Genes and Biochemical Characterization of Three Novel Chlorophyllase Isozymes from Brassica oleracea

• Published in: Journal of agricultural and food chemistry 2010-08, Vol.58 (15), p.8651-8657
• Main Authors: Lee, Guan-Chiun, Chepyshko, Hanna, Chen, Hsiu-Hui, Chu, Chih-Chieh, Chou, Yi-Fan, Akoh, Casimir C, Shaw, Jei-Fu
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; biochemical compounds; Biological and medical sciences; Brassica - chemistry; Brassica - enzymology; Brassica - genetics; Brassica oleracea; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - metabolism; Chemical Aspects of Biotechnology/Molecular Biology; chlorophyll; chlorophyllase; Cloning, Molecular; enzyme activity; Enzyme Stability; Food industries; Fruit and vegetable industries; Fundamental and applied biological sciences. Psychology; genes; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; isozymes; Kinetics; Molecular Sequence Data; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; postharvest physiology; recombinant DNA; Sequence Homology, Amino Acid
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf1016384

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.