CHEMICAL BIOLOGY GROUP

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CHEMICAL BIOLOGY GROUP
Dr.P.Gautam, Professor, Centre for
Biotechnology, Anna University, Chennai -600
025. pgautam_at_annauniv.edu gpennathur_at_gmail.com ww
w.annauniv.edu/biotech/boc/
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.
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INTRODUCTION TO LIPASES

• 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.

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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 gt 10 carbon atoms can be regarded as
lipase substrates, with trioleoylglycerol being
the standard substrate.
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The Alpha / Beta Fold Lipases
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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.
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Chromatograms of rac-Ibuprofen and Ester
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Docking ibuprofen to CRL
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What and Where Scales in Simulations
continuum
mesoscale
Monte Carlo
10-6 S
Time Scale
molecular
dynamics
domain
10-8 S
quantum
?
exp(-
E/kT)
chemistry
10-12 S
F MA
10-10 M
10-8 M
10-6 M
10-4 M

Length Scale
Courtsey , Smith, University of Utah
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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

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How MD Simulations Work The Force Field
bond stretch
torsional
intermolecular interactions
valence angle bend
intramolecular nonbonded
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WHY ONE TYPE OF SUBSTRATE IS PREFERED OVER THE
OTHER AT THAT PARTICULAR pH?
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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/
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Results
pH based Molecular dynamics simulation of Candida
rugosa lipase
Simulated structures (blue red) superimposed
on to the crystal structure (green).
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CONCLUSION
The extent to which the flap opens is dependent
on pH. Thereby explaining the differential
selectivity at the two different pH states.
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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 flaps
conformation such as to correlate it to the
experimental result.
Protein Engineering V16(12), (2003), 1017-1024.
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Solvent hydrophobicity in the interfacial
activation of Candida rugosa lipase.
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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.

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Both lids are shown. Fall in exposure of binding
site (yellow) seen here
Structures after 3, 7, 10ns. Simulated structures
in red
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Future Work
Computational Lead
Cherukuvada et al (2005) PLoS Comput. Biol
Setting up SM-FRET
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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
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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