Understanding biosynthesis ofcomplex metabolites

using computational biology

D. MohantyNational Institute of Immunology

New Delhi

O OCH3

CH3

N

OOHO

H3C

O

O OH3C

O

OH

OCH3

CH3

H3CO

OH

H3C

O

O

O

OH

O

O

HO OH

O

NMe2

HO

O

OMeOH

OH OH

NH

O

O

O

O

OH OHO

O

O

O

O

OH

O

H3C

HCH3

O

OHO

O

CH3

O

O O

CH3H3C

H3C

OH

HO

OH3C

CH3

N

S

CH3

HO

OH

OH

Domains Involved in Protein-Protein

Interactions

Substrate Specificity of Catalytic Domains

PKS NRPS Modifying Domains

Acyl Transferase

(AT)

Adenylation

(A)

Acyl CoA Synthetases

(ACS)

Keto Synthases

(KS)

Condensation

(C)

Glycosyl Transferases

(GTr)

Chalcone Synthases

(CHS)

N-Acyl Transferases

(NAT)

KS – ACP

AT – ACP

A – PCP

C – PCP

PapA5 – ACP

Levels of Functional AnnotationSequence based methods: Fundamental for functional annotation

Drawback: Cannot predict substrate specificity

PCPSSCoA

SCoA

Amino acid Coumarate

Oxyluciferin

Amino acyl PCP

Fatty acid Acyl CoA

Coumaroyl CoA

Luciferin

Model protein based on known structure of a similar protein

Range of possible substrates

Find the substrate which

binds to the model protein

Computational Chemistry

D F G H Y K L M V C

Knowledge Based Approach

Homology modeling

Knowledge Based ApproachSequence/structure information for large number of proteins with known

specificity

Predicting substrate specificity of new members of the familyusing evolutionary information

Design of novel proteinswith altered specificity.

Design of novel polyketides/nonribisomalpeptides

In silico identification of PKS/NRPS products

Sequence-Product correlation

Predictive rules

BIOSYNTHESIS OF A MODULAR PKS

KS:KETOSYNTHASE; AT:ACYL TRANSFERASE; DH:DEHYDRATASE; ER:ENOYL REDUCTASE; KR:KETOREDUCTASE; ACP:ACYL CARRIER PROTEEIN; TE:THIOESTERASE

rifamycin B(antituberculosis)erythromycinA antibacterial)

rapamycin(immunosuppressant)

O OCH3

CH3

N

OOHO

H3C

O

O OH3C

O

OH

OCH3

CH3

H3CO

OH

H3C

O

O

O

OH

O

O

HO OH

O

NMe2

HO

O

OMeOH

OH OH

NH

O

O

O

O

OH OHO

O

O

O

O

OH

POSSIBLE STARTER AND EXTENDER UNITS

FAS

HO SCoA

O O

SCoA

O

Malonyl CoA

Acetyl CoA

CoAS

O

CoAS

O

O

SCoA

Butyryl CoA

Benzoyl CoA

Isobutyryl CoA

H3C SCoA

O O

Acetoacetyl CoA

HO SCoA

O O

HO SCoA

O O

Malonyl CoA

Methylmalonyl CoASCoA

O

Acetyl CoA

SCoA

O

Acetyl CoA

PKS

SCoA

O

Propionyl CoA

ACYL TRANSFERASE (AT) DOMAIN

Involved in selection of starter and extender units during Biosynthesis of Fatty acids and Polyketides

PKSDB

Yadav G, Gokhale R.S and Mohanty D. (2003) Nucl. Acids Res. 31:3654-3658

Table 3 (a)

Yadav, G., Gokhale, R. S. and Mohanty, D. (2003)

J. Mol. Biol. 328, 335-363.

Trivedi et al. Mol Cell 2005, 17: 1-13

Substrate Specificity of AT domains of PKS

•KS domain dendrogram•Different sub-families of KS domain sequences show distinct clustering.•How to Quantify this difference?

Classification of KS sequence into subfamilies: Modular or Iterative ?

Identification of residues in KS which control number of iterations

Threading of sequence onto known structural folds

Homology Models based on highest scoring structural templates

Active site residue extraction and analyses

Cavity Analyses of various models in terms of :

Volume Hydrophobicity Topology

Comparison across iterative PKS subfamilies

Analyses of KS domains from iterative PKS

The E.coli KAS-II

Catalytic Pocket

Plots of iterative KS model cavity parameters

01020

30405060

708090

0 10 20 30 40 50 60

hydrophobicity (A F M Y L W V I )

%u

nsa

tura

tio

n

Are we analyzing the correct cavity?

Saturation of ProductVs

Hydrophobicity of CLRs

Cavity Volume Vs No. of Iterations

Shapes of Active Sites of Iterative PKSs

MSAS NAPTHOPYRONE

107 108 131 132 133 134 135 138 193 198 202 205 206 207 229 265 268 269 270 271 272 273 276 279 308 309 313 342 398 399 400 401

1KAS G I P F F V P I A G F A R A F D H M T S P P G A P A A L F G F G

AVILA C T A - - - - - L - - P G A Y D T N G I M A G Q R L M E F G Y G

CALORS T A Y T G I A V Q L A G T Y D T D G I M A G Q Q L V E F G Y G

MSAP N S P W M G I A I - - P G L Y D T N G I M A Q Q P V T E Y G Y G

MSAT N S A W M G I A L L L A G A Y D T N G I M A A Q P L T E Y G Y G

MSPG N S A H M G V A L L L A G A Y D T L G I M A A Q S L T E Y G Y S

MSPP G V A Y - - - V L - P T R V Y D T N G I M A S Q P L T E Y G Y G

THNGL F Y T Y F I T V M I L G Q F Y S A V S I T H G Q Q A T E F S A A

THNCL - T T Y Y I T V M I L G Q F Y S A I S I T H G Q Q A T E F S A A

THNND - T T Y Y I T V M I L G Q F Y S A I S I T H G Q Q A T E F S A A

AFMEL - T T Y F I P N L N L G H F Y S A V S I T R V Q Q A V E F S A A

WANA F Y T Y F I P N L N L G H F Y S A V S I T R V Q Q A V E F S A A

NAPATT S T Y F I P N L N L G H F Y C S I T R P H D Q Q A T E F S A A

AFLAT - T T Y F I T N I G L G F F Y S S E S M T R V Q Q V V E F S A A

AFTOX - T T Y F I T N I G L G F F Y S S E S M T R V Q Q V V E F S A A

STERG F H T Y F I T N I G L G F F Y S S M T R P F A Q Q V V E F S A A

ENDY2 S T P F T L A L S E F V G A F D G L T R P E G Q A V A K M G F G

ENCV T T A F T L A L S E F T G A F D G I T R P E G H A V T K M G F G

ENPKS S T P F T L A L S E F T G A F D G I T R P E G H A L T K M G F G

ENKIT T T G D T L A L S E F T G A F D G I T R P E G H A V T K M G F G

ENCRZ T T P F T L A L S E F T G A F D G I T R P E G Y A V T K M G F G

ENGHA T L P F T L A L S E F T G A F D G I T R P E G Y A V T K M G F G

C1027 T L P F T L A L S E F T G A F D G I T R P E G Y A V T K M G F G

ENMEGS T P F T L A L S E F T G A F D G I T R P E G Y S V T K M G F G

ENECO T L P F T L A L S E F T G A F D G I T R P E G Y R V T K M G F G

ENMADT L P F S L A L S E F T G A F D G I T R P E G Y E V T K M G F G

