Identification of regions of the tomato gamma-glutamyl kinase that are involved in allosteric regulation by proline.
The first step of proline biosynthesis is catalyzed by gamma-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.
Fujita
Tomomichi
T
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47906-1392, USA.
Maggio
Albino
A
Garcia-Rios
Mario
M
Stauffacher
Cynthia
C
Bressan
Ray A
RA
Csonka
Laszlo N
LN
D000483:Alleles; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D018551:Solanum lycopersicum / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry* / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D010455:Peptides; D011392:Proline; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase