Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon.
Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress.
Takahara
Kentaro
K
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Japan.
Akashi
Kinya
K
Yokota
Akiho
A
D000123:Acetyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis / Q000187:drug effects / Q000528:radiation effects; D002736:Chloroplasts / Q000378:metabolism; D002956:Citrulline / Q000096:biosynthesis*; D029773:Citrullus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003681:Dehydration; D018698:Glutamic Acid / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014162:Transfection
D018076:DNA, Complementary; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C417971:dip-1 protein, Citrullus lanatus; D049452:Green Fluorescent Proteins; C021951:N(delta)-acetylornithine; D002956:Citrulline; D018698:Glutamic Acid; D009952:Ornithine; D000123:Acetyltransferases; C020646:glutamate acetyltransferase; D000581:Amidohydrolases; C018732:N-acetylornithine deacetylase