Technology, C. R. Trisaia, S. S. Jonica Km-419-500, Rotondella (MT), Italy.
**
Present address: Dept. of Natural Sciences, Texas A&M International
University, Laredo TX 7804.
To whom correspondence should be addressed. Tel.: 765-494-4969;
Fax: 765-496-1496; E-mail: lcsonka@bilbo.bio.purdue.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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