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A bifunctional plant enzyme: an isopentenyl diphosphate hydrolase fused to a dipeptidyl peptidase III


Karačić, Zrinka; Vukelić, Bojana; Ho, Gabrielle H.; Jozić, Iva; Sučec, Iva; Salopek-Sondi, Branka; Kozlović, Marija; Brenner, Steven E.; Ludwig-Müller, Jutta; Abramić, Marija
A bifunctional plant enzyme: an isopentenyl diphosphate hydrolase fused to a dipeptidyl peptidase III // Novel enzymes 2016
Groningen, Nizozemska, 2016. str. 92-92 (poster, međunarodna recenzija, sažetak, znanstveni)


Naslov
A bifunctional plant enzyme: an isopentenyl diphosphate hydrolase fused to a dipeptidyl peptidase III

Autori
Karačić, Zrinka ; Vukelić, Bojana ; Ho, Gabrielle H. ; Jozić, Iva ; Sučec, Iva ; Salopek-Sondi, Branka ; Kozlović, Marija ; Brenner, Steven E. ; Ludwig-Müller, Jutta ; Abramić, Marija

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Novel enzymes 2016 / - , 2016, 92-92

Skup
Novel Enzymes 2016

Mjesto i datum
Groningen, Nizozemska, 10.-14.10.2016

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Plant biochemistry, Nudix hydrolase, dipeptidyl-peptidase III, isopentenyl diphosphate

Sažetak
A novel enzyme with dual activity - an isopentenyl diphosphate (IPP) hydrolase from the Nudix family fused to a dipeptidyl peptidase III (DPP III) domain has been discovered in Physcomitrella patens and Arabidopsis thaliana and biochemically characterized.1 Heterologously expressed Physcomitrella patens and Arabidopsis thaliana Nudix-DPP III (PpND and AtND) proteins showed peptidase activity against the preferred artificial substrate of DPPs III. The Nudix domain of these proteins was presumed to be a Nudix hydrolase (phosphatase), since it contains a functional Nudix box motif. Based on sequence similarity with isopentenyl diphosphate isomerase (also a Nudix fold protein), we recognized IPP as a possible substrate. Indeed, in a screen of 73 potential Nudix hydrolase substrates, both PpND and AtND showed preference for IPP, producing isopentenyl phosphate (IP) as a final product. With both phosphatase and peptidase activity confirmed by enzyme assays, we used site- directed mutagenesis to investigate structure- activity relationships. In PpND, Glu92 and Glu592 were identified as putative catalytic residues. Point mutations of each Glu92 and Glu592 to Ala caused an absence of phosphatase and peptidase activity, respectively. Our results confirm the presence of two separate active sites, although both domains are needed for proper folding and activity of this enzyme. Separate domains produced as recombinant proteins were either insoluble or unstable. The exclusive presence of this fusion protein in plants is still unclear and may have an adaptive role for life on land. The physiological role of the phosphatase might be in the regulation of the pool of IPP, the isoprenoid building block, in plant cell cytoplasm, while DPP III is supposed to be involved in protein catabolism. Functional role of this dual enzyme is being investigated on Physcomitrella patens mutants. From a biotechnological point of view, this enzyme might be useful in microbial isopentenol biofuel production. References 1 Karačić, Z. et al. (2016), A novel plant enzyme with dual activity: an atypical Nudix hydrolase and a dipeptidyl peptidase III, Biological Chemistry (in print), DOI: 10.1515/hsz-2016-0141

Izvorni jezik
Engleski

Znanstvena područja
Kemija



POVEZANOST RADA


Projekt / tema
HRZZ-IP-2013-11-7235 - Povezanost fleksibilnosti, aktivnosti i strukture u porodici dipeptidil-peptidaza III (Sanja Tomić, )

Ustanove
Institut "Ruđer Bošković", Zagreb