ENMIC T T P F T L A L S E F A G A F D G M T R P E G Y A V T K M G F G

ENESP T L P F S L V L S E F L G A F D G G T R P D G Q E V T K M G F G

ENCIR S L A D S L A L S E F T G A F D G I T R E A R - A L T K M G F G

BIKGF C A A F T A T L M W L A S F Y N C T P L F V S L P V A E Y G A S

BIKAV - T T Y Y I P N M N L G H F Y S S I T R P L A Q Q A V E F S A A

CMPC M T T Y S A T A I T E L N M Y D T T G I T M H Q P A Q E F G F G

LOVS M T T Y S A T A I T E L S M Y D T T G I T M H Q P A Q E F G F G

LOVGFM C Q Y G A T A I M E L S M Y D T P G L T M A Q P A R E F G F G

FUMONW G G G - - D V - - M V A Y D G M S M P S H - S V L E F G I G

ACTIVE SITE RESIDUES OF ITERATIVE KS DOMAINS

CORRECT ORDER OF ORFs WITHIN A PKS BIOSYNTHETIC CLUSTER

Simocyclinone PKS

Mupirocin PKS cluster

ROLE OF LINKERS

INTERMODULAR DOCKING INTERACTIONS

Pairs tested 66

Both charged Interactions 20

One charged Interaction 11

At least One Good Interaction 23

Percentage of Total 82%

Both Bad Interactions 0

Cognate – Non Cognate Differentiation (An example):

Pair 1 Pair 2 Interface

Ery1-C D E Ery2-N K R Ery 1-2 (cognate)

D-K E-R + +

Ery2-C D K Ery3-N R D Ery 2-3 (cognate)

D-R K-D + +

Ery 1-3

Noncognate

D-R E-D + -

PREDICTION OF ORF ORDER USING LINKER INTERACTIONS

The Spinosyn Biosynthetic clusterORFs: 1 2 3 4 5

ORDER Interface 1

Interface 2

Interface 3

Interface 4

TOTAL:

Good – Bad - Neutral

1-2-3-4-5 . . + + + + + + 6 0 2

1-2-4-3-5 . . + + + - + - 4 2 2

1-3-2-4-5 . . - - + + + + 4 2 2

1-3-4-2-5 . . + + - + + - 4 2 2

1-4-3-2-5 . . - + + + + - 4 2 2

1-4-2-3-5 . . + - - - + - 2 4 2Charged Interaction (+) Bad Interaction(-) Neutral Interaction (.)

PKS ORFs Combinations PredictionGood Bad Neutral Better Equal Worse

Albicidin 1 1 YesAmphotericin 6 24 6 0 4 0 3 20 YesAnsamitocin 4 2 2 1 3 1 0 0 NoAscomycin 3 1 YesAvermectin 4 2 1 1 4 0 1 0 YesBleomycin 1 1 YesBorrelidin 6 24 5 1 4 2 5 16 Yes

CFA 2 1 YesCompactin 1 1 YesEpothilone 4 2 0 0 6 0 1 0 Yes

Erythromycin 3 1 YesGeldanamycin 3 1 Yes

Lankacidin cplxLankamycin 3 1 YesLeinamycin 2 1 YesLovastatin 1 1 Yes

Megalomycin 3 1 YesMicrocystin 1 1 YesMonensin 8 720 10 1 3 0 720 0 NoMupirocin 3 cplx

Mycolactone 3 1 YesMyxalamid 5 6 0 0 8 0 3 2 Yes

Myxothiazol 3 1 Yesnanchangmycin 9 5040 13 1 2 480 240 4320 Yes

Niddamycin 5 6 4 1 3 1 3 2 YesNystatin 6 24 6 0 4 0 3 20 Yes

Oleandomycin 3 1 YesPederin 2 1 Yes

Pikromycin 4 2 3 0 3 0 0 1 YesPimaricin 5 6 6 0 2 0 1 4 Yes

Pyoluteorin 2 2 NoRapamycin 3 1 YesRifamycin 5 6 2 0 6 0 2 3 YesSoraphen 2 1 YesSpinosad 5 6 6 0 2 0 0 5 Yes

Stigmatellin 9 5040 2 5 9 768 1056 3216 YesTylactone 5 6 1 1 6 4 1 0 No

Vicenistatin 4 2 5 0 1 0 1 0 YesYersiniabactin 1 1 Yes

Cognate Non Cognate

ORF ORDER PREDICTION FOR PKS CLUSTERS

Naturally occurring peptides produced by NRPSs.

