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Structure/function relationships in D-aspartate oxidase, the key enzime for the modulation of D-aspartate - Gianluca Molla

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Structure/function relationships in D-aspartate oxidase, the key enzime for the modulation of D-aspartate - Gianluca Molla

  1. 1. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 1 Como - 2016 Structure/function relationships in D-aspartate oxidase, the key enzyme for the modulation of D-Aspartate in the brain GIANLUCA MOLLA Università degli Studi dell’Insubria The Protein Factory
  2. 2. Introduction In PubMed: you can find 1829 papers with the “structure-function relationships” words in the title Protein Structure Role of protein residues Validation of molecular model Enzyme properties Predict function Find a rational for observed properties
  3. 3. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 3 Flavoproteins In vitro evolution of enzymes Protein engineering Protein crystallogarphyCloning of novel flavoproteins and enzymes Rapid kinetics Structural bioinfromatics Biocatalysis
  4. 4. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 4 FAD Flavoproteins Aerobic metabolism Soil detoxification Phototropism Signal transduction Photosynthesis Fetus developement Control of enzyme function Bioluminescence Apoptosis Membrane electron chains
  5. 5. Introduction D-Aspartate oxidase Catalyzed reaction Localization/Phylogenesis Biochemistry Cloning and heterologous expression Cofactor binding Thermal stability Kinetic parameters Substrate specificity Substrate binding and active site architecture Model of 3D-structure DASPO as a novel drug target Classical amino acid oxidase inhibitors Screening of natural compounds Virtual screening of chemical libraries Outline
  6. 6. Physiological endogenous co-agonist for synaptic N-Methyl- D-Aspartate receptors at central excitatory Synapses (CNS) D-amino acids in mammalian brain Brain development Synaptic transmission Synaptic plasticity ? D-Aspartate D-Serine Altered D-Ser concentration Altered NMDAR activity Disturbed glutamatergic neurotransmission Pathology (e.g. schizophrenia)
  7. 7. Regional and temporal distribution of D-aspartate Temporal distribution of D-Asp supports an important role in the development and neurogenesis of the brain Central nervous system + endocrine (and neuroendocrine) system pineal gland, pituitary gland, pancreas, ovaries, testes, thyroid gland, parathyroid gland, hypothalamus, gastrointestinal tract and adrenal glands. Rat adrenal gland Rat pancreas Rat (mouse) retinae Rat (pig) testis Rat salivary glands Katane et al., 2010 human gastric juice human spermatozoa human follicular fluid human salivary glands Embryonic day 12 caudal forebrain, midbrain and hindbrain in neuroblasts At post-natal day 0 D-Asp immunoreactivity is reduced Between E14 and E20 D-Asp extends to the whole brain including the cerebral cortex. In cytoplasm and processes of neuroblasts axons. Sakai et al., 1998 Errico et at., 2015 Post-natal day 28 D-Asp almost disappears Zones playing major role in regulating development Human prefrontal cortex: E14 60% D-Asp, 40% L-Asp)
