Molecular, structural, and phylogenetic characterization of two chlorophyllase isoforms in Pachira macrocarpa

Abstract

Chlorophyllase (Chlase) was one of first plant enzymes to be characterized biochemically, but its phylogenetic position and tertiary structure remain unknown. In this study, two isoforms of the Chlase gene, PmCLH1 and PmCLH2, were cloned from Pachira macrocarpa (Pm), and they shared 84 % identity in amino acid sequences. An unrooted phylogenetic tree classified 38 selected PmCLH homologous sequences into four clades, and PmCLHs were placed in the same clade as Arabidopsis AtCLH1. Moreover, the recombinant PmCLHs exhibited chlorophyll degradation activity in vitro. Tertiary structure modeling revealed that the catalytic triad of PmCLHs and AtCLHs were located on surface-exposed loops and clustered as a catalytic domain. Expressions of PmCLHs were not transcriptionally active in all tissues, and PmCLH1 and PmCLH2 were more abundant in young and old leaves, respectively. Multi-band patterns were observed in both young and old leaves of P. macrocarpa according to a Western blot analysis, suggesting that posttranslational modification was required for PmCLH maturation. In conclusion, we present the first report on the evolution and tertiary structural modeling of Chlase. We also provide evidence that mRNA and protein levels of PmCLHs are unequally expressed in young and old leaves of P. macrocarpa.