1. In silico identification of novel biosynthetic pathways in Mycobacteria Tuberculosis Research – An Indian Perspective (TRIP) AstraZeneca India 20 Oct 2005 D. Mohanty National Institute of Immunology New Delhi

2. Phosphatidyl inositol mannoside Mycobactin Mycolic acids Sulfolipid Trehelose MYCOBCATERIUM TUBERCULOSIS Mycoketide PDIM

4. Genes Metabolites

5. Domains Involved in Protein-Protein Interactions Substrate Specificity of Catalytic Domains PKSNRPSModifying 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

6. Levels of Functional Annotation Sequence based methods: Fundamental for functional annotation Drawback: Cannot predict substrate specificity

7. PCPS SCoA Amino acid Coumarate Oxyluciferin Amino acyl PCP Fatty acidAcyl CoA Coumaroyl CoA Luciferin

8. 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

9. Applications of comparative modeling. The potential uses of a comparative model depend on its accuracy. This in turn depends significantly on the sequence identity between the target and the template structure on which the model was based.

10. Knowledge Based Approach Sequence/structure information for large number of proteins with known specificity Predicting substrate specificity of new members of the family using evolutionary information Design of novel proteins with altered specificity. Design of novel polyketides/nonribisomal peptides In silico identification of PKS/NRPS products Sequence-Product correlation Predictive rules

11. Phthiocerol dimycocerosates (PDIMs)  GENETIC STUDIES: pps CLUSTER REQUIRED FOR PDIMs BIOSYNTHESIS  FadD KNOCK-OUTS DISRUPT PDIM BIOSYNTHESIS KOLLATUKUDY et al. MOL. MICROBIOL. (1997) 24, 263-270 COX et al. NATURE (1999) 402, 79-83 CAMACHO et al. J. BIOL. CHEM. (2001) 276, 19845 mas 2929 fadD26 ppsA ppsB ppsD ppsC ppsE drrA drrB drrC papA5 fadD28 mmpL7 26.751 kb 6.333 kb 44.199 kb tesA FUNCTIONAL IMPORTANCE OF PPS CLUSTER

12. POSSIBLE STARTER AND EXTENDER UNITS FAS Malonyl CoA Acetyl CoA Butyryl CoA Benzoyl CoA Isobutyryl CoA Acetoacetyl CoA Malonyl CoA Methylmalonyl CoA Acetyl CoA PKS Propionyl CoA ACYL TRANSFERASE (AT) DOMAIN Involved in selection of starter and extender units during Biosynthesis of Fatty acids and Polyketides

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

15. Table 3 (a)

16. 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

17. RETROBIOSYNTHESIS OF PDIM (16 ORFS) mmpL 7 tesA Rv2929 fadD26ppsA ppsB ppsCppsDppsE drrABC papA5 mas fadD28 PCPAC P KSATKRAC P KSATAT KRKR AC P KSATKRDHDH ERAC P KSATKRKR DHDH ACPKSKS ATAC P KSATKRDHDH ERER CERER 3 (n-C 16 -C 20 acid) + 3 Malonyl-CoA + 10 Methyl Malonyl-CoA + NADPH + ATP + CoASH PapA5 R 2 = -H, -CH 3 ATP PPi AMP FadD26 R 3 = -CH 2 -CH 3 or -CH 3 R 4 = -OCH 3 or mycocerosic acids phthiocerol PpsA-E Mas PDIM R 1 = -(CH 2 ) 3-7 -CH3 TRIVEDI et al., Molecular Cell (2005) 17 631-643. CELL-FREE RECONSTITUTION OF 26 CATALYTIC STEPS

18. CAT Fold CAT NRPS CRAT BAHD PapA5 E2p PREDICT STRUCTURAL FOLD AND CORRELATE WITH KNOWN CHEMISTRY

19. Threading or Fold Recognition Proteins often adopt similar folds despite no significant sequence or functional similarity. For many proteins there will be suitable template structures in PDB. Unfortunately, lack of sequence similarity will mean that many of these are undetected by sequence-only comparison done in homology modelling.

20. NRPS Domains Con D-X L-X Cyc Epi B A H D C R A T CAT N R P S CAT Superfamily

21. 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

22. Mbt BIOSYNTHETIC GENE CLUSTER Quadri et al., Chem Biol. 1998 Nov;5(11):631-45. De Voss et al., Proc Natl Acad Sci U S A. 2000 Feb 1;97(3):1252-7. GENETIC LOCUS INVOLVED IN TAILORING THE MYCOBACTIN PEPTIDIC CORE TO PRODUCE FUCTIONAL SIDEROPHORE NOT KNOWN?  NON-RIBOSOMAL PEPTIDE SYNTHASES (NRPSs) NON-RIBOSOMAL PEPTIDE SYNTHASES (NRPSs)  POLYKETIDE SYNTHASES (PKSs) POLYKETIDE SYNTHASES (PKSs) BIOCHEMICAL AND GENETIC STUDIES SUGGESTED INVOLVEMENT OF THIS CLUSTER IN BIOSYNTHESIS OF MYCOBACTIN CORE MYCOBACTIN CORE

23. IRON-DEPENDENT TRANSCRIPTIONAL PROFILING USING MICROARRAYS Rodriguez & Smith; Mol Microbiol. 2003 Mar;47(6):1485-94. IDENTIFIED NUMBER OF GENES Rv1347cacp fadD33 fadE14 (SIMILARITY TO PROTEINS INVOLVED IN  -OXIDATION) (HOMOLOGY WITH HISTONE ACETYL TRANSFERASE)

24. 120160200240280320360400440480 m/z, amu 0 10 20 30 40 50 60 70 80 90 100 Rel. Int. (%) 475.32 295.23 144.14 431.45 251.24 367.33 181.15 197.17 1 2 3 MS/MS of 475.32 1 2 3 100150200250300350400450500550600 m/z, amu 0 10 20 30 40 50 60 70 80 90 100 Rel. Int. (%) 369.26 433.32 266.24 477.31 187.11 295.16 251.17 142.09 234.14 3 2 1 MS/MS of 477.31 1 2 3 II III BIOCHEMICAL PATHWAY INVOLVED IN TAILORING MYCOBACTIN CORE  Fatty acids are transferred as acyl-S-enzyme intermediates by Rv1347c  Novel acyl-ACP dehydrogenase generates unsaturation in the lipidic chain

25. Aryl-N-acetyl transferaseRv1347c (1YK3)

26. Rv1347c + CoA (Transformed) Myristic Acid (C-14) (Ligand) Docking (AutoDock) 29 member cluster 30 member cluster (COO - Flipped) Docking of Myristic Acid

27. Rhizobactin Ornibactin Acinetoferrin Aerobactin Nocobactin Mycobactin Pyoverdin P. putida (Pyoverdin) M. bovis (Mycobactin R = C17-C20) M. tuberculosis (Mycobactin R = C17-C20) M. smegmatis (Mycobactin R = C9-C19) M. Aviam (Mycobactin R= C11-C18) E. coli (Aerobactin) B. cepacia R18194 B (Ornibactin) N. farcinia (Nocobactin) S. meliloti B (Rhizobactin) V. fisheri (Aerobactin) V. mimicus (Aerobactin) B. cepacia R18194 A (Ornibactin) B. cepacia R1808 (Ornibactin) Acenobacter sp. (Acinetoferrin) S. meliloti A (Rhizobactin) A B C

28. LPLPVFLCALM. bovis (Mycobactin)Acyl (R = C17-C20) LPLPVFLCALM. tuberculosis (Mycobactin )Acyl ( R = C17-C20) MPLPVFLCAL M. smegmatis (Mycobactin )Acyl ( R = C9-C19) LSLPVFFCSLM. Aviam (Mycobactin )Acyl ( R= C11-C18) LALPVCHLHTB. cepacia R1808 (Ornibactin)Acyl (2-ene) WRMKCGSYICAcenobacter sp. (Acinetoferrin)Acyl (2-ene) WPIRTGYACCS. meliloti A (Rhizobactin)Acyl (2-ene)* LQLRTLHLAA E. coli (Aerobactin)Acetyl FSIGPCALNLN. farcinia (Nocobactin)Acetyl CTLPLQNLSIS. meliloti B (Rhizobactin) Acetyl* INMRSLRIVI V. fisheri (Aerobactin) Acetyl INMRSLLFLLV. mimicus (Aerobactin) Acetyl IALSVAYMQLP. putida (Pyoverdin)Formyl LHWSLGYMLIB. cepacia R18194 A (Ornibactin) Acyl (b-OH)/Formyl? LHWSLGYMLMB. cepacia R18194 B (Ornibactin)Acyl (b-OH)/Formyl? * Predicted specificity based on these two positions. ? Unable to differentiate between two substrate based on these two positions.

29. mbt-2 Rv1347c acp fadD33 fadE14 Low Iron Concentration Mycobactin coreFatty acyl-ACP DidehydroxymycobactinMycobactin mbt-1 B C D E F G H I J A mbtG IdeR Fe- box BIOSYNTHETIC SCHEME FOR AMPHPHILIC MYCOBACTIN NEW GENETIC LOCUS INVOLVED IN SIDEROPHORE BIOSYNTHESIS

30. Patterns in Networks: Network Motifs

31. ACKNOWLEDGEMENTS COMPUTATIONAL BIOLOGY GROUP CHEMICAL BIOLOGY GROUP KNOWLEDGE-BASED COMPUTATIONAL APPROACH RECONSTRUCTION OF METABOLIC PATHWAYS Dr. S.K. Basu, Director, NII BTIS, DBT, India Rajesh S. Gokhale Gitanjali Yadav Md. Zeeshan Ansari Pankaj Kamra

32. Cinnamate Coumarate Caffeate Ferulate Sinapate 3,4-DMC Substrates of Coumarate CoA Ligases Coenzyme A Coumarate CoA Ligase

33. Substrates of NRPS Adenylation domain of NRPS PCP Domain

34. Acetic acid n ~ 4 - 8 : Medium chain fatty acid n ~ 5 -11: Long chain fatty acid n > 11 : Very Long chain fatty acid Coenzyme A Fatty acid CoA Ligase Substrates 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.