CHEMICAL BIOLOGY GROUP

RESEARCH INTERESTSRESEARCH INTERESTSIsolation 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.edugpennathur@gmail.com

www.annauniv.edu/biotech/boc/

• 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

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.

The Alpha / Beta Fold Lipases

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.

Mechanism of Action of Lipases

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

p58

Reactions Using Candida Rugosa LipaseReactions Using Candida Rugosa Lipase

Chromatograms of rac-Ibuprofen and Chromatograms of rac-Ibuprofen and EsterEster

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

F = MA

exp(-E/kT)

domain

quantumchemistry

moleculardynamics

Monte Carlo

mesoscale continuum

What and Where: Scales in Simulations

Length Scale

Tim

e S

cale

10-10 M 10-8 M 10-6 M 10-4 M

10-12 S

10-8 S

10-6 S

Courtsey , Smith, University of Utah

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

How MD Simulations Work: The Force Field

intermolecularinteractions

intramolecularnonbonded

torsional

bond stretch

valence anglebend

Cluster Computing Environment

• 16 nodes

• Presently runs on RHL 9.0

• Software for MD simulations

GROMACS 3.0

www.gromacs.org

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 hoursTime taken

7.25.6pH

Results of enzymatic hydrolysis using Candida rugosa lipase

0.69900.00000.6990Ile 406

3.31091.30364.6145Arg 405

1.22320.31171.5349Lys 404

0.00000.87370.8737Phe 403

2.18210.00002.1821Gln 402

0.00000.00000.0000Gln 182

0.00000.00000.0000Asp 181

2.45130.31712.7684Lys 180

0.00000.00000.0000Leu 179

0.00000.00000.0000Gly 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/

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

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.

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.

Solvent hydrophobicity in the interfacial activation of Candida rugosa lipase.

J. Biochem. Mol. Bio & Biophys Vol 4, pp 293 2000.

Protein Octane Flap at C8-H2O interface

after 4ns of simulations

Simulated flapClosed flap

H2O

RMSD of flap after 4ns = 10.2 Å

Structure of Psuedomonas Aueroginsa Lipase

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.

Both lids are shown. Fall in exposure of binding site (yellow) seen here

Structures after 3, 7, 10ns. Simulated structures in red

Setting up SM-FRET

Computational LeadCherukuvada et al (2005)PLoS Comput. Biol

Future Work

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

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