  8. 8. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 9 Metabolism of D-aspartate Serine racemase D-amino acid oxidase D-Aspartate oxidase First activity was reported in rabbit kidney by Still 1949 2010 2015 Aspartate Racemase EC 5.1.1.13 (?) L-aspartate D-aspartate
  9. 9. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 10 Reaction catalyzed by D-aspartate oxidase D-Aspartate Sketching chemical compounds: www.emolecules.com/ E-FAD E-FADH2 + + Imino aspartate Oxaloacetate H2O NH3 + + Imino aspartate O2 H2O2E-FADE-FADH2 + + N-Methyl-D-Aspartate Oxaloacetate + Methylamine CH3NH2+
  10. 10. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 11 Phylogenetic tree of DASPO Orthologs Ancestral gene http://phylomedb.org/ DASPO activity found in homogenates from cephalopods, gastropods, fishes, amphibians, birds, mammals, yeast Orthologs NO DASPO in bacteria or plants Paralogs Ancestral gene
  11. 11. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 12 Distribution genevisible.com/search Nebion-geneinvestigator: high performance search engine for gene expression that integrates thousands of manually curated, well described public microarray and RNAseq experiments (Katane et al., 2010) DASPO and it’s substrate D-Asp are complementary present in mammalian tissues After birth Kidney - liver - Cerebrum DASPO D-Asp Katane, 2010
  12. 12. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 13 Interactome http://string-db.org/ PEX5 (peroxisomal biogenesis factor 5) The product of this gene binds to the C- terminal PTS1 peroxisomal targeting signal. PEX5 Plays an essential role in peroxisomal protein import. DASPO is localized in peroxisomes (like the homolog DAAO) Peroxisomal catalase eliminates toxic H2O2 which is produced during the reoxidation of the cofactor od FAD
  13. 13. Introduction D-Aspartate oxidase Catalyzed reaction Localization/Phylogenesis Biochemistry Cloning and heterologous expression Cofactor binding Thermal stability Kinetic parameters Substrate specificity Substrate binding and active site architecture Model of 3D-structure DASPO as a novel drug target Classical amino acid oxidase inhibitors Screening of natural compounds Virtual screening of chemical libraries Outline
  14. 14. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 15 10 20 30 40 50 60 70 80 90 100 110 120 | | | | | | | | | | | | OXDD_BOVIN MDTVRIAVVGAGVMGLSTAVCISKMVPGCSITVISDKFTPETTSDVAAGMLIPPTYPDTPIQKQKQWFKETFDHLFAIVNSAEAEDAGVILVSGWQIFQSIPTEEVPYWADVVLGFRKMT OXDD_PIG MDTVRIAVVGAGVMGLSTAVCIFKLVPGCSITVISDKFTPETTSDVAAGMLIPPVYPDTPIHKQKQWFKDTFDHLFAIANSAEAKDAGVLLVSGWQIFQSAPTEEVPFWADVVLGFRKMT OXDD_HUMAN MDTARIAVVGAGVVGLSTAVCISKLVPRCSVTIISDKFTPDTTSDVAAGMLIPHTYPDTPIHTQKQWFRETFNHLFAIANSAEAGDAGVHLVSGWQIFQSTPTEEVPFWADVVLGFRKMT OXDD_MOUSE MDTVCIAVVGAGVIGLSTAACISQLVPGCTVTVISDRFTPDTTSNVAAGMLIPHTCADTPVPTQKRWFRETFEHLSEIAKSAEAADAGVHLVSGWQIFRSVPAEEVPFWADVVLGFRKMT D3ZDM7_RAT MDTVRIAVVGAGVIGLSTAACVSQLVPRCSVTVISDRFTPDTTSNVAAGMLIPPTYPDTPVPTLKRWFRETFQHLSEIARSAEAVDAGIHLVSGWQIFRSVPTEEVPFWADVVLGFREMT 130 140 150 160 170 180 190 200 210 220 230 240 | | | | | | | | | | | | OXDD_BOVIN KDELKKFPQHVFGHAFTTLKCEGPAYLPWLQKRVKGNGGLILTRRIEDLWELHPSFDIVVNCSGLGSRQLAGDSKIFPVRGQVLKVQAPWVKHFIRDSSGLTYIYPGVSNVTLGGTRQKG OXDD_PIG KNELKKFPQHVCGQAFTTLKYEGPTYLPWLEKRVKGSGGLVLTRRVEDLWELHPSFDIVVNCSGLGSKQLVGDMDIFPVRGQVLKVQAPWVKHFIRDGSGLTYIYPGLANVTLGGTRQKG OXDD_HUMAN EAELKKFPQYVFGQAFTTLKCECPAYLPWLEKRIKGSGGWTLTRRIEDLWELHPSFDIVVNCSGLGSRQLAGDSKIFPVRGQVLQVQAPWVEHFIRDGSGLTYIYPGTSHVTLGGTRQKG OXDD_MOUSE EAELKRFPQYVFGQAFTTLKCETSAYLPWLERRIKGSGGLLLTRRIEDLWELQPSFDIVVNCSGLGSRRLVGDPMISPVRGQVLQARAPWVKHFIRDGGGLTYVYPGMSYVTLGGTRQKG D3ZDM7_RAT EAELKRFPQYEFGQAFTTLKCETSAYLPWLEKRIKGSGGLLLTRRIEDLWELQPSFDIVVNCSGLGSRRLVGDATVSPVRGQVLQAQAPWVKHFIRDGGGLTYVYPGTSYVTLGGSRQTG 250 260 270 280 290 300 310 320 330 340 | | | | | | | | | | OXDD_BOVIN DWNLSPDAEISKEILSRCCALEPSLRGAYDLREKVGLRPTRPSVRLEKELLAQDSRRLPVVHHYGHGSGGIAMHWGTALEATRLVNECVQVLRTPAPKSKL OXDD_PIG DWNLSPNAEISKQILSRCCALEPSLRGACDIREKVGLRPSRPGVRLEKELLVQGSQRLPVVHNYGHGSGGIAMHWGTALEAARLVSECVQALRTPAPKSKL OXDD_HUMAN DWNLSPDAENSREILSRCCALEPSLHGACNIREKVGLRPYRPGVRLQTELLARDGQRLPVVHHYGHGSGGISVHWGTALEAARLVSECVHALRTPIPKSNL OXDD_MOUSE DWNRSPDAELSREIFSRCCTLEPSLHRAYDIKEKVGLRPSRPGVRLQKEILVRGQQTLPVVHNYGHGSGGISVHWGSALEATRLVMECIHTLRTPASLSKL D3ZDM7_RAT DWNLSPDAELSREIFSRCCALEPSLHRACDIKEKVGLRPSRPGVRLQKEILVRGEQRLPVVHNYGHGSGGISVHWGSALEATRLVMECVHTLRTPASLSKL Sequence alignment Mammalian DASPOs sequence identity: 75-91% GXGXXG------------------D Dinucleotide binding motif (Wierenga sequence) PST1-prexisomal targeting sequence Rat 91.2% 80.4% Mouse 81.8% Human
  15. 15. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 16 10 20 30 40 50 60 70 80 90 100 110 120 | | | | | | | | | | | | DDO1xx0 ----------------------------MDTARIAVVGAGVVGLSTAVCISKLVPRCSVTIISDKFTPDTTSDVAAGMLIPHTYPDTPIHTQKQWFRETFNHLFAIANSAEAGDAGVHLV DDO2xx1 ----------------------------MDTARIAVVGAGVVGLSTAVCISKLVPRCSVTIISDKFTPDTTSDVAAGMLIPHTYPDTPIHTQKQWFRETFNHLFAIANSAEAGDAGVHLV 130 140 150 160 170 180 190 200 210 220 230 240 | | | | | | | | | | | | DDO1xx0 SGWQIFQSTPTEEVPFWADVVLGFRKMTEAELKKFPQYVFGQAFTTLKCECPAYLPWLEKRIKGSGGWTLTRRIEDLWELHPSFDIVVNCSGLGSRQLAGDSKIFPVRGQVLQVQAPWVE DDO2xx1 S--------------------------------G---------------------------IKGSGGWTLTRRIEDLWELHPSFDIVVNCSGLGSRQLAGDSKIFPVRGQVLQVQAPWVE 250 260 270 280 290 300 310 320 330 340 350 360 | | | | | | | | | | | | DDO1xx0 