The interconversion of ACC deaminase and D-cysteine desulfhydrase by directed mutagenesis.
Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. These enzymes cleave ACC, the immediate precursor of ethylene in plants, into ammonia and alpha-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. In the work reported here, it was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather it utilizes D: -cysteine as a substrate, and in fact encodes a D: -cysteine desulfhydrase. Kinetic characterization of the tomato enzyme indicates that it is similar to other, previously characterized, D: -cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering only two amino acid residues within the predicted active site served to change the enzyme from D: -cysteine desulfhydrase to ACC deaminase. Conversely, by altering two amino acid residues at the same positions within the active site of ACC deaminase from Pseudomonas putida UW4 the enzyme was converted into D: -cysteine desulfhydrase. Therefore, it is possible that a change in these two residues may have occurred in an ancestral protein to result in two different enzymatic activities.
Todorovic
Biljana
B
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada. btodorov@sciborg.uwaterloo.ca
Glick
Bernard R
BR
D000595:Amino Acid Sequence; D019755:Carbon-Carbon Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003542:Cystathionine gamma-Lyase / Q000378:metabolism*; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D040681:Structural Homology, Protein
D050505:Mutant Proteins; D011994:Recombinant Proteins; C033634:1-aminocyclopropane-1-carboxylate deaminase; D019755:Carbon-Carbon Lyases; D003542:Cystathionine gamma-Lyase