Genes and biochemical characterization of three novel chlorophyllase isozymes from Brassica oleracea

Guan Chiun Lee, Hanna Chepyshko, Hsiu Hui Chen, Chih Chieh Chu, Yi Fan Chou, Casimir C. Akoh, Jei Fu Shaw

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli (Brassica oleracea) florets and characterized. The amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GXSXG). However, only BoCLH3 lacked the His residue which is the component of the catalytic triad (Ser-His-Asp). N-terminal sequences of BoCLH1 and BoCLH2 were predicted to have typical signal sequences for the chloroplast, whereas the plasma membrane-targeting sequence was identified in BoCLH3. The predicted molecular masses of BoCLH1, 2, and 3 were 34.7, 35.3, and 23.5 kDa, respectively. The recombinant BoCLHs were successfully expressed in Escherichia coli for the biochemical characterization. The recombinant BoCLH3 showed very low chlorophyllase activity possibly due to its incomplete catalytic triad. BoCLH1 and BoCLH2 showed significant differences in biochemical properties such as pH stability and temperature optimum. Kinetic analysis revealed that BoCLH1 preferably hydrolyzed Mg-free chlorophyll, while BoCLH2 hydrolyzed both chlorophyll and Mg-free chlorophyll at a similar level. Different characteristics between BoCLH1 and BoCLH2 implied that they may have different physiological functions in broccoli. The catalytic triad of recombinant BoCLH2 was identified as Ser141, His247, and Asp170 by site-directed mutagenesis. It suggested that the three broccoli chlorophyllase isozymes were serine hydrolases.

Original languageEnglish
Pages (from-to)8651-8657
Number of pages7
JournalJournal of Agricultural and Food Chemistry
Volume58
Issue number15
DOIs
Publication statusPublished - 2010 Aug 11

Fingerprint

chlorophyllase
Brassica
broccoli
Chlorophyll
Brassica oleracea
Isoenzymes
isozymes
Genes
chlorophyll
Complementary DNA
Mutagenesis
genes
site-directed mutagenesis
Molecular mass
florets
Hydrolases
Cell membranes
Protein Sorting Signals
hydrolases
signal peptide

Keywords

  • Broccoli (Brassica oleracea)
  • Chlorophyllase
  • isozyme

ASJC Scopus subject areas

  • Chemistry(all)
  • Agricultural and Biological Sciences(all)

Cite this

Genes and biochemical characterization of three novel chlorophyllase isozymes from Brassica oleracea. / Lee, Guan Chiun; Chepyshko, Hanna; Chen, Hsiu Hui; Chu, Chih Chieh; Chou, Yi Fan; Akoh, Casimir C.; Shaw, Jei Fu.

In: Journal of Agricultural and Food Chemistry, Vol. 58, No. 15, 11.08.2010, p. 8651-8657.

Research output: Contribution to journalArticle

Lee, Guan Chiun ; Chepyshko, Hanna ; Chen, Hsiu Hui ; Chu, Chih Chieh ; Chou, Yi Fan ; Akoh, Casimir C. ; Shaw, Jei Fu. / Genes and biochemical characterization of three novel chlorophyllase isozymes from Brassica oleracea. In: Journal of Agricultural and Food Chemistry. 2010 ; Vol. 58, No. 15. pp. 8651-8657.
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abstract = "Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli (Brassica oleracea) florets and characterized. The amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GXSXG). However, only BoCLH3 lacked the His residue which is the component of the catalytic triad (Ser-His-Asp). N-terminal sequences of BoCLH1 and BoCLH2 were predicted to have typical signal sequences for the chloroplast, whereas the plasma membrane-targeting sequence was identified in BoCLH3. The predicted molecular masses of BoCLH1, 2, and 3 were 34.7, 35.3, and 23.5 kDa, respectively. The recombinant BoCLHs were successfully expressed in Escherichia coli for the biochemical characterization. The recombinant BoCLH3 showed very low chlorophyllase activity possibly due to its incomplete catalytic triad. BoCLH1 and BoCLH2 showed significant differences in biochemical properties such as pH stability and temperature optimum. Kinetic analysis revealed that BoCLH1 preferably hydrolyzed Mg-free chlorophyll, while BoCLH2 hydrolyzed both chlorophyll and Mg-free chlorophyll at a similar level. Different characteristics between BoCLH1 and BoCLH2 implied that they may have different physiological functions in broccoli. The catalytic triad of recombinant BoCLH2 was identified as Ser141, His247, and Asp170 by site-directed mutagenesis. It suggested that the three broccoli chlorophyllase isozymes were serine hydrolases.",
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