Molecular modeling of enzyme-substrate interactions and reaction mechansims in peroxidases and aldo-keto reductases Carme Rovira ICREA / Parc Científic de Barcelona Barcelona
What we do Molecular (atomistic) simulation Application of classical methodologies (based on Newton s equations) to investigate the properties of matter from an atomic or molecular basis. oxygen active center myoglobin
Techniques Classical molecular dynamics Interaction energy from MM E = E bond + E ang + E dih + E vdw + E elec + Ab initio molecular dynamics Interaction energy from QM E = min E ρ(r) [ ρ(r), { R }] I QM/MM CPMD QM MM computationally intense calculations Other: Docking, structure alignment, sequence alignment,...
Projects in MN Substrate recognition and catalysis in 1. Heme peroxidases and catalases N N (protection against oxidative damage) N N Fe O O - O O - heme 2. Aldo-keto reductases (reduction of carbonyl compounds) all trans retynaldehyde retynaldehyde
1. Heme peroxidases Oxidation of substrates using hydrogen peroxide H 2 O 2 + 2S red 2H 2 O + 2S ox (S red / S ox = reduced / oxidized substrate) His42 Role: Defense against pathogens His170 heme Applications: Biotechnology and biocatalysis Horseradish peroxidase (HRP) Berglund et al. Nature 2002 (PDB code 1h58)
Catalase-peroxidases (KatG) heme peroxidases Bifunctional enzyme peroxidase: H 2 O 2 + 2S red 2H 2 O + 2S ox catalase: 2 H 2 O 2 2 H 2 O + O 2 His Arg heme His Asp
KatG as antitubercular target heme peroxidases KATG is responsible for INH activation (Zhang et al. Nature. 1992 358, 591-3) KATG mutations cause drug resistance to INH (Altamirano et al. J. Infect. Dis. 1994 169, 162-5) First X-Ray structures (2003-2004) Isoniazid (INH) Open questions How does the enzyme operate? Where does INH bind? INH activation mechanism?
Reaction intermediates heme peroxidases Compound I intermediate Tyr238-O - Tyr238-OH ph heme heme P. Vidossich et al. JACS 129, 13436 (2007). O + typical peroxidase intermediate Alfonso-Prieto et al. JACS 129, 4193 (2007).
Reaction intermediates heme peroxidases Compound I intermediate Tyr238-O - Tyr238-OH ph heme Trp330 heme P. Vidossich et al. JACS 129, 13436 (2007). Uncommon electronic structure (ph dependent) Possible relation with enzyme bifunctionality (peroxidase and catalase reactions occur at low/high ph)
Catalase reaction mechanism heme peroxidases ~ 150-250 QM atoms 10 ps AIMD, 10 5 h MN (4 months, 64/128 procs). Reaction free energy landscape products path A path B reactants t Two different pathways (useful for catalase inhibitor design) (Alfonso-Prieto et al. JACS 2009, 131, 11751-61)
2. Mechanisms of substrate specificity in aldo-keto reductases (AKRs) Reduction of carbonyl compounds (aldehydes and ketones) AKR -C=O -C-OH Substrates: lipids, steroids, cathecolamines, prostaglandins, retinoids,...
Human small intestine aldose reductase (AKR1B10) New findings: 1. Detected in large amounts in certain types of lung cancer possible diagnosis marker 2. Activity with retinaldehyde (a precursor of retinoic acid, RA, an anticancer agent) Up-regulation of AKR1B10 activity in cancer could be related with decrease of RA levels Fukumoto et al. Clin. Cancer Res. 11, 1776 (2005) Sheetz & King, J. Am. Med. Assoc. 288, 2579 (2002) Gallego et al. Biochem. J. 2006, 399, 101 Seeking for AKRB10 inhibitors - understanding the basis of substrate recognition
Kinetic data aldo-keto reductases The activity of AKR1B10 has been evaluated with substrates of different sizes. OH O 1 Gliceraldehyde (1) OH ze si 9 cis retinaldehyde (2) All trans retinaldehyde (3) O 2 O 3 (O. Gallego, J. Ruiz, J. Farrés, X. Parés, UAB)
Kinetic data aldo-keto reductases (O. Gallego, J. Ruiz, J. Farrés, X. Parés, UAB) K cat, min 1 AKR1B10 Aldose reductase (65% homology to AKR1B10 and involved in diabetes) AKR1B1 D,L-Glyceraldehyde 35 ± 1 31 ± 1 9-cis-retinaldehyde 0.9 ± 0.1 0.7 ± 0.1 all-trans-retinaldehyde 27 ± 1 035± 0.35 001 0.01 What are the structural determinants of AKR1B10 specificity for all trans retinaldehyde?
Initial structure for the calculations aldo-keto reductases loop B loop C loop A AKR1B10 structure in complex with iha non specific inhibitor (I. Fita, IRB-CSIC)
Calculations aldo-keto reductases Inhibitor (I) Substrate (S) 1) Remove I 2) Docking of S 3) MD AKR1B1/S cis AKR1B10/S cis AKR1B1/S trans AKR1B10/S trans Programs: AUTODOCK, NAMD 100 ns MD, 10 5 h MN (2 months, 128/256 procs).
Results AKR1B10 in complex with retinaldehyde Flipping of Lys125 side chain + conformational change of loop A No such change is observed in AKR1B1 (Leu125) Lys125 Lys125 is the key residue (confirmed by site directed mutagenesis) all-trans-retinaldehyde 9-cis-retinaldehyde Gallego et al. PNAS, 104, 20764 (2007); Ruiz et al. Chem. Biol. Inter. 178, 171 (2009) loop A
Coworkers Mercedes Alfonso-Prieto (PCB, Barcelona) Pietro Vidossich (PCB, Barcelona) Albert Ardèvol (PCB, Barcelona) Experimental collaborators: Ignacio Fita (IRB-CSIC, Barcelona) Peter C. Loewen (U. Manitoba, Canada) Xavier Parés, Jaume Farrés (UAB, Barcelona)