Substrates of NRPS

Acetyl Medium Long Coumarate Luciferase NRPSW H H I H F

* V * Y G D

T * * S F *

S G * S F *

Y * * P * *

G * * * A *

V G G G G G

G G G A G G

T A G G G *

W Y Y Y Y Y

W G G G G G

G P P P S V

* * * V A *

K

95%

K

83%

K

93%

K

92%

K

100%

K

95%

Consensus Active Site Patterns Of Six Subfamilies

* No consensus

Lys 517

Gly 324

3.02 Å

4.82 Å

Active site pattern

Specificity determining residues (SDR)

Products 22

ORFs 74

C 181

A 151

M 14

T 161

Te 22

NRPSDB

Active site residues of Adenylation domains

SEARCHNRPS

Ansari MZ, Yadav, G., Gokhale, R. S. and Mohanty, D. (2004) Nucl. Acids Res. 32:W405-13

.

Genetic algorithm for 250 runs. Grid size: 22.5 X 22.5 X 22.5 (Å)3Cluster rmsd :1.7ÅMajor cluster: 223Minor cluster: 27

How good are models at such low sequence homology ?

Crystal structure of the long chain CoA ligase (1V26)

Model of the same protein based on 1AMU

Ligand Rmsd = 2.3 Å 10/18

Glycosyltransferases (GT), enzymes that transfer sugars to other molecules.

R=nucleoside nucleoside monophosphate

R’ = Sugar, Lipid, Protein, DNA Secondary metabolites

K X calic1P Dallose tylo3A dehydrovancosamine balhi3A Lepivancosamine chlor3A Lvancosamine vanco1A Lvancosamine vanco3A Dglucose balhi2A Dglucose chlor2A Dglucose vanco2A Dglucose vanco4A NAcGlucosamine a40926.1A Nacetylglucosamine teico1A Nacetylglucosamine teico2A Ldehydrovancosamine balhi1A Lepivancosamine chlor1A Mannose a40926.2A mannose teico3F X avila4F X everni4F X avila2F X everni3F X avila3F X everni2J X nanch2H Dglucosamine butiroH X genta2H X tobra1H X genta1H X tobra2G 3OcarbamoylDmannose bleo2G Lgulose bleo1H Ldihydrostreptose streptoJ X nanch3O Dglucosamine mitoI Dglucose rebecC Dmycosamine amphoterC Dmycosamine nystaC mycosamine candiC Dmycosamine pimaC mycosamine ce108C mycosamine rimoP Lmycarose tylo2E LoleandrosylLolendrose avermK X calic2K X calic4K X calic3B X nogal3B Lrhamnose elloraT Ldigitoxose jadoQ Dfucofuranose gilvoF X everni1N Dolivose urdam1R angolosamine mederN Dolivose lando1N Dolivose lando5R Lrhodinose granaS X heda2L ChromoseAD chromo2S X heda1L DolivosylDolivose mithra3M Lnoviose novoM Lnoviose clorobioM Lnoviose coumerP TriOmethylrhamnose spino1N Dolivose lando2N Dolivose lando6N Dolivose lando4N Dolivose urdam3N Lrhodinose urdam4J 4OmethylLRhodinose nanch1D vicenisamine vicenB X aclaci1B daunosamine daunomyB X adriaB daunosamine daunoruB X aclaci2B X nogal2P Lmegosamine megalo3P Ddesosamine erythro2P Ddesosamine megalo2P Ddesosamine oleando1P Loleandrose oleando2P Dmycaminose tylo1P Ddesosamine methymycinP Ddesosamine pikroP Lmycarose erythro1P Lmycarose megalo1P Dforosamine spino2K sugar c1027N Lrhodinose urdam2N Lrhodinose lando3N Lrhodinose lando7L DolivosylDolivose mithra4B X nogal1F X avila1F X everni5L ChromoseAD chromo1L Doliose mithra2L Dolivose chromo3L Dolivose chromo4L Dolivose mithra1

Vancomycin group

Orthosomycin group

Aminoglycoside AB

Hybrid NRPS-PKS

Polyene macrolide AB

Enediyne group

Aminocoumarin AB

Angucycline AB

Anthracycline group

Macrolide group

Aureolic acid AB

GTr sequences cluster according to substrate specificity

Prediction accuracy = 77%

N-Domain

C-Domain

Linker

DVV and its binding residues

TYD and its binding residues

CGTDLMMLQMPPPELTGDDPYNTSGSDMLGKRVPRLQSAGVPGFMT

TRGELGSEVFHSGTVSRGEIGSEVHASGUA

1RRVBest MatchQuery

VVV

CTS

8

GGG

9

TTS

10

RRR

11

GGG

12

DDD

13

EEE

15

LIM

55

MML

59

MGG

60

LLK

61

QKR

62

MLV

65

PPP

66

PPR

67

PLL

68

EEQ

73

LKS

76

TSA

80

GGG

102

DDV

103

DPP

141

PKG

143

YYF

166

LLI

218

GGG

245

SSS

246

EEE

293

294

FFH

296

HGA

309

SSS

311

GGG

313

TTU

314

VLA

317

NNM

331

TTT

332

Acceptor Binding Residues

Donor Binding Residues

Identifying substrate (donor/acceptor) binding residues

MurG

GtfA

Benchmark the Prediction Accuracy

% identity ~ 20%

Correctly predicted approximately the same site and 50%

of the donor binding residues

C

C

GtfD

% identity ~ 55%

Correctly predicted the same site and 90% of the donor binding residues

C

Organization of SEARCHGTr and its backend database GTrDB

Kamra P, Gokhale RS and Mohanty D (2005) Nucl. Acids Res., In Press

amino acid

CAT Fold

O2NHC

OH

CH

NHO

CHCl3

O H3C C

O

S CoA

H2C

NNH

His

H

O2NHC

OH

CH

NHO

CHCl3

O CH3C

O

HS CoA

CAT

O R C

O

S CoA

H2C

NNH

His

H

O C HS CoA

CRAT

O

-O

N+H3C CH3

CH3

O

-O

N+H3C CH3

CH3

O

R

S

PCP 1

O

CHNH2

AA 1

S

PCP 2

O

CHNH2

AA 2

S

PCP 2

O

CHNH

AA 2 O

NH2

AA 1

C

S

PCP 1

O

CHNH2

AA 1

S

PCP 2

O

CHNH2

S

PCP 2

O

CHNH

O

NH2

AA 1

Cy

X H X H

S

PCP 2

O

CHN

X

NH2

AA 1

Cy

S

PCP 1

O

CNH2

AA 1H

ES

PCP 1

O

CNH2

AA 1H

R OH H3C C

O

S CoA CH3C

O

HS CoAR O

OH

OH

OH

R OH

CAT

NRPS

CRAT

BAHD

CH2)18(

O

(H2C)12

O

(H2C)12

OO

H3C

CH2)18(

OHHOH3CS

CH2)12(

O

ACP

SH

ACP

PapA5

O

SH S C

O

CH3

E2-Lys S CoA

H3C C

O

S CoA

O

SH SH

E2-Lys

H2C

NNH

His

E2

H

E2p

PREDICT STRUCTURAL FOLDAND CORRELATE WITH

KNOWN CHEMISTRY

NRPS Domains

ConD-XL-X

Cyc

Epi

BAHD

CRAT

CAT

NRPS

CAT Superfamily

MAS ACP(Homology Model)

PapA5(Crystal Structure)

Protein Docking

PapA5

WT R234E R312E

Identification of crucial residues involved in protein-protein interaction

Mutational studies of these crucial residues

Trivedi et. al. Mol. Cell. (2005) 17, 631-643

Acknowledgements

Gitanjali YadavMd. Zeeshan AnsariPankaj Kamra

Dr. Rajesh S. GokhaleChemical Biology Group

Dr. S.K. Basu, Director, NII

BTIS, DBT, India

Cinnamate CoumarateCaffeate

Ferulate Sinapate 3,4-DMC

Substrates of Coumarate CoA Ligases Coenzyme A

Coumarate CoALigase

Substrates of NRPS

Adenylation domain of NRPS

PCP Domain

Acetic acid

n ~ 4 - 8 : Medium chain fatty acid

n ~ 5 -11: Long chain fatty acidn > 11 : Very Long chain fatty acid

Coenzyme AFatty acid CoA

LigaseSubstrates of Fatty Acid CoA Ligases

Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria

Trivedi, O.A., Arora, P., Sridharan, V., Tickoo, R., Mohanty, D. and Gokhale, R.S. 2004 Nature 428:441.