Biochemical characterization of a dual enzymatic activity hydrolase from moss Physcomitrella patens

Abstract

Nudix-dipeptidil-peptidaza III je biljni enzim s dvojnom hidrolaznom aktivnošću: dipeptidil-peptidaznom (DPP III) i fosfataznom (Nudix). Biokemijski su okarakterizirani homolozi iz mahovine (Physcomitrella patens) i uročnjaka (Arabidopsis thaliana). C-terminalna domena oba homologa predstavlja atipičnu DPP III s obzirom na aktivno mjesto (pentapeptid HEXXH umjesto heksapeptida HEXXGH u tipičnim DPP III), te je slabije aktivna prema sintetskim supstratima u usporedbi s tipičnim, do sada istraženim DPP III. N-terminalna domena je član nadporodice Nudix proteina. Utvrđeno je da Nudix domena ima fosfataznu aktivnost prema supstratima izopentenil-difosfatu i dimetilalil-difosfatu, prekursorima u sintezi izoprenoida. Funkcionalnim istraživanjima na mutantama mahovine P. patens koje ne eksprimiraju ovaj enzim pokazano je da on sudjeluje u metabolizmu biljnih hormona citokinina. Preliminarni rezultati analize ekspresije gena u mahovini u prisutnosti stresora sugeriraju da enzim Nudix-DPP III sudjeluje u odgovoru biljaka na stres (sušu, solni i oksidacijski stres). Zaključno, predložen je naziv izopentenil-difosfat-fosfohidrolaza za novopronađenu enzimsku aktivnost.Nudix-dipeptidyl peptidase III is a plant enzyme with dual hydrolase activity: dipeptidylpeptidase III (DPP III) and phosphatase (Nudix). Homologues from moss (Physcomitrella patens) and angiosperm (Arabidopsis thaliana) were biochemically characterized. C-terminal domain of both homologues represents an atypical DPP III considering the active site (pentapeptide HEXXH instead of hexapeptide HEXXGH in typical DPPs III), and it is less active towards synthetic supstrates than typical, formerly characterized DPPs III. N-terminal domain is a member of Nudix superfamily. It was established that it has phosphatase activity towards isopentenyl-diphosphate and dimethylallyl-diphosphate, precursors in isoprenoid sythesis. Functional research on moss P. patens knock-out mutants showed that this enzyme is involved in cytokinin metabolism. Preliminary analysis of gene expression in the presence of stressors suggests that Nudix-DPP III enzyme is involved in plant response to stress (drought, salt and oxidative stress). Finally, the name isopentenyl diphosphate phosphohydrolase is suggested for the newly identified enzyme activity