Characterization of Three L-Asparaginases from Maritime Pine (Pinus pinaster Ait.)

Abstract

Asparaginases (ASPG, EC 3.5.1.1) catalyze the hydrolysis of the amide group of L-asparagine producing L-aspartate and ammonium. Three ASPG, PpASPG1, PpASPG2, and PpASPG3, have been identified in the transcriptome of maritime pine (Pinus pinaster Ait.) that were transiently expressed in Nicotiana benthamiana by agroinfection. The three recombinant proteins were processed in planta to active enzymes and it was found that all mature forms exhibited double activity asparaginase/isoaspartyl dipeptidase but only PpASPG1 was able to catalyze efficiently L-asparagine hydrolysis. PpASPG1 contains a variable region of 77 amino acids that is critical for proteolytic processing of the precursor and is retained in the mature enzyme. Furthermore, the functional analysis of deletion mutants demonstrated that this protein fragment is required for specific recognition of the substrate and favors enzyme stability. Potassium has a limited effect on the activation of maritime pine ASPG what is consistent with the lack of a critical residue essential for interaction of cation. Taken together, the results presented here highlight the specific features of ASPG from conifers when compared to the enzymes from angiosperms.

Identification of Metabolic Pathways Differentially Regulated in Somatic and Zygotic Embryos of Maritime Pine

“…In fact, the release of urea and ornithine through the action of ARG and subsequent catabolism, in coordination with the activity of P5CDH, could provide not only ammonium but also glutamate for GS1b activity ( Cañas et al, 2008 ). Asparagine is also important in N storage and mobilization in pine ( Canales et al, 2012 ), and the observed decrease in the abundance of ASPG transcripts is consistent with the homeostasis of asparagine ( Van Kerckhoven et al, 2017 ; Figure 7 ).…”