HFIRDGSGLTYIYPGTSHVTLGGTRQKGDWNLSPDAENSREILSRCCALEPSLHGACNIREKVGLRPYRPGVRLQTELLARDGQRLPVVHHYGHGSGGISVHWGTALEAARLVSECVHALRTPIPKSNL DDO2xx1 HFIRDGSGLTYIYPGTSHVTLGGTRQKGDWNLSPDAENSREILSRCCALEPSLHGACNIREKVGLRPYRPGVRLQTELLARDGQRLPVVHHYGHGSGGISVHWGTALEAARLVSECVHALRTPIPKSNL Mammalian DASPOs sequences DASPO cDNA was cloned from: human, mouse, bovine, pig, octopus, yeast 2 forms: DDO1 and DDO2 (alternative splicing) DDO1  341AA DDO2 is a deletion variant (59AA missing)  282AA. Recombinant DDO2 is insoluble (IB) 10 20 30 40 50 60 70 80 90 100 110 120 | | | | | | | | | | | | katane MDTVCIAVVGAGVIGLSTAACISQLVPGCTVTVISDRFTPDTTSNVAAGMLIPHTYADTPVPTQKRWFRETFEHLSEIAKSAEAADAGVHLVSGWQIFHSVPAEEVPFWADVVLGFRKMT OXDD_MOUSE MDTVCIAVVGAGVIGLSTAACISQLVPGCTVTVISDRFTPDTTSNVAAGMLIPHTCADTPVPTQKRWFRETFEHLSEIAKSAEAADAGVHLVSGWQIFRSVPAEEVPFWADVVLGFRKMT 130 140 150 160 170 180 190 200 210 220 230 240 | | | | | | | | | | | | katane EAELKRFPQYVFGQAFTTLKCETSAYLPWLERRIKGSGGLLLTWRIEDLWELQPSFDIVVNCSGLGSRRLVGDPMISPVRGQVLQARAPWVKHFIRDGGGLTYVYPGMSYVTLGGTRQKG OXDD_MOUSE EAELKRFPQYVFGQAFTTLKCETSAYLPWLERRIKGSGGLLLTRRIEDLWELQPSFDIVVNCSGLGSRRLVGDPMISPVRGQVLQARAPWVKHFIRDGGGLTYVYPGMSYVTLGGTRQKG 250 260 270 280 290 300 310 320 330 340 | | | | | | | | | | katane DWNRSPDAELSREIFSRCCTLEPSLHRAYDIKEKVGLRPSRPGVRLQKEILVRGQQTLPVVHNYGHGSGGISVHWGSALEATRLVMECIHTLRTPASLSKL OXDD_MOUSE DWNRSPDAELSREIFSRCCTLEPSLHRAYDIKEKVGLRPSRPGVRLQKEILVRGQQTLPVVHNYGHGSGGISVHWGSALEATRLVMECIHTLRTPASLSKL mDDO01 mDDO02 mDDO01 mDDO02 mDDO01 mDDO02 Mouse DASPO cDNA 2 different sequences - 3 AA substitutions (position 56, 99, 164)  341AA mDDO01: Y H W mDDO02: C R R Human: Y Q R Human DASPO cDNA
  16. 16. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 17 DASPO heterologous expression N-term His-Tag Chemical lysis Crude extract n.a. Katane et al., 2007 hDASPO Bl21(de3) Setoyama et al., 1997 0.01 mM IPTG OD600=0.6 IPTG 37°C 20h Sonicationheat treatment 55°C 15 minphenyl sepharose  DEAE sepharose 2.8 mg/LpET11 Bl21(de3)pLysS N-term GHS-Tag N-term His-Tag 0.1 mM IPTG OD600=0.5 26°C 16h Chemical lysis+ Affinity chromatography 0.40 mg/L 0.07 mg/L Bl21(de3)pLysS Katane et al., 2008 mDASPO N-term His-Tag n.a. Katane et al., 2015 Bl21(de3)pLysS ratDASPO 0.5 mM IPTG @ OD600=0.5 26°C 16h N-term His-Tag Katane et al., 2015 Bl21(de3)pLysS 0.1 mM IPTG OD600=0.5 26°C 16h Chemical lysis+ Affinity chromatography Chemical lysis+ Affinity chromatography Partly IB 0.01 mM IPTG OD600=0.5 30°C 20h N-term His-Tag Katane et al., 2015 Bl21(de3)pLysS ?? mM IPTG OD600=0.5 IPTG Chemical lysis+ Affinity chromatography Most IB n.a. N-term His-Tag Katane et al., 2013 Bl21(de3)pLysS 0.5(?) mM IPTG OD600=0.5 Chemical lysis+ Affinity chromatography 2.3 mg/L Bovine GI724 Negri et al., 1999 0.1 mg/mL Trp@OD600=0.5 37°C 16h Sonicationphenyl sepharose  S15 strong cation exchanger 10 mg/LTrp promoter Strain InductionCostruct Purification Yield n.a.
