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Shrestha et al., 1
Modelling of pyruvate decarboxylases
from ethanol producing bacteria
Anjala Shrestha1, Srisuda Dhamwichukorn1, Ekachai Jenwitheesuk2*
1 Joint Graduate School of Energy and Environment, KMUTT, Thailand 2National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Thailand
2Shrestha et al.,
IntroductionIntroductionPyruvate decarboxylase (PDC, EC 4.1.1.1)Pyruvate decarboxylase (PDC, EC 4.1.1.1) Common in plants and fungi but very rare in Common in plants and fungi but very rare in
prokaryotes and absent in animals prokaryotes and absent in animals (Konig,1998 ).(Konig,1998 ).
Non-oxidative Decarboxylation of 2-keto acid to Non-oxidative Decarboxylation of 2-keto acid to aldehydesaldehydes
Carboligation with aldehyde to form chiral 2-Carboligation with aldehyde to form chiral 2-hydroxyketones hydroxyketones (Pohl, 2004)(Pohl, 2004)
Carboligation with benzaldehyde to form R-Carboligation with benzaldehyde to form R-phenylacetylcarbinol (R-PAC)phenylacetylcarbinol (R-PAC)
Shrestha et al., 3
Catalysis steps of PDC
Thiamin diphosphate (ThDP) and Mg2+ ions as cofactors.
Shrestha et al., 4
PDC possessing microorganismsPDC possessing microorganisms Gram-negative- Gram-negative- Zymomonas mobilis Zymomonas mobilis (ZmPDC) (ZmPDC)
--Zymobacter palmaeZymobacter palmae (ZpPDC) (ZpPDC) Gram-positive - Gram-positive -Sarcina ventriculiSarcina ventriculi (SvPDC) (SvPDC)
Possess Pyruvate decarboxylase and alcohol Possess Pyruvate decarboxylase and alcohol dehydrogenase (adh, EC 1.1.1.1) dehydrogenase (adh, EC 1.1.1.1)
Z. mobilisZ. mobilis ATCC29291ATCC29291 PDB ID: 1zpd PDB ID: 1zpd (Dobritzsch, (Dobritzsch, 1998)1998)
Shrestha et al., 5
•Objective of studyObjective of studyTo generate To generate 3D structures of the ZpPDC and 3D structures of the ZpPDC and
SvPDC using homology modelling SvPDC using homology modelling techniquetechnique
Enzyme-substrate interactionsEnzyme-substrate interactionsSubunit-subunit interfaces that might be Subunit-subunit interfaces that might be
related to the different biochemical related to the different biochemical characteristics.characteristics.
Shrestha et al., 6
MethodologyMethodology
Predicted 3D-structuresPredicted 3D-structures
Model assessmentModel assessment
Discrete Optimized Protein Energy
Discrete Optimized Protein Energy
Residue-specific All-atoms Conditional Probability Discriminatory Function
Residue-specific All-atoms Conditional Probability Discriminatory Function
Modeller Modeller 9v3 9v3 (Sali, 1993)(Sali, 1993)
7Shrestha et al.,
Energy minimization by the NAMD program without Energy minimization by the NAMD program without water molecules water molecules (Phillips, 2005)(Phillips, 2005)
Protein interface analysis by Protein-Protein interaction Protein interface analysis by Protein-Protein interaction
server server (Reynolds, 2009)(Reynolds, 2009)
PROCHECK version 3.5.4 PROCHECK version 3.5.4 (Morris,1992)(Morris,1992)
Docking of cofactors by superimposition with the Docking of cofactors by superimposition with the template by DeepView template by DeepView (SWISS-PdbViewer, v. 3.7)(SWISS-PdbViewer, v. 3.7)
Mol_Volume version 1 Mol_Volume version 1 (Kalé, 1999),(Kalé, 1999), R_PROBE-2.0 Å. R_PROBE-2.0 Å.
