Fruit ripening-associated leucylaminopeptidase with cysteinylglycine dipeptidase activity from durian suggests its involvement in glutathione recycling

Background: Durian (Durio zibethinus L.) is a highly popular fruit in Thailand and several other Southeast Asian countries. It is abundant in essential nutrients and sulphur-containing compounds such as glutathione (GSH) and gamma-glutamylcysteine (gamma-EC). Cysteinylglycine (Cys-Gly) is produced by GSH catabolism and occurs in durian fruit pulp. Cysteine (Cys) is a precursor of sulphur-containing volatiles generated during fruit ripening. The aforementioned substances contribute to the strong odour and flavour of the ripe fruit. However, the genes encoding plant Cys-Gly dipeptidases are unknown. The aim of this study was to measure leucylaminopeptidase (LAP) activity in durian fruit pulp. Results: We identified DzLAP1 and DzLAP2, which the former was highly expressed in the fruit pulp. DzLAP1 was expressed at various ripening stages and in response to ethephon/1-MCP treatment. Hence, DzLAP1 is active at the early stages of fruit ripening. DzLAP1 is a metalloenzyme similar to 63 kDa in size. It is activated by Mg2+ or Mn2+ and, like other LAPs, its optimal alkaline pH is 9.5. Kinetic studies revealed that DzLAP1 has K-m = 1.62 mM for its preferred substrate Cys-Gly. DzLAP1-GFP was localised to the cytosol and targeted the plastids. In planta Cys-Gly hydrolysis was confirmed for Nicotiana benthamiana leaves co-infiltrated with Cys-Gly and expressing DzLAP1. Conclusions: DzLAP1 has Cys-Gly dipeptidase activity in the gamma-glutamyl cycle. The present study revealed that the LAPs account for the high sulphur-containing compound levels identified in fully ripened durian fruit pulp.

Automated summary

The study identified DzLAP1 as the main leucylaminopeptidase (LAP) responsible for LAP activity in durian fruit pulp, with DzLAP1 being active in the early stages of fruit ripening. DzLAP1 is a metalloenzyme similar to other LAPs, and shows activity towards its preferred substrate Cys-Gly.