1. CHEMICAL BIOLOGY GROUP RESEARCH INTERESTS Isolation and Characterization of Microorganisms for Lipases and Biosurfactants. Development of HTS Assays for Lipases Bioinformatics and Molecular Dynamics of Lipases. Protein structure prediction using knowledge based methods. Dr.P.Gautam, Professor, Centre for Biotechnology, Anna University, Chennai -600 025. pgautam@annauniv.edu gpennathur@gmail.com www.annauniv.edu/biotech/boc/

3. Similar to seriene proteases has Ser-His-Glu/Asp acids as active site triad, 534 aa. Lipases catalyses the hydrolysis of triacylglycerides (fats) to di, mono acylglycerols, glycerol and free fatty acids. Used in preparation of many food stuffs and in the pharmaceutical industry for chiral specific synthesis of racemic mixtures. Lipases work in aqueous and non-aqueous environments. Candida rugosa lipase INTRODUCTION TO LIPASES

4. Lipases are simply defined as carboxylesterases catalyzing the hydrolysis (and synthesis) of long-chain acylglycerols. There is no strict definition available for the term "long-chain," Glycerolesters with an acyl chain length of > 10 carbon atoms can be regarded as lipase substrates, with trioleoylglycerol being the standard substrate.

5. The Alpha / Beta Fold Lipases

6. Ribbon structures of Pseudomonas glumae lipase (a), Candida rugosa lipase (b), Candida antarctica B lipase (c), Humicola lanuginosa lipase (d), Fusarium solani pisi cutinase (e), and Human pancreatic lipase (f). The tentatively placed lid or lids are coloured white.

7. Mechanism of Action of Lipases

8. The chemical structure of 2- (4 iso-butylphenyl) propionic acid (racemic ibuprofen) showing the stereogenic center and the R- and S- configuration. p58

9. Reactions Using Candida Rugosa Lipase

10. Chromatograms of rac-Ibuprofen and Ester

11. Docking ibuprofen to CRL Three potential a bonds (dotted lines) were observed between the docked S(+)Ibuprofen and Candida rugosa lipase Biocatalysis Biotransformation Vol 17 pp 475-486. In contrast to the S(+) isomer the R(-) forms no hydrogen bonds with the active site residues

13.  F = MA exp(-  E/kT) domain quantum chemistry molecular dynamics Monte Carlo mesoscale continuum What and Where: Scales in Simulations Length Scale Time Scale 10 -10 M10 -8 M10 -6 M10 -4 M 10 -12 S 10 -8 S 10 -6 S Courtsey, Smith, University of Utah

14. Motivation: Why atomistic MD simulations?  Mechanisms  MD simulations provide a molecular level picture of structure and dynamics  property/structure relationships  What if?  Experiments often do not provide the molecular level information available from simulations  Simulators and experimentalists can have a synergistic relationship, leading to new insights into Function

15. How MD Simulations Work: The Force Field intermolecular interactions intramolecular nonbonded torsional bond stretch valence angle bend

16. Cluster Computing Environment 16 nodes Presently runs on RHL 9.0 Software for MD simulations GROMACS 3.0 www.gromacs.org

17. WHY ONE TYPE OF SUBSTRATE IS PREFERED OVER THE OTHER AT THAT PARTICULAR pH? 9%48% Methyl ester 49%5%Butyl ester 48 hours5.5 hours Time taken 7.25.6 pH Results of enzymatic hydrolysis using Candida rugosa lipase

18. 0.69900.00000.6990Ile 406 3.31091.30364.6145Arg 405 1.22320.31171.5349Lys 404 0.00000.8737 Phe 403 2.18210.00002.1821Gln 402 0.0000 Gln 182 0.0000 Asp 181 2.45130.31712.7684Lys 180 0.0000 Leu 179 0.0000 Gly 178 Side chainBackboneTotal accessibilityAmino acids and position Solvent accessibility of CRL Lysine 180 and 404 are in a buried pocket in both open and closed forms of CRL Software package WHATIF was used for the above calculation. http://www.cmbi.kun.nl/whatif/

19. pH based Molecular dynamics simulation of Candida rugosa lipase Lysines 180 & 404 Protonated Lysines 180 & 404 deprotonated Simulated structures (blue & red) superimposed on to the crystal structure (green). Results

20. The extent to which the flap opens is dependent on pH. Thereby explaining the differential selectivity at the two different pH states. pH 5.6 The flap after simulation comes towards a closed conformation. Thus facilitating a less bulkier group, the methyl ester of ibuprofen to diffuse in to the active site of CRL. pH 7.2 CONCLUSION The flap after simulation comes to a “more open” conformation with respect to the energy minimized conformation, facilitating a bulkier, butly ester of ibuprofen to diffuse in to the active of CRL.

21. Importance of buried Lysines Simulations conducted by only protonating the histidines based on the results given by pKa predictor, that all lysines are protonated immaterial of their pH 5.6 or 7.2 pH 5.6 pH 7.2 There is no change in the in the flap’s conformation such as to correlate it to the experimental result. Histidines ND1 and NE2 sites were protonated as a function of pH. Protein Engineering V16(12), (2003), 1017-1024.

22. Solvent hydrophobicity in the interfacial activation of Candida rugosa lipase. J. Biochem. Mol. Bio & Biophys Vol 4, pp 293 2000.

23. Protein Octane Flap at C8-H 2 O interface after 4ns of simulations S imulated flap Closed flap H2OH2O RMSD of flap after 4ns = 10.2 Å

25. Structure of Psuedomonas Aueroginsa Lipase

26. Domain Movement in Lipases: Evidence for a double-lid in Pseudomonas aeruginosa lipase from MD Simulations ●Pseudomonas aeruginosa lipase (PDB Identifier: 1EX9) is the prototype of the bacterial I.1 lipase family. ●The open form of this lipase has been crystallized and a helix (125-148) and its surrounding loops have been identified as the lid. ●We have performed molecular dynamics (MD) simulations of this lipase in water for 10 ns and observe that the lid closes during the simulation time scale. ●From the analysis of domain movements we conclude that this lipase has two lids: the second lid, which encompasses residues 200-230, contains a loop-helix-loop motif. ●We also show that movement of each lid is independent of that of the other.

27. Both lids are shown. Fall in exposure of binding site (yellow) seen here Structures after 3, 7, 10ns. Simulated structures in red

28. Setting up SM-FRET Computational Lead Cherukuvada et al (2005) PLoS Comput. Biol Future Work

29. Acknowledgement Department of Science and Technology (DST) Govt. of India. Council of scientific and industrial research (CSIR) Govt. of India Department of Biotechnology- Biotechnology Information Services (DBT-BTIS) Govt. of India

30. When I was a boy of fourteen, my father was so ignorant I could hardly stand to have the old man around. But when I got to be twenty-one, I was astonished at how much he had learned in seven years. ~ Mark Twain, "Old Times on the Mississippi" Atlantic Monthly, 1874