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