Prekomjerna ekspresija i biokemijska karakterizacija proteina dipeptidil-peptidaze III iz termofilne bakterije Caldithrix abyssi

master's thesis

• Title (english): Overexpression and biochemical characterization of protein dipeptidyl-peptidase III from thermophilic bacteria Caldithrix abyssi
• Author: Iva Sučec
• Mentor: Mihaela Matovina (mentor)
• Committee member: Jasmina Rokov Plavec (predsjednik povjerenstva)
• Committee member: Branimir Bertoša (član povjerenstva)
• Committee member: Nives Galić (član povjerenstva)
• Committee member: Marko Močibob (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2018-07-17, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Dipeptidil-peptidaze III (DPPIII) su egzopeptidaze koje prema MEROPS bazi podataka pripadaju obitelji
metalopeptidaza M49. Kako bi se okarakterizirala porodica DPPIII i kod termofilnih organizmima, za
ovaj rad odabran je član porodice DPPIII iz anaerobne, termofilne bakterije Caldithrix abyssi koja se
može naći u morskim hidrotermalnim izvorima. Primijećeno je da prvi motiv aktivnog mjesta proteina
CaDPPIII sadrži pentapeptidni motiv 'HEISH' umjesto karakterističnog heksapeptidnog motiva
'HEXXGH', dok je drugi motiv potpuno očuvan.
Provedena je heterologna ekspresija kodirajućeg slijeda proteina DPPIII s histidinskim i
glutation-S-transferaznim privjeskom. Rekombinantni protein pročišćen je afinitetnom
kromatografijom i gel-filtracijom. Proteinu CaDPPIII-His6 određena je izoelektrična točka (pI = 6,12), te
je nativnom gel-elektroforezom i izoelektričnim fokusiranjem pokazano postojanje dva različita
strukturna oblika. Ispitivanje sintetskih supstrata peptidaza, pokazalo je značajnu hidrolitičku
aktivnost enzima prema Arg-Arg-2-naftilamidu (Arg2-2NA), Gly-Arg-2NA, Phe-Arg-2NA i Pro-Arg-2NA.
Ioni metala nemaju značajan utjecaj na peptidaznu aktivnost prema supstratu Gly-Arg-2NA. Najveća
peptidazna aktivnost pokazana je kod pH = 7,0 i kod temperatura od 55 ˚C do 65 ˚C. Potpunu inhibiciju
enzimske aktivnosti uzrokovali su kelirajući agensi, dok je djelomična inhibicija pokazana sa sulfhidrilblokirajućim
reagensima. Kinetička mjerenja pokazala su da CaDPPIII kao supstrat preferira Arg2-2NA
kao i drugi okarakterizirani enzimi ove porodice. U usporedbi s humanim, kvaščevim i enzimom iz
Bacteroides thetaiotaomicron, Km enzima CaDPPIII je znatno veći, dok je konstanta brzine reakcije
prvog reda koja odgovara pretvorbi supstrata u produkt (kcat) usporediva s istom konstantom enzima
iz B. thetaiotaomicron.
Dobiveni rezultati potvrđuju da je protein CaDPPIII član porodice DPPIII.

Abstract (english)

Dipeptidyl-peptidases III (DPPsIII) are exopeptidases that are classified as M49 family of
metallopeptidases in MEROPS database. To better understand DPPsIII from thermophilic organisms,
a protein from anaerobic, thermophilic bacteria Caldithrix abyssi was chosen for this thesis. These
bacteria can be found in Mid-Atlantic Ridge hydrothermal vents. Protein CaDPPIII has a modified
pentapeptide 'HEISH' active site motif, instead of characteristic 'HEXXGH' while the second motif is
conserved.
The gene for CaDPPIII with His-tag and GST-tag was heterologously expressed. The
recombinant protein was purified with affinity and size exclusion chromatography. Isoelectric point
(pI =6,12) was determined and two structural forms were detected by native electrophoresis and
isoelectric focusing. Among various synthetic peptidase substrates examined, CaDPPIII-His6 preferred
Arg-Arg-2-naphthylamide (Arg2-2NA), Gly-Arg-2NA, Phe-Arg-2NA and Pro-Arg-2NA. No significant
enhancement in peptidase activity was observed in the presence of metal ions. Its hydrolytic activity
was highest at pH = 7,0, and at 55 ˚C to 65 ˚C. Chelating agents completely inhibited the enzyme, while
the sulfhydryl reagents inhibited it partially. Kinetic measurements showed that preferred substrate
is Arg2-2NA, a characteristic DPPIII substrate. Constant Km is substantially higher for this enzyme
compared to human, yeast and bacterial enzyme. First-order rate constant (kcat) is comparable with
the same constant of the bacterial enzyme.
Obtained results confirm CaDPPIII as a member of DPPIII family.

• Keywords: Caldithrix abyssi; dipeptidil-peptidaza III; hidrolaza; metalopeptidaza
• Keywords (english): Caldithrix abyssi; dipeptidyl peptidase III; hydrolase; metallopeptidase
• Language: croatian
• URN:NBN: urn:nbn:hr:217:951364
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-09-04 10:31:26