Biochemical diversity among the 1-amino-cyclopropane-1-carboxylate synthase isozymes encoded by the Arabidopsis gene family.
1-Amino-cyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) is the key enzyme in the ethylene biosynthetic pathway in plants. The completion of the Arabidopsis genome sequence revealed the presence of twelve putative ACS genes, ACS1-12, dispersed among five chromosomes. ACS1-5 have been previously characterized. However, ACS1 is enzymatically inactive whereas ACS3 is a pseudogene. Complementation analysis with the Escherichia coli aminotransferase mutant DL39 shows that ACS10 and 12 encode aminotransferases. The remaining eight genes are authentic ACS genes and together with ACS1 constitute the Arabidopsis ACS gene family. All genes, except ACS3, are transcriptionally active and differentially expressed during Arabidopsis growth and development. IAA induces all ACS genes, except ACS7 and ACS9; CHX enhances the expression of all functional ACS genes. The ACS genes were expressed in E. coli, purified to homogeneity by affinity chromatography, and biochemically characterized. The quality of the recombinant proteins was verified by N-terminal amino acid sequence and MALDI-TOF mass spectrometry. The analysis shows that all ACS isozymes function as dimers and have an optimum pH, ranging between 7.3 and 8.2. Their Km values for AdoMet range from 8.3 to 45 microm, whereas their kcat values vary from 0.19 to 4.82 s-1 per monomer. Their Ki values for AVG and sinefungin vary from 0.019 to 0.80 microm and 0.15 to 12 microm, respectively. The results indicate that the Arabidopsis ACS isozymes are biochemically distinct. It is proposed that biochemically diverse ACS isozymes function in unique cellular environments for the biosynthesis of C2H4, permitting the signaling molecule to exert its unique effects in a tissue- or cell-specific fashion.
Yamagami
Takeshi
T
Plant Gene Expression Center, United States Department of Agriculture, Albany, California 94710, USA.
Tsuchisaka
Atsunari
A
Yamada
Kayoko
K
Haddon
William F
WF
Harden
Leslie A
LA
Theologis
Athanasios
A
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D007527:Isoenzymes; D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase