Modelling of pyruvate decarboxylases from ethanol producing bacteria

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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

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IntroductionPyruvate decarboxylase (PDC, EC
4.1.1.1)

• Common in plants and fungi but very rare in
  prokaryotes and absent in animals (Konig,1998 ).
• Non-oxidative Decarboxylation of 2-keto acid to
  aldehydes
• Carboligation with aldehyde to form chiral
  2-hydroxyketones (Pohl, 2004)
• Carboligation with benzaldehyde to form
  R-phenylacetylcarbinol (R-PAC)

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• Catalysis steps of PDC
• Thiamin diphosphate (ThDP) and Mg2 ions as
  cofactors.

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PDC possessing microorganisms Gram-negative-
Zymomonas mobilis (ZmPDC) -Zymobacter palmae
(ZpPDC) Gram-positive -Sarcina ventriculi
(SvPDC) Possess Pyruvate decarboxylase and
alcohol dehydrogenase (adh, EC 1.1.1.1)
Z. mobilis ATCC29291 PDB ID 1zpd (Dobritzsch,
1998)
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Objective of studyTo generate 3D structures of
the ZpPDC and SvPDC using homology modelling
techniqueEnzyme-substrate interactionsSubunit
-subunit interfaces that might be related to the
different biochemical characteristics.
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Methodology
Predicted 3D-structures
Model assessment
Discrete Optimized Protein Energy
Residue-specific All-atoms Conditional
Probability Discriminatory Function
Modeller 9v3 (Sali, 1993)
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• Energy minimization by the NAMD program without
  water molecules (Phillips, 2005)
• Protein interface analysis by Protein-Protein
  interaction server (Reynolds, 2009)
• PROCHECK version 3.5.4 (Morris,1992)
• Docking of cofactors by superimposition with the
  template by DeepView (SWISS-PdbViewer, v. 3.7)
• Mol_Volume version 1 (Kalé, 1999), R_PROBE-2.0 Å.

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Protein-ligand docking by Autodock 4 (Morris,
1998)
Autodock graphical user interface (Autodock
tools 1.4.6)
Protein, Ligand structures Grid box (40 points on
each side with distance of 0.375 Å)
AutoGrid version 4
Affinity Maps for each atom types of ligands
Ki exp (?G X 1000)/ (Rcal X TK) ?G
Intermolecular energy Internal energy of ligand
Rcal1.98719 cal K-1 mol-1 TK298.15 K,
Kd1/Ki
Autodock version4
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Results and Discussions Homology models
assessment
Ca ZpPDC and ZmPDC 2.2.7 Å (Picture A)
SvPDC and EcIPDC 2.93 Å (Picture B)
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Ramachandran Plot
ZpPDC
SvPDC Residue 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
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Secondary Structures

• a/ß topology common to all thiamine dependent
  enzymes.

ZpPDC-PYR
ZpPDC-R
ZpPDC-PP
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Interface Analysis of Models

• H-bonds
• SvPDC model (30)
• ZpPDC model (44)
• Interface areas
• ZpPDC 3587 Å2
• SvPDC 3284 Å2

R20.75
Conformational changes upon substrate binding
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Binding modes of the cofactors
Residues within 4 Å of ThDP
X-ray crystal str. of ZmPDC
Homology model of ZpPDC
Homology model of SvPDC
Blue Acidic Yellow Polar Red Basic Grey
Hydrophobic
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Substrate binding sites of the homology models
Asp26, His113, His114, Tyr290, Thr388 and
Glu473 in ZmPDC
Ala in EcIPDC ScPDC
Tyr384 in SvPDC
SvPDC Thr288
Conversion of Thr288 to ZmPDC analogue would
decrease active site volume and subsequently
decrease Km value for pyruvate.
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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 .
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Substrate activation of homology models

• SvPDC model suggests that conformational changes
  occurs upon substrate binding at regulatory site
  similar to ScPDC form-B (Lu, 2000).

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Affinity of ZpPDC and SvPDC towards other 2-keto
acids
Linear relationship between molecular volume of
substrate and experimental Km value for ZmPDC
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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
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Conclusion

• Homology models of PDCs of Z. palmae and S.
  ventriculi -- to explore the structural
  similarities and differences among bacterial PDCs
  at the atomic level.
• Similar in cofactor binding modes, substrate
  binding residues, and active site volume.
• Mechanism of allosteric activation shown by SvPDC
  is similar to ScPDC form-B which needs further
  experimental verification.
• Preference of ZpPDC and SvPDC enzymes for
  aliphatic 2-ketoacids.

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Acknowledgment

• Joint Graduate School of Energy and Environment
  (JGSEE), King Mongkuts 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.