  17. 17. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 18 Molecular mass and oligomerization state Source n. res Da Oligomerization State Asterotremella humicola (yeast) n.a. ~40000 tetramer Octopus vulgaris n.a. ~ 37000 monomer Mus musculus 341 37546 n.a. Bos taurus 341 37660 monomer Sus scrofa (Pig) 341 37316 tetramer Rattus norvegicus (Rat) 341 37543 n.a. Homo sapiens 341 37535 n.a. R. gracilis (yeast) 368 40076 Dimer Sus scrofa (Pig) 347 39336 Conc. Dependent Rattus norvegicus (Rat) 346 38820 Monomer Homo sapiens 347 39474 Dimer 80% sequence identity between mammalian DAAOs Few changes at the interface result into a different oligomerization state DASPODAAO Related to enzyme stability Monomeric yeast DAAO mutants show an increase in ~5°C lower Tm values than the dimeric wild-type DAAO Oligomerization in flavoprotein oxidases is: Not related to an allosteric behavior (yeast DAAO/human DAAO/Bacillus glycine oxidase) Not related to enzyme activity
  18. 18. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 19 Cofactor binding Flavin adenine dinucleotide 454 nm380 nm
  19. 19. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 20 μM nM 38% %[HOLOPROTEIN](active) [DASPO] = 1 μM 73% 92% 8% Protein Ligand Kd (M) Human DAAO free 8x10-6 Human DAAO Benzoate 0.3x10-6 [Holoprotein]= [Apoprotein][FAD]/Kd peroxisome [DASPO] [FAD] [substrate] Cofactor binding Kd (M) Affinity DASPO DAAO <50% active DASPO (at 1 μM [DASPO])
  20. 20. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 21 Cofactor binding - structure - Elongated conformation of FAD - Conserved amino acid residues at a few crucial positions. - part of the Rossmann fold xhxhGxGxxGxxxhxxh(x)8hxhE(D) - N-terminal part of the sequence (dinucleotide-binding motif) Dym et al., 2001 Flavoproteins Ferredoxin reductase FR P-cresol methylhydr oxylase, PCMH Pyruvate oxidase PO Single membered families glutathione reductase GR1 glutathione reductase GR2 DASPO belongs to GR2 family Gly2-close contact of the main chain to the pyrophosphate of FAD Gly3- close packing of the helix with the β-sheet. Gly1-tight turn of the main chain
  21. 21. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 22 Cofactor binding - structure Yeast DAAO Mutation Y238R results in a 10-fold weaker binding of the coenzyme. human DAAO Different conformation of residues hydrophobic stretch on si-face of the flavin ring (47–51, VAAGL). Kawazoe et al, 2006 https://projects.biotec.tu-dresden.de/plip-web/plip/index mDASPO Mutation of S308 results into minor change in FAD affinity. mDASPO Mutation R237A results into slightly decrease in FAD affinity (Katane et al., 2010). Need for 3D structure
  22. 22. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 23 Cofactor binding - structure Luca Mollica - Gianluca Tomasello, Varese, 2015 Kd = koff/kon + FAD kon koff Affinity is correlated to the association/dissociation rate constants In flavooxidases: a (partial) protein unfolding is requested for FAD dissociation Differences in the stability/rigidity of the FBD result into a different Kd Molecular Dynamics of FAD dissociation of yeast DAAO
  23. 23. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 24 Human DASPO stability As a general rule ligands stabilize flavooxidases Enzyme Source Free E Tm (°C) E+ligand Tm (°C) Glycine oxidase Bacillus subtilis 56.9 D-amino acid oxidase Rhodotorula gracilis 55.9 D-amino acid oxidase Homo sapiens 51.8 55.4 D-aspartate oxidase Homo sapiens DASPO+Tartrate DASPO+Tartrate+FAD DASPO+FAD DASPO
  24. 24. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 25 pH Human DASPO Temperature and pH profile Katane et al., 2015 pH of CHO peroxisomes (6.9-7.1) (like the cytoplasm) Temperature
  25. 25. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 26 hDASPO Kinetic parameters on D-Asparate Apparent kinetic parameters on D-Aspartate Human DASPO is the mammalian DASPO with best kinetic parameters Mouse DASPO is the less kinetically efficient mammalian DASPOs (and DAAOs) Katane 2015, Varese 2016 What is the local (in vivo) concentration of D-Asp (or NMDA)?
