A novel plant enzyme with dual activity: an atypical Nudix hydrolase and a dipeptidyl peptidase III

Abstract

In a search for plant homologues of dipeptidyl peptidase III (DPP III) family, we found a predicted protein from the moss Physcomitrella patens (UniProt entry: A9TLP4), which shared 61% sequence identity with the Arabidopsis thaliana uncharacterized protein, designated Nudix hydrolase 3. Both proteins contained all conserved regions of the DPP III family, but instead of the characteristic hexapeptide HEXXGH zinc-binding motif, they possessed a pentapeptide HEXXH, and at the N-terminus, a Nudix box, a hallmark of Nudix hydrolases, known to act upon a variety of nucleoside diphosphate derivatives. To investigate their biochemical properties, we expressed heterologously and purified Physcomitrella (PpND) and Arabidopsis (AtND) protein. Both hydrolyzed, with comparable catalytic efficiency, the isopentenyl diphosphate (IPP), a universal precursor for the biosynthesis of isoprenoid compounds. In addition, PpND dephosphorylated four purine nucleotides (ADP, dGDP, dGTP, and 8-oxo-dATP) with strong preference for oxidized dATP. Furthermore, PpND and AtND showed DPP III activity against dipeptidyl-2-arylamide substrates, which they cleaved with different specificity. This is the first report of a dual activity enzyme, highly conserved in land plants, which catalyses the hydrolysis of a peptide bond and of a phosphate bond, acting both as a dipeptidyl peptidase III and an atypical Nudix hydrolase.

The evolution of function within the Nudix homology clan

“…Enzyme‐catalyzed hydrolysis among Nudix hydrolases usually occurs at a phosphorus atom participating in a pyrophosphate linkage, although there are enzymes that perform nucleophilic substitution at the carbon atom of some sugars (e.g., GDP‐sugar glycosyl hydrolases) . Furthermore, some Nudix hydrolases contain additional and distinct protein domains that perform other enzymatic functions . Nudix protein hydrolase activity thus results in either one or two phosphorylated products [Fig.…”

“…Transcriptional regulation and calcium channel gating activities were also reported for Nudix homology proteins. Noncatalytic Nudix homology domains are typically part of a multidomain protein and bind small molecules or interact with other protein domains …”

“…38 Furthermore, some Nudix hydrolases contain additional and distinct protein domains that perform other enzymatic functions. 39 Nudix protein hydrolase activity thus results in either one or two phosphorylated products [ Fig. 2(a)].…”

“…Noncatalytic Nudix homology domains are typically part of a multidomain protein and bind small molecules or interact with other protein domains. 20,39 It is challenging to assign specific function to an uncharacterized member of this clan. Nudix pyrophosphohydrolases have been characterized for an enormous diversity of functions, including activity on capped RNA, 23,48 (deoxy)nucleoside di-and triphosphates, 49,50 Ap n As, 29,51 NDP-sugars, 52,53 and coenzymes such as thiamin pyrophosphate, CoA, and NADH.…”

Transglycosidases and Fungal Cell Wall β-(1,3)-Glucan Branching

“…A few to mention, tyrosine hydroxylase from Toxoplasma gondii can utilize either phenylalanine or tyrosine to generate L-DOPA [17], 3β,20α-hydroxy steroid oxidoreductase from calf fetal RBC [18], a novel plant enzyme showing dipeptidyl peptidase III and an atypical Nudix hydrolase activities cleaving peptide and phosphate linkages, respectively [19], peroxiredoxin possessing peroxidase and catalase activity [20], neopullulanase showing hydrolysis and transglycosylation at α-(1,4)-and α-(1,6)-glucosidic linkages [21] and AmiA in Chlamydia with amidase and carboxypeptidase activity [22]. But our current findings along with these examples raise the question, what is the biological relevence of such dual activity?…”