Genes and Biochemical Characterization of Three Novel Chlorophyllase Isozymes fromBrassica oleracea

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.

Three transcripts of EDS1-like genes respond differently to Vitis flexuosa infection

“…The G-X-S-X-G motif, which is highly conserved, is found in may lipases and esterases. This motif contains an active site residue of serine in the serine hydrolases family genes (Baudoin et al 1997;Bezier et al 2002;Reina et al 2007;Lee et al 2010). The L-family amino acid sequences contain the signature sequences of…”

A novel recombinant chlorophyllase from cyanobacterium Cyanothece sp. ATCC 51142 for the production of bacteriochlorophyllide a

“…The activity assays were performed according to the report of Lee et al. . Three substrates—BChl a from R. sphaeroides , Chl a from Anacystis nidulans algae, and Chl b from spinach—were purchased from Sigma Co. (St. Louis, MO, USA).…”

“…The deduced amino acid sequences of all cloned Chlase genes share a highly conserved lipase motif GXSXG , with the middle serine functioning as an active site , indicating that the Chlases belong to the serine hydrolase family . Although numerous studies have focused on Chlases in the plant kingdom, no study has investigated Chlase‐related activities of photosynthetic bacteria.…”

Quality Maintenance of Postharvest Horticultural Crops by Stress Treatments and Approach for the Elucidation of Its Mechanism

“…The expression of all Chlase genes, BoCLH1, BoCLH2, and BoCLH3 (Lee et al, 2010), increased immediately after UV-B treatment, whereas the expression of BoPPH showed a small reduction with the treatment. The expressions of BoCLH2 and BoCLH3 in broccoli florets with UV-B treatment were more up-regulated than in the control during storage at 15°C.…”

“…Chl degradation has been suggested to have the following putative pathways, as shown in Figure 1. An early step of Chl a degradation seems to be the removal of phytol and the formation of chlorophyllide (Chlide) a by chlorophyllase (Amir-Shapira et al, 1987;Holden, 1961;Lee et al, 2010;McFeeters et al, 1971;Mínguez-Mosquera et al, 1994;Shimokawa et al, 1978;Tsuchiya et al, 1997) followed by the removal of Mg 2+ by Mgdechelating substances (MDS), which are heat-stable and low-molecular-weight compounds (Costa et al, 2002;Kunieda et al, 2005;Shioi et al, 1996;Suzuki et al, 2005). Finally, pheophorbide (Pheide) a is degraded to fluorescent Chl catabolites, which are primary colorless catabolites, via a red Chl catabolite, by both Pheide a oxygenase and red Chl catabolite reductase (Hörtensteiner and Lee, 2007;Matile et al, 1999).…”