Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: finding of a lipase motif and the induction by methyl jasmonate.
Chlorophyllase (Chlase) is the first enzyme involved in chlorophyll (Chl) degradation and catalyzes the hydrolysis of ester bond to yield chlorophyllide and phytol. In the present study, we isolated the Chlase cDNA. We synthesized degenerate oligo DNA probes based on the internal amino acid sequences of purified Chlase from Chenopodium album, screened the C. album cDNA library, and cloned a cDNA (CaCLH, C. album chlorophyll-chlorophyllido hydrolase). The deduced amino acid sequence (347 aa residues) had a lipase motif overlapping with an ATP/GTP-binding motif (P-loop). CaCLH possibly was localized in the extraplastidic part of the cell, because a putative signal sequence for endoplasmic reticulum is at the N terminus. The amino acid sequence shared 37% identity with a function-unknown gene whose mRNA is inducible by coronatine and methyl jasmonate (MeJA) in Arabidopsis thaliana (AtCLH1). We expressed the gene products of AtCLH1 and of CaCLH in Escherichia coli, and they similarly exhibited Chlase activity. Moreover, we isolated another full-length cDNA based on an Arabidopsis genomic fragment and expressed it in E. coli, demonstrating the presence of the second Arabidopsis CLH gene (AtCLH2). No typical feature of signal sequence was identified in AtCLH1, whereas AtCLH2 had a typical signal sequence for chloroplast. AtCLH1 mRNA was induced rapidly by a treatment of MeJA, which is known to promote senescence and Chl degradation in plants, and a high mRNA level was maintained up to 9 h. AtCLH2, however, did not respond to MeJA.
Tsuchiya
T
T
Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.
Ohta
H
H
Okawa
K
K
Iwamatsu
A
A
Shimada
H
H
Masuda
T
T
Takamiya
K
K
D000085:Acetates / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D019663:Chenopodiaceae / Q000201:enzymology / Q000235:genetics*; D002734:Chlorophyll / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology*; D004790:Enzyme Induction; D015723:Gene Library; D017343:Genes, Plant; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D054883:Oxylipins; D018515:Plant Leaves / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000085:Acetates; D003517:Cyclopentanes; D007527:Isoenzymes; D054883:Oxylipins; D011994:Recombinant Proteins; D002734:Chlorophyll; C072239:methyl jasmonate; D002265:Carboxylic Ester Hydrolases; C019875:chlorophyllase