  26. 26. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 27 Kinetic mechanism - Rapid (pre-steady state) kinetics D-Aspartate + + Oxaloacetate O2 NH3H2O + Reductive Half- Reaction Oxidative Half- Reaction Oxidized DASPO Reduced DASPO
  27. 27. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 28 Enzyme Monitored Turnover Kinetic mechanism - Rapid (pre-steady state) kinetics Mathematical transformation of absorbance changes in vo vs [O2] Michaelis-Menten kinetics @ different [D-Asp] Determination of “real” kinetic parameters Bovine DASPO: kcat= 11.1 s-1, Km,D-Asp= 2.2 mM, Km,O2= 0.17 mM The affinity for O2 is higher in comparison with human DAAO Human DAAO: kcat = 14.7 s-1 Km,D-Asp= 8.2 mM, Km,O2= 1.2 mM Human DASPO is very fast
  28. 28. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 29 Kinetic mechanism - Rapid (pre-steady state) kinetics hDASPO has a different rate limiting step in comparison with bovine DASPO and hDAAO Bovine DASPO: kred= 180 s-1 , apparent Kd = 40 mM Human DAAO: kred= 180 s-1 , apparent Kd = 12.7 mM Reductive Half-Reaction Analysis of the “first” part of the reaction Exponential decay interpolation Abs@450nm
  29. 29. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 30 Substrate specificity The activity profile among mammalian DASPOs is similar Is DASPO actually a NMDA oxidase? Activity on Gly or D-Ser: <0.1% Activity vs D-Glu and D-Asn very low Activity vs D-Asp and NMDA Katane et al, 2015; Negri et al., 2007
  30. 30. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 31 Homology modelling - I-Tasser web server Template: human DAAO in complex with inhibitor pdb code: 3g3e 38% http://zhanglab.ccmb.med.umich.edu/I-TASSER/
  31. 31. Comparison of the models of mammalian DASPOs Human, rat and mouse DASPO are almost supeimposable (RMSD = 0.41Å) NO significant differences of the active site Sequence/conformation differences in 2 loops Differences in these loops result into differences in substrate binding 210 220 230 240 | | | | OXDD_BOVIN WVKHFIRDSSGLTYIYPGVSNVTLGGTRQKG OXDD_PIG WVKHFIRDGSGLTYIYPGLANVTLGGTRQKG OXDD_HUMAN WVEHFIRDGSGLTYIYPGTSHVTLGGTRQKG OXDD_MOUSE WVKHFIRDGGGLTYVYPGMSYVTLGGTRQKG D3ZDM7_RAT WVKHFIRDGGGLTYVYPGTSYVTLGGSRQTG 40 50 60 70 | | | | OXDD_BOVIN PETTSDVAAGMLIPPTYPDTPIQKQKQWFKETFDHLFAIV OXDD_PIG PETTSDVAAGMLIPPVYPDTPIHKQKQWFKDTFDHLFAIA OXDD_HUMAN PDTTSDVAAGMLIPHTYPDTPIHTQKQWFRETFNHLFAIA OXDD_MOUSE PDTTSNVAAGMLIPHTCADTPVPTQKRWFRETFEHLSEIA D3ZDM7_RAT PDTTSNVAAGMLIPPTYPDTPVPTLKRWFRETFQHLSEIA
  32. 32. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 33 Structure of human DASPO active site Arg278 Human DASPO (model) in complex with D-Asp (docked) Human DAAO in complex with imino serine (Experimental structure) Arg283 Tyr223 Tyr228 Arg237 Tyr224 Ser308 Gly313 Leu215Arg216 RCSB databank http://www.rcsb.org/pdb/home/home.do
  33. 33. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 34 Substrate binding Arg278 Tyr223 Arg237 Ser308 Arg216Human DASPO (model) in complex with D-Asp (docked) Katane et al., 2011
  34. 34. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 35 Yeast DAAO in complex with D-3F-Ala (Experimental structure) D-Alanine D-Aspartate DAAO Km Kcat Cat. Eff. Km Kcat Cat. Eff. WT 0.8 5000 6100 18 29 1.6 M213R 17.8 (22X ) 630 (8X ) 35.4 (172X ) 2 (8X ) 235 (8X ) 118 (74X ) Yeast DAAO M213R in complex with D-Asp (Model) Substrate binding Sacchi et al., 2002
  35. 35. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 36 Large sidechain: steric hindrance Small sidechain: increase affinity for substrate Arg278 Tyr223 Arg237 Ser308 Arg216Human DASPO (model) in complex with D-Asp (docked) Substrate binding Katane et al., 2008
  36. 36. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 37 Catalytical mechanism The chemical step of catalysis in amino acid oxidases is the direct transfer of an hydride (hydride transfer mechanism) Requirements: overlapping of reacting orbitals (orbital steering)
  37. 37. 1. Generation of all of the possible combinations between the ligand and the protein (System states/conformations) 2. Estimation of the interaction energy of each state 3. Ranking of the states based on their energies (lowest = best) Search for an energy minimum of the system: ligand + protein Energy States Search for the best mode of interaction between 2 molecules Molecular docking
  38. 38. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 39 Substrate specificity Arg237 Arg216Tyr223 Arg278 Ser30 8 Arg237 Arg216Tyr223 Arg278 Ser308 Arg237 Arg216Tyr223 Arg278 Ser308 Arg237 Arg216Tyr223 Arg278 Ser308 PDBeChem database The Chemical Component Dictionary. http://www.ebi.ac.uk/pdbe-srv/pdbechem/ Avogadro http://avogadro.cc/wiki/Main_Page Molecule editor and visualizer designed for cross-platform
  39. 39. Introduction D-Aspartate oxidase Catalyzed reaction Localization/Phylogenesis Biochemistry Cloning and heterologous expression Cofactor binding Thermal stability Kinetic parameters Substrate specificity Substrate binding and active site architecture Model of 3D-structure DASPO as a novel drug target Classical amino acid oxidase inhibitors Screening of natural compounds Virtual screening of chemical libraries Outline
  40. 40. 1. Investigation of classical amino acid oxidase inhibitors 2. Screening of natural compound collections 3. In silico and in vitro screening of large chemical libraries of potential ligands Strategies to identify DASPO ligands/potential lead compounds for the design of novel DASPO inibitors Binding parameters: - IC50: the functional strength of the inhibitor (determined at a single [S]) - Ki: the inhibition constant - Kd: physical interaction between the protein and the ligand Km is the concentration of substrate at which enzyme activity is at half maximal 𝐾𝑖 = 𝐼 𝐼𝐶50 1 + 𝑆 𝐾𝑚 Cheng-Prusoff correlation D-Aspartate oxidase as a novel drug target Elevated levels DDO mRNA in prefrontal cortex of schizophrenic patients  low [D-Asp] D’Aniello et al., 2005 Errico et al., 2013 DASPO represents a novel target for potential drugs Treatment of: mental pathologies (schizophrenia) Infertility
  41. 41. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 42 D-Aspartate oxidase inhibitors Binding of ligands at the active site: Changes of FAD absorbance spectrum Fluorescence (emission spectrum of protein or cofactor) CD signal (changes in structure) Binding of 6-Chloro-1,2-benzisoxazol-3(2H)-one (CBIO)
  42. 42. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 43 D-Aspartate oxidase inhibitors Ligand MW (Da) Rat DASPO (mM) Mouse DASPO (mM) Human DASPO (mM) Human DAAO (mM) Malonate 104.1 1.56 1.22 0.15 NO Tartrate 148.1 0.47 2.09 0.87 (Kd) NO Aminooxyacetic acid 91.1 1.42 1.92 1.49 Anthranilate 137.1 n.a. (Kd) 0.04 (Kd) Benzoate 69.5 IC50) 10.2 (Kd)(?) 0.007 (Kd) Crotonate 25.3 IC50) CBIO 169.6 n.a. (Kd) Canonical ligands of DASPO have a quite high Kd: they cannot be lead compounds in drug discovery Common chemical features: Carboxyl group(s) (or equivalent) Hydrogen bond donor/acceptor Ability to form staking aromatic/pi interactions D-Asp
  43. 43. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 44 Discovery of novel DASPO inhibitors - screening of natural compounds - Katane et al., 2010 257 natural compounds of microbial origin Thiolactomycin Screening for inhibition of activity Mouse DASPO 20 mM D-Asp
  44. 44. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 45 Discovery of novel D-aspartate oxidase inhibitors - screening of natural compounds TLM is more effective than classical inhibitors Ki 0.7 mM 1.6 mM 1.9 mM 6.4 mM IC50: 2.0 mM 5.3 mM 8.7 mM (mixed inhibition) TLM inhibition is higher at low [FAD] (3 uM) TLM interferes with FAD binding IC50: 0.5 mM TLM is not a good human DASPO (IC50>10 mM) or mammalian DAAO inhibitor At low [FAD] TLM is a good (IC50=4.7 mM) mammalian DAAO inhibitor [FAD] = 60 μM
  45. 45. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 46 R237, R278, G49, M50Interacting residues: R237, A48 Tautomer II Tautomer I Variant R237A (30% residual activity) Lower inhibition efficiency of TLM Protein Large Excess [FAD] (mM) 3X Kd [FAD] (mM) WT 2.0 0.51 R237A 4.81 (2.5X) 1.24 (2.4X) A48, A49, M50: hydrophobic stretch that interacts with FAD (human DAAO, Kawazoe et al., 2006) R237 important for substrate binding Discovery of novel D-aspartate oxidase inhibitors - screening of natural compounds Dual effect of TLM: on substrate and on FAD binding Absence of carboxyl group: TLM binds differently vs D-Aspartate Very low potency as inhibitor
  46. 46. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 47 Database of commercially available compounds (Namiki HTS integrated database) Homology models of human DASPO (based on human DAAO) + Molecular dynamics simulations 5 representative structures of models Discovery of novel D-aspartate oxidase inhibitors - HTP screening - Katane et al., 2015 Molecular docking 1050 compounds in silico screening for chemicostructural similarities to reference compounds 4 million molecules 2499 compounds malonic acid benzoic acid 3-hydroxyquinolin-2(1H)-one 192 compounds 38 compounds Filter to remove the compounds having reactive functional groups cluster analysis Visual inspection: compounds presumed to form interaction with Arg278 10 compounds aldehyde and acyl halide
  47. 47. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 48 Discovery of novel D-aspartate oxidase inhibitors - HTP screening IC50 determination Demonstration of physical interaction Reference compounds
  48. 48. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 49 Discovery of novel D-aspartate oxidase inhibitors - HTP screening Specific differences in the structure of mammalian DASPOs 1.5X 10X 4.4X 2X 2X 8X Compound 2 (5-aminonicotinic acid) is the most promising human DASPO inhibitor In vitro inhibition of DASPO activity In vivo inhibition of DASPO activity
  49. 49. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 50 Discovery of novel D-aspartate oxidase inhibitors - HTP screening NO NO NO NO Carboxyl Reference compound 5-ANA YES Amino NO NO Partial Partial NO NO NO NO Pyridine nitrogen
  50. 50. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 51 Docking of 5ANA hDASPO-D-AsphDASPO-5-ANA Protein Predicted Binding Energy (kcal/mol) Predicted Kd (uM) D-Aspartate -4.8 310 5-ANA -6.1 34 The carboxyl group is essential for interaction with DASPO (Arg278). The amino group is an important determinant of its interaction with DASPO (Ser308). The Pyridine nitrogen is essential for its interaction with DASPO (Arg216?) Arg237 Arg216 Tyr223 Arg278 Ser308 Aromatic stacking with FAD
  51. 51. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 52 Can we increase the ligand affinity? Ki (μM) Estimated binding energy (kcal/mol) Best inhibitor of DASPO by Katane, 2015 3.8 -7.4 0.74 LE (-ΔG/HAC) (kcal/mol/HAC) Discovery of novel D-aspartate oxidase inhibitors - HTP screening Best inhibitor of DASPO by Duplantier, 2009 0.76 -8.5 0.71 3-hydroxyquinolin-2(1H)-one Increase affinity means increase ligand size How? Filling the upper part of the active site (also part of the active site entrance). Best inhibitor of DAAO by Terry-Lorenzo, 2014 0.007 -11.1 0.53 What we learned? Best inhibitor of DASPO by Katane, 2010 700 -4.3 0.31
  52. 52. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 53 Conclusions 2. Mammalian DASPOs share very similar sequence and (possibly) structure But the mouse DASPO seems more close to hDAAO than hDASPO 4. Probably, in vivo, the activity of the enzyme is much lower than expected The peroxisome pH is far from the optimal pH of DASPO Actual in vivo [D-Aspartate] is lower than enzyme Km? Need for the experimental 3D structure of the enzymes Functional data suggest a different role/expression of the proteins (?) 1. Mammalian DASPOs and DAAOs are similar (structure/sequence/function) b. Different kinetic parameters a. Different FAD affinity Is mDASPO FAD affinity increased by the presence of ligands? hDASPO is by far more efficient than other amino acid oxidases Absence of an active site lid? Which is the reason of the low efficiency of mouse DASPO? c. Different interactions with inhibitors 3. Mammalian DASPOs show some very different properties
  53. 53. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 54 Flavoproteins
  54. 54. Como 2016 Gianluca Molla - Università degli Studi dell’Insubria 55 Lab of Functional Post-Genomic and Protein Engineering Loredano Pollegioni Silvia Sacchi Luciano Piubelli Gianluca Molla Laura Caldinelli Elena Rosini Pamela Cappelletti Roberta Melis Fabio Tonin Paolo Motta Giulia Murtas Acknowledgements

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