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Thank you for your attention.
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ZmPDC 1 --MSYTVGTYLAERLVQIGLKHHFAVAGDYNLVLLDNL
LLNKNMEQVYCCNELNCGFSAE 58 GYARAKGAAAAVVTYSVZpPDC
1 ---MYTVGMYLAERLAQIGLKHHFAVAGDYNLVLLDQLLLNKDM
EQVYCCNELNCGFSAE 57 GYARARGAAAAIVTFSVSvPDC 1
--MKITIAEYLLKRLKEVNVEHMFGVPGDYNLGFLDYVEDSKDIEWVGSC
NELNAGYAAD 58 GYARLRGFGVILTTYGVEcIPDC 1
MRTPYCVADYLLDRLTDCGADHLFGVPGDYNLQFLDHVIDSPDICWVGCA
NELNASYAAD 60 GYARCKGFAALLTTFGVScPDC 1
-MSEITLGKYLFERLKQVNVNTVFGLPGDFNLSLLDKIYEVEGMRWAGNA
NELNAAYAAD 59 GYARIKGMSCIITTFGV
?
Pyra4
Pyrß4 Pyra5 Pyra6
Pyrß5 Pyra7 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
Pyrß6 Pyra8 Ra1
Rß1 ZmPDC 151
VIKTALRE-KKPVYLEIACNIASMPCA 176
APGPASALFNDEASDEA-SLNAAVEETLKFIAXRDKVAVLVGSKLRAAG
ZpPDC 150 VIRTALRE-RKPAYLEIACNVAGAECV 175
RPGPINSLLRELEVDQT-SVTAAVDAAVEWLQDRQNVVMLVGSKLRAAA
SvPDC 150 VIESIYKY-QLPGYIELPVDIVSKEIE 175
IDEMK-PLNLTMRSNEK-TLEKFVNDVKEMVASSKGQHILADYEVLRAK
EcIPDC 152 VLTTMLRE-RRPGYLMLPADVAKKAAT 177
PPVNA-LTHKQAHADSA-CLKAFRDAAENKLAMSKRTALLADFLVLRHG
ScPDC 152 CIRTTYVT-QRPVYLGLPANLVDLNVP 177
AKLLQTPIDMSLKPNDAESEKEVIDTILALVKDAKNPVILADACCSRHD
Ra2 Rß2
Rß3 Ra3 Ra4 Rß4 ZmPDC 225
AEEAAVKFADA 235 LGGAVATMAAA-KSFFPEENPHYIGTSWGEVSYP
GVEKTMKEADAVIALAPVFNDYSTTG 294 WZpPDC 224
AEKQAVALADR 234 LGCAVTIMAAE-KGFFPEDHPNFRGLYWGEVSSE
GAQELVENADAILCLAPVFNDYATVG 293 WSvPDC 223
AEKELEGFINE 233 AKIPVNTLSIG-KTAVSESNPYFAGLFSGETSSD
LVKELCKASDIVLLFGVKFIDTTTAG 292 FEcIPDC 225
LKHALQKWVKE 235 VPMAHATMLMG-KGIFDERQAGFYGTYSGSASTG
AVKEAIEGADTVLCVGTRFTDTLTAG 294 FScPDC 227
VKAETKKLIDL 237 TQFPAFVTPMG-KGSIDEQHPRYGGVYVGTLSKP
EVKEAVESADLILSVGALLSDFNTGS 296 F
Rß5 Rß6 Rß7 Ra5 Ra6
PPa1 ZmPDC
296 TDIPDPKKLVLAEPRSVVVNGIRFPSVHLKDYLTRLAQKVSKKTGA
LDFFKSLNAGELK 354 KAAPADPSAPLVNAEIAZpPDC 295
NSWPKGDNVMVMDTDRVTFAGQSFEGLSLSTFAAALAEKAPSRPATTQGT
QAP----VL 349 GIEAAEPNAPLTNDEMTSvPDC 294
RYINKDVKMIEIGLTDCRIGETIYTGLYIKDVIKALTD------AKIKFH
NDVKVEREA 346 VEKFVPTDAKLTQDRYFEcIPDC 296
THQLTPAQTIEVQPHAARVGDVWFTGIPMNQAIETLVEL-----CKQHVH
AGLMSSSSG 349 AIPFPQPDGSLTQENFWScPDC 298
SYSYKTKNIVEFHSDHMKIRNATFPGVQMKFVLQKLLTTIAD--AAKGYK
PVAVPARTP 354 ANAAVPASTPLKQEWMW
PPß1 PPa2 PPß2
PPa3 PPß3 /PPa4ZmPDC 372
RQVEALLTPNTTVIAETGDSWFNAQRMKLPNGARVEYEMQWGH 414
IGWSVPAAFGYAVGA----PERRNILMVGDGSFZpPDC 367
RQIQSLITSDTTLTAETGDSWFNASRMPIPGGARVELEMQWGH 409
IGWSVPSAFGNAVGS----PERRHIMMVGDGSFSvPDC 364
KQMEAFLKPNDVLVGETGTSYSGACNMRFPEGSSFVGQGSWMS 406
IGYATPAVLGTHLAD----KSRRNILLSGDGSFEcIPDC 367
RTLQTFIRPGDIILADQGTSAFGAIDLRLPADVNFIVQPLWGS 409
IGYTLAAAFGAQTAC----PNRRVIVLTGDGAAScPDC 372
NQLGNFLQEGDVVIAETGTSAFGINQTTFPNNTYGISQVLWGS 414
IGFTTGATLGAAFAAEEIDPKKRVILFIGDGSL
?
-???-

PPa5 PPß4 PPa6 PPa7
PPa8 PPß5 PPa9ZmPDC 444
QLTAQEVAQMVRLKLPVIIFLINNYGY 470
TIEVMIHDG--PYNNIKNWDYAGLMEVFNGNGGYDSGAGKGLKAKTGGE
ZpPDC 439 QLTAQEVAQMIRYEIPVIIFLINNRGY 465
VIEIAIHDG--PYNYIKNWNYAGLIDVFND----EDGHGLGLKASTGAE
SvPDC 436 QLTVQEVSTMIRQKLNTVLFVVNNDGY 462
TIERLIHGPEREYNHIQMWQYAELVKTLATE---RDIQPTCFKVTTEKE
EcIPDC 439 QLTIQELGSMLRDKQHPIILVLNNEGY 465
TVERAIHGAEQRYNDIALWNWTHIPQALS-----LDPQSECWRVSEAEQ
ScPDC 448 QLTVQEISTMIRWGLKPYLFVLNNDGY 474
TIEKLIHGPKAQYNEIQGWDHLSLLPTFG------AKDYETHRVATTGE
-----------------------? ? ??
PPß6 PPa10
.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