Shrestha et al., 8
Protein-ligand docking by Autodock 4 Protein-ligand docking by Autodock 4 (Morris, 1998)(Morris, 1998)
Autodock graphical user interface
(Autodock tools 1.4.6)
AutoGrid version 4
Autodock version4
Ki= exp [(∆G X 1000)/ (Rcal X TK)
∆G= Intermolecular energy + Internal energy of ligand
Rcal=1.98719 cal K-1 mol-1
TK=298.15 K, Kd=1/Ki
Protein, Ligand structures
Grid box (40 points on each side with distance of 0.375 Å)
Affinity Maps for each atom types of ligands
Shrestha et al., 9
Cα : ZpPDC and ZmPDC 2.2.7 Å (Picture A)
SvPDC and EcIPDC 2.93 Å (Picture B)
Results and Discussions Homology models assessment
Shrestha et al.,Shrestha et al., 1010
Ramachandran PlotRamachandran Plot
ZpPDC SvPDCResidue in most favoured regions 879 (90.4%) 882 (88.6%) Residue in additional allowed regions 86 (8.8%) 103 (10.3)Residues in generously allowed regions 5 (0.5%) 7 (0.7%)Residues in disallowed regions 2 (0.2%) 4 (0.4%)Overall PROCHECK (-1.0 to - 0.05) -0.32 -0.33
Shrestha et al., 11
Secondary StructuresSecondary Structures
ZpPDC-PYR ZpPDC-R ZpPDC-PP
α/β topology common to all thiamine dependent enzymes.
Shrestha et al., 12
Interface Analysis of ModelsInterface Analysis of Models
H-bonds
SvPDC model (30)
ZpPDC model (44)
Interface areas
ZpPDC= 3587 Å2
SvPDC = 3284 Å2
R2=0.75
Conformational changes upon substrate binding
Shrestha et al., 13
Binding modes of the cofactors
Blue: Acidic; Yellow: Polar
Red: Basic; Grey: Hydrophobic
Residues within 4 Å of ThDP
X-ray crystal str. of ZmPDC
Homology model of ZpPDC
Homology model of SvPDC
Shrestha et al., 14
Substrate binding sites of the homology Substrate binding sites of the homology modelsmodels
Asp26*, His113*, His114*, Tyr290, Thr388 and Glu473 in ZmPDC
SvPDC: Thr288
Conversion of Thr288 to ZmPDC analogue would decrease active site volume and subsequently decrease Km value for pyruvate.
Tyr384 in SvPDC
Ala in EcIPDC & ScPDC
Shrestha et al., 15
Pyruvamide interacts with ThDP and side chains of Asp26, His113, Glu468 of ZpPDC, and Asp27, His114 of SvPDC. The distances between pyruvamide and these structures are within 4 Å .
Shrestha et al., 16
Substrate activation of homology modelsSubstrate activation of homology models
SvPDC model suggests that conformational changes occurs upon substrate binding at regulatory site similar to ScPDC form-B (Lu, 2000).
Shrestha et al., 17
Affinity of ZpPDC and SvPDC towards other 2-Affinity of ZpPDC and SvPDC towards other 2-
keto acidsketo acids
0
2
4
6
8
10
12
14
400 500 600 700 800
Molecular Volume (Å3)
Expe
rimen
tal K
m (m
M) o
f Zm
PDC
Linear relationship between molecular volume of substrate and experimental Km value for ZmPDC
Shrestha et al., 18
The larger 2-keto acids bind with higher dissociation constant (Kd =1/Ki) than those of the smaller 2-keto acids to both homology models.
Ile472 and Thr388 of ZmPDC
Substrate binding preferences of the ZpPDC and SvPDC enzymes are similar to ZmPDC
19Shrestha et al.,
ConclusionConclusion Homology models of PDCs of Homology models of PDCs of Z. palmaeZ. palmae and and S. S.
ventriculiventriculi -- to explore the structural similarities and -- to explore the structural similarities and differences among bacterial PDCs at the atomic level.differences among bacterial PDCs at the atomic level.
Similar in cofactor binding modes, substrate binding Similar in cofactor binding modes, substrate binding
residues, and active site volume. residues, and active site volume.
Mechanism of allosteric activation shown by SvPDC is Mechanism of allosteric activation shown by SvPDC is similar to ScPDC form-B which needs further similar to ScPDC form-B which needs further experimental verification.experimental verification.
Preference of ZpPDC and SvPDC enzymes for aliphatic Preference of ZpPDC and SvPDC enzymes for aliphatic 2-ketoacids.2-ketoacids.
Shrestha et al., 20
AcknowledgmentAcknowledgment
• Joint Graduate School of Energy and Environment (JGSEE), King Mongkut’s University of Technology Thonburi, Bangkok, Thailand.
• Grateful to Philip Shaw, Sissades Tongsima, Pavita Tipsombatboon, Alisa Wilantho and Wanwimon Mokmak for their technical support and valuable comments.
Shrestha et al., 21
Thank you for your attention.
Shrestha et al., 22
ZmPDC 1 --MSYTVGTYLAERLVQIGLKHHFAVAGDYNLVLLDNLLLNKNMEQVYCCNELNCGFSAE 58 GYARAKGAAAAVVTYSVZpPDC 1 ---MYTVGMYLAERLAQIGLKHHFAVAGDYNLVLLDQLLLNKDMEQVYCCNELNCGFSAE 57 GYARARGAAAAIVTFSVSvPDC 1 --MKITIAEYLLKRLKEVNVEHMFGVPGDYNLGFLDYVEDSKDIEWVGSCNELNAGYAAD 58 GYARLRGFGVILTTYGVEcIPDC 1 MRTPYCVADYLLDRLTDCGADHLFGVPGDYNLQFLDHVIDSPDICWVGCANELNASYAAD 60 GYARCKGFAALLTTFGVScPDC 1 -MSEITLGKYLFERLKQVNVNTVFGLPGDFNLSLLDKIYEVEGMRWAGNANELNAAYAAD 59 GYARIKGMSCIITTFGV ▲ Pyrα4 Pyrβ4 Pyrα5 Pyrα6 Pyrβ5 Pyrα7 ZmPDC 76 G-ALSAFDAIGGAYAENLPVILISGAPNNNDHAAGHVLHHALG 117 KTDYHYQLEMAKNITAAAEAIYTPEEAPAKIDHZpPDC 75 G-AISAMNAIGGAYAENLPVILISGSPNTNDYGTGHILHHTIG 116 TTDYNYQLEMVKHVTCARESIVSAEEAPAKIDHSvPDC 76 G-SLSAINATTGSFAENVPVLHISGVPSALVQQNRKLVHHSTA 117 RGEFDTFERMFREITEFQSIISEYN-AAEEIDREcIPDC 78 G-ELSAMNGIAGSYAEHVPVLHIVGAPGTAAQQRGELLHHTLG 119 DGEFRHFYHMSEPITVAQAVLTEQN-ACYEIDRScPDC 77 G-ELSALNGIAGSYAEHVGVLHVVGVPSISAQAKQLLLHHTLG 118 NGDFTVFHRMSANISETTAMITDIATAPAEIDR
Pyrβ6 Pyrα8 Rα1 Rβ1 ZmPDC 151 VIKTALRE-KKPVYLEIACNIASMPCA 176 APGPASALFNDEASDEA-SLNAAVEETLKFIAXRDKVAVLVGSKLRAAGZpPDC 150 VIRTALRE-RKPAYLEIACNVAGAECV 175 RPGPINSLLRELEVDQT-SVTAAVDAAVEWLQDRQNVVMLVGSKLRAAASvPDC 150 VIESIYKY-QLPGYIELPVDIVSKEIE 175 IDEMK-PLNLTMRSNEK-TLEKFVNDVKEMVASSKGQHILADYEVLRAKEcIPDC 152 VLTTMLRE-RRPGYLMLPADVAKKAAT 177 PPVNA-LTHKQAHADSA-CLKAFRDAAENKLAMSKRTALLADFLVLRHGScPDC 152 CIRTTYVT-QRPVYLGLPANLVDLNVP 177 AKLLQTPIDMSLKPNDAESEKEVIDTILALVKDAKNPVILADACCSRHD
Rα2 Rβ2 Rβ3 Rα3 Rα4 Rβ4 ZmPDC 225 AEEAAVKFADA 235 LGGAVATMAAA-KSFFPEENPHYIGTSWGEVSYPGVEKTMKEADAVIALAPVFNDYSTTG 294 WZpPDC 224 AEKQAVALADR 234 LGCAVTIMAAE-KGFFPEDHPNFRGLYWGEVSSEGAQELVENADAILCLAPVFNDYATVG 293 WSvPDC 223 AEKELEGFINE 233 AKIPVNTLSIG-KTAVSESNPYFAGLFSGETSSDLVKELCKASDIVLLFGVKFIDTTTAG 292 FEcIPDC 225 LKHALQKWVKE 235 VPMAHATMLMG-KGIFDERQAGFYGTYSGSASTGAVKEAIEGADTVLCVGTRFTDTLTAG 294 FScPDC 227 VKAETKKLIDL 237 TQFPAFVTPMG-KGSIDEQHPRYGGVYVGTLSKPEVKEAVESADLILSVGALLSDFNTGS 296 F
Rβ5 Rβ6 Rβ7 Rα5 Rα6 PPα1 ZmPDC 296 TDIPDPKKLVLAEPRSVVVNGIRFPSVHLKDYLTRLAQKVSKKTGALDFFKSLNAGELK 354 KAAPADPSAPLVNAEIAZpPDC 295 NSWPKGDNVMVMDTDRVTFAGQSFEGLSLSTFAAALAEKAPSRPATTQGTQAP----VL 349 GIEAAEPNAPLTNDEMTSvPDC 294 RYINKDVKMIEIGLTDCRIGETIYTGLYIKDVIKALTD------AKIKFHNDVKVEREA 346 VEKFVPTDAKLTQDRYFEcIPDC 296 THQLTPAQTIEVQPHAARVGDVWFTGIPMNQAIETLVEL-----CKQHVHAGLMSSSSG 349 AIPFPQPDGSLTQENFWScPDC 298 SYSYKTKNIVEFHSDHMKIRNATFPGVQMKFVLQKLLTTIAD--AAKGYKPVAVPARTP 354 ANAAVPASTPLKQEWMW
PPβ1 PPα2 PPβ2 PPα3 PPβ3 /PPα4ZmPDC 372 RQVEALLTPNTTVIAETGDSWFNAQRMKLPNGARVEYEMQWGH 414 IGWSVPAAFGYAVGA----PERRNILMVGDGSFZpPDC 367 RQIQSLITSDTTLTAETGDSWFNASRMPIPGGARVELEMQWGH 409 IGWSVPSAFGNAVGS----PERRHIMMVGDGSFSvPDC 364 KQMEAFLKPNDVLVGETGTSYSGACNMRFPEGSSFVGQGSWMS 406 IGYATPAVLGTHLAD----KSRRNILLSGDGSFEcIPDC 367 RTLQTFIRPGDIILADQGTSAFGAIDLRLPADVNFIVQPLWGS 409 IGYTLAAAFGAQTAC----PNRRVIVLTGDGAAScPDC 372 NQLGNFLQEGDVVIAETGTSAFGINQTTFPNNTYGISQVLWGS 414 IGFTTGATLGAAFAAEEIDPKKRVILFIGDGSL ▲ -▲▲▲- PPα5 PPβ4 PPα6 PPα7 PPα8 PPβ5 PPα9ZmPDC 444 QLTAQEVAQMVRLKLPVIIFLINNYGY 470 TIEVMIHDG--PYNNIKNWDYAGLMEVFNGNGGYDSGAGKGLKAKTGGEZpPDC 439 QLTAQEVAQMIRYEIPVIIFLINNRGY 465 VIEIAIHDG--PYNYIKNWNYAGLIDVFND----EDGHGLGLKASTGAESvPDC 436 QLTVQEVSTMIRQKLNTVLFVVNNDGY 462 TIERLIHGPEREYNHIQMWQYAELVKTLATE---RDIQPTCFKVTTEKEEcIPDC 439 QLTIQELGSMLRDKQHPIILVLNNEGY 465 TVERAIHGAEQRYNDIALWNWTHIPQALS-----LDPQSECWRVSEAEQScPDC 448 QLTVQEISTMIRWGLKPYLFVLNNDGY 474 TIEKLIHGPKAQYNEIQGWDHLSLLPTFG------AKDYETHRVATTGE -----------------------▲ ▲ ▲▲ PPβ6 PPα10 .ZmPDC 518 LAEAIKVALAN 528 -TDGPTLIECFIGREDCTEELVKWGKRVAAANSRKPVNKLL 568ZpPDC 509 LEGAIKKALDN 519 -RRGPTLIECNIAQDDCTETLIAWGKRVAATNSRKPQA--- 556SvPDC 509 LAAAMEEINKG 519 -TEGIAFVEVVMDKMDAPKSLRQEASLFSSQNNY------- 552EcIPDC 510 LADVLEKVAH- 519 -HERLSLIEVMLPKADIPPLLGALTKALEACNNA------- 552ScPDC 518 WDKLTQDKSFN 528 DNSKIRMIEIMLPVFDAPQNLVEQAKLTAATNAKQ------ 563