1. Biochemical and Biophysical
Characterization of AnAEst, a
novel SGNH hydrolase
Kiranmayee Bakshy
Indian Institute of Technology Madras,
Chennai, India
PI: Dr. Manoj Narayanan
1

2. Contents
 Comparative analysis (structural and
functional evolution)
 Biochemical characterization (functional and
kinetic analysis)
 Biophysical characterization (thermal stability
– structure function relationship)
2

3. Tools set
• Sequence and structural homology searches DALI, HHPRED, PDB
• Sequence and structural alignment tools FATCAT, TCOFFEE,
MULTIPROT
• Molecular visualization tools PyMol, RasMol, SwissPDB
• Protein over expression in E.coli BL21, rosetta strains
• Protein purification using Ni-NTA column chromatography followed by
size exclusion chromatography
• Site-directed mutagenesis (Stratagene) for active site mutants
generation
• Enzyme activity assay methods: titrimetry, HPLC, colorimetry,
spectrophotometry, fluorimetry, zymography, TLC
• Biophysical techniques: DSC, far and near UV CD, fluorescence
spectroscopy and Trp fluorescence quenching studies, ANS binding
studies, fourth derivative spectra
• Crystallization: hanging drop method; using Hampton crystallization
screens
• Ligands used for co-crystallization: AEBSF, PMSF, imidazole, acetate
3

4. 4
Serine hydrolases
Structural classification of Serine hydrolases
Beta proteins Alpha/beta proteins
Trypsin-like serine proteases
Crotonase-like (Seq-10821; Str-59)
Methylesterase C-domain (Seq-1586; Str-2)
Subtilisin-like (Seq-6532; Str-160)
α/β Hydrolase (Seq-28102; Str-750)
Flavodoxin-like (SGNH hydrolases)
Gariev, IA. and Varfolomeev, SD. (2006) Bioinformatics 22, 2574-2576
(Seq-5119; Str-13)
 In 1995, Upton and Buckley identified new class of lipolytic enzymes
 In 2003, this class has been named GDSL group of serine lipases/esterases
SGNH hydrolases are widely spread across all taxa

5. 5
α/β hydrolase fold SGNH hydrolase fold
Structural comparison
P. aeroginosa lipase
PDB ID 1EX9 PDB ID 1IVN
E. coli TAP
• Compact fold performs multiple functions
• Biochemical studies available for very few of them

6. 6
Flavodoxin fold substantially different from the canonical α/β hydrolase fold
N
C
β3 β1
β2
β4β5
α1 α2
α3α4α5
α6 α7
NuAc
H
Topological differences
SGNH hydrolase fold
E. coli TAP
Canonical α/β hydrolase fold
P. aeroginosa lipase
N
C
α1
α2α3α4α5
α6
β1β2β3 β4β5β6β7β8
NuAc
H
α-helix
β-strand
Blue colour - insertions with respect to SGNH hydrolase fold
Mala and Takeuchi, Anal Chem Insights (2008),3, 9–19
Akoh,CC et al., Progress in Lipid Res. (2004), 43, 534–552

7. Structure based sequence alignment
7
α/β hydrolases
SGNH hydrolases
SGNH family members can be identified only from these four blocks

8. 8
Comparative analysis of SGNH
hydrolases

9. 9
S.No. PDB ID ENZYME FUNCTION SOURCE OLIGOMERIZATION
1. 1IVN
Thioesterase I/Protease
I/Lysophospholiase L1
Escherichia coli Monomer
2. 1WAB
Platelet-activating factor
acetylhydrolase
Bos taurus Dimer
3. 2VPT Carbohydrate esterase Clostridium thermocellum Dimer
4. 1DEO Rhamnogalacturonan acetylesterase Aspergillus aculeatus Monomer
5. 1FLC
Haemagglutinin-esterase-fusion
glycoprotein
Influenza C virus Trimer
6. 1ESC Hydrolase (Serine esterase) Streptomyces scabies Dimer
7. 1Z8H Putative lipase
Anabaena sp. Strain PCC
7120
Dimer
8. 3BZW Putative lipase
Bacteroides
thetaiotaomicron
Trimer
9. 2HSJ Putative platelet activating factor Streptococcus pneumonia Tetramer
10. 2APJ Carbohydrate esterase Arabidopsis thaliana Monomer
11. 1ZMB Acetylxylan esterase
Clostridium
acetobutylicum
Dimer
12. 2O14 Hypothetical protein Bacillus subtilis Monomer
13. 1YZF Lipase/Acylhydrolase Enterococcus faecalis Monomer
Structurally characterized SGNH hydrolases
Diverse functions can be observed among the 6 well characterized members

10. 10
Structure based sequence alignment of
SGNH hydrolases
 SGNH hydrolases share a very low sequence identity
 Catalytic residues are structurally conserved

11. 11
RGAE-TAP MsAct-TAP
AnAEst-TAP Bt12063b-TAP
Loop 1
Loop 2
Tertiary structural variations around the active site cleft can
be implicated to diverse substrate specificity
Structural basis for diversity in substrate
specificity

12. 12
Highly conserved tertiary structures and
catalytic site
Well conserved tertiary structures in spite of the presence of highly variant
primary structure
TAP
SsEst
Active site
rmsd ranges from 1.5-3.2 Å

13. 13
Structural basis for diversity in quaternary
structure
The diversity in oligomerization and substrate specificity can be attributed
to specific secondary structural insertions
Side-by-side (II type) dimer
Back-to-back (X3 type) dimer
α-helix
β-strand
Blue colour - insertions with respect to E.coli TAP

14. Conclusions
 Flavodoxin fold is substantially different from the canonical α/β
hydrolase fold - hence the name SGNH hydrolase fold
 SGNH family members can be identified only from the four
conserved sequence blocks
 SGNH hydrolases share a very low sequence identity and the
catalytic residues are structurally well conserved
 Tertiary structures are well conserved in spite of the presence of
highly variant primary and quaternary structure
 The diversity in oligomerization and substrate specