Proučavanje dioksigenaza ovisnih o željezu računalnim metodama

doctoral thesis

• Title (english): Computational studies of iron dependent dioxygenases
• Author: Hrvoje Brkić
• Mentor: Sanja Tomić (mentor)
• Committee member: Borislav Kovačević (predsjednik povjerenstva)
• Committee member: Sanja Tomić (član povjerenstva)
• Committee member: Nenad Pavin (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Physics); Zagreb
• Defense date and country: 2014-03-21, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Physics
• Universal decimal classification (UDC): 53 - Physics

Abstract

Tema ove doktorske disertacije je teorijsko proučavanje dioksigenaza, enzima koji pomoću iona željeza kataliziraju ugradnju oba atoma iz molekule kisika u supstrat. Proučavana su dva enzima Acetilaceton dioksigenaza iz Acinetobacter Jonsoii (Dke1) te ljudska 3,4-dioksigenaza 3-hidroksiantranilne kiseline (3HAO). Trodimenzionalne strukture navedenih enzima, određene rendgenskom strukturnom analizom i pohranjene u PDB bazi, korištene su kao ishodište pri modeliranju. Parametri koji nedostaju u AMBER polju sila (raspodjela naboja na ionu željeza i ligandima koji ga koordiniraju, duljine veza, iznosi kutova te pripadajuće barijere), a nužni su za simulacije, određeni su kvantno mehaničkim računima (QM) na sustavu Dke1 a uspješno su primijenjeni i na 3HAO sustavu. Stabilnost i ponašanje enzima u približno fiziološkim uvjetima proučeni su primjenom empirijskih metoda molekulskog modeliranja. Tim pristupom je analizirana stabilnost sustava, zastupljenost molekula vode u aktivnom mjestu, mreža vodikovih veza, interakcije pojedinih aminokiselinskih ostataka tijekom simulacija, njihovi srednji kvadratni pomaci tj. fluktuacije te putevi ulaska/izlaska molekula vode, reaktanata, produkata i metalnog kofaktora u enzim... Dana su teorijska pojašnjenja smanjene katalitičke aktivnosti enzima, promjene u transportu metala, molekula vode, supstrata i reaktanata do aktivnog mjesta enzima te promijene međusobnih interakcija aminokiselinskih ostataka uslijed točkastih mutacija. Pomoću QM računa utvrđen je reakcijski mehanizam na modelnom sustavu, a pomoću hibridnog kvantno mehaničkog-molekulsko mehaničkog (QM/MM) računa taj je mehanizam potvrđen u realnom okruženju enzima.

Abstract (english)

Subject of this doctoral thesis is theoretical study of dioxygenasis, non-hem iron dependent enzymes that incorporate both molecular oxygen atoms into substrate. Two enzymes were studied: Acetylacetone dioxygenasis form Acinetobacter Jonsoii (Dke1), and human 3,4-dioxygenasis of 3-hydroxyanthranilic acid (3HAO). Three dimensional structures of these enzymes, determined by x-ray crystallography and stored to the Protein Data Bank (www.ebi.ac.uk/pdbe ; codes 3BAL and 2QNK respectively), were used as starting points in the molecular modelling. Parameters required in MD simulations of the systems, but missing in the AMBER force field (i.e. charge distribution on iron ion and coordinating residues, bond lengths, angles, dihedrals and their barriers), were determined using quantum mechanical (QM) calculations. Stability and behaviour of the enzymes in nearly physiological conditions were studied by applying empirical methods of molecular modelling. Results of these simulations revealed: water population in the active site, hydrogen bond networks, amino acid residues interactions, protein flexibility… Besides, the pathways for trafficking different ligands (water, reactants, products and metal cofactor) from the enzyme environment to the active site were determined. Impact of point mutations to the hydrogen bond network and to the active site water accessibility was determined in Dke1. The reduced enzyme activity of the mutants was studied in correlation with trafficking of the metal ion, water, substrate and reactants to the enzyme active site, as well as in corelation with the amino acid residues interactions. Using hybrid quantum-mechanics/molecular-mechanics (QM/MM) calculations the reaction mechanism for 3HAO was determined. On the basis of the MD results, we proposed explanation of the reduced enzyme activity of the mutants.

• Keywords: metaloenzimi; molekulska dinamika; polje sila; semiempirijski računi; kvantno mehanički računi; diketon dioksigenaza; 3; 4-dioksigenaza 3-hidroksiantranilne kiseline
• Keywords (english): metalloenzymes; molecular dynamics; force field; semiempirical calculations; quantum mechanical calculations; diketon dioxygenases; dioxygenasis of 3-hydroxy anthranilic acid; dioxygenase reaction mechanism; quantum mechanics/molecular mechanics calculations
• Language: croatian
• URN:NBN: urn:nbn:hr:217:363487
• Study programme: Title: Physics; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje fizika (doktor/doktorica znanosti, područje prirodnih znanosti, polje fizika)
• Type of resource: Text
• Extent: 117 str.
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-08-25 07:45:39