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

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

Triarylborane Dyes as a Novel Non‐Covalent and Non‐Inhibitive Fluorimetric Markers for DPP III Enzyme

Novel dyes were prepared by simple “click CuAAC” attachment of a triarylborane–alkyne to the azide side chain of an amino acid yielding triarylborane dye 1 which was conjugated with pyrene (dye 2) forming a triarylborane–pyrene FRET pair. In contrast to previous cationic triarylboranes, the novel neutral dyes interact only with proteins, while their affinity to DNA/RNA is completely abolished. Both the reference triarylborane amino acid and triarylborane–pyrene conjugate bind to BSA and the hDPP III enzyme with high affinities, exhibiting a strong (up to 100‐fold) fluorescence increase, whereby the triarylborane–pyrene conjugate additionally retained FRET upon binding to the protein. Furthermore, the triarylborane dyes, upon binding to the hDPP III enzyme, did not impair its enzymatic activity under a wide range of experimental conditions, thus being the first non‐covalent fluorimetric markers for hDPP III, also applicable during enzymatic reactions with hDPP III substrates

Coumarin derivatives act as novel inhibitors of human dipeptidyl peptidase III: combined in vitro and in silico study

Dipeptidyl peptidase III (DPP III), a zinc- dependent exopeptidase, is a member of the metalloproteinase family M49 with distribution detected in almost all forms of life. Although the physiological role of human DPP III (hDPP III) is not yet fully elucidated, its involvement in pathophysiological processes such as mammalian pain modulation, blood pressure regulation, and cancer processes, underscores the need to find new hDPP III inhibitors. In this research, five series of structurally different coumarin derivatives were studied to provide a relationship between their inhibitory profile toward hDPP III combining an in vitro assay with an in silico molecular modeling study. The experimental results showed that 26 of the 40 tested compounds exhibited hDPP III inhibitory activity at a concentration of 10 µM. Compound 12 (3-benzoyl-7-hydroxy-2H-chromen-2-one) proved to be the most potent inhibitor with IC50 value of 1.10 μM. QSAR modeling indicates that the presence of larger substituents with double and triple bonds and aromatic hydroxyl groups on coumarin derivatives increases their inhibitory activity. Docking predicts that 12 binds to the region of inter-domain cleft of hDPP III while binding mode analysis obtained by MD simulations revealed the importance of 7- OH group on the coumarin core as well as enzyme residues Ile315, Ser317, Glu329, Phe381, Pro387, and Ile390 for the mechanism of the binding pattern and compound 12 stabilization. The present investigation, for the first time, provides an insight into the inhibitory effect of coumarin derivatives on this human metalloproteinase