Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2013
Publication Date: 12/13/2013
Citation: Savary, B.J., Vasu, P., Cameron, R.G., Mccollum, T.G., Nunez, A. 2013. Structural Characterization of the Thermally-Tolerant Pectin Methylesterase Purified from Citrus sinensis Fruit and Its Gene Sequence. Journal of Agricultural and Food Chemistry. 61:12711-12719. Interpretive Summary: Enzymes accumulated in plant cell walls serve diverse physiological functions including metabolism, polysaccharide structure modification, and molecular communication in interactions with other organisms. Pectin methylesterases are economically important enzymes for their impact on quality and processing properties of fruit and vegetable food products. We have now purified TT-PME to homogeneity from sweet orange finisher pulp and determined the complete corresponding nucleic acid sequence. Purified TT-PME was observed by SDS-PAGE as two doublet bands with molecular masses of approximately 46,000 Da and 56,000 Da. Direct Edman sequencing from these proteins showed a common N-terminal peptide. De novo sequencing of eight TT-PME tryptic peptides determined by MALDI-TOF/TOF mass spectrometry provided additional internal sequences. TT-PME did not correspond to any previously reported Citrus spp. PME sequence. Our results show Citrus TT-PME is a distinctive new isoform with phylogenetic relationship closer to PME isoforms in other species rather than to previously described Citrus PME genes.
Technical Abstract: Despite the longstanding importance for the thermally-tolerant pectin methylesterase (TT-PME) activity in citrus juice processing and product quality, unequivocal identification of the protein and its corresponding gene has remained elusive. We purified TT-PME from sweet orange [Citrus sinensis (L.) Osbeck] finisher pulp and determined the corresponding mRNA sequence (1710 base pair) by cDNA cloning. An optimized purification scheme provided 20-fold increased enzyme yield over previous reports. SDS-PAGE showed two principal glycoisoforms. Their molecular masses determined by MALDI-TOF MS were approximately 47,900 Da and 53,000 Da. De novo peptide sequences generated from TT-PME by MALDI TOF/TOF MS enabled identification of anonymous Citrus EST cDNA accessions. These facilitated the subsequent cloning and complete sequencing of tt-pme cDNA obtained from a fruit mRNA library by RT- and RLM-RACE PCR. Our results show Citrus TT-PME is a novel isoform with sequence identity closer to the glycosylated kiwifruit PME than to the previously described Citrus thermally-labile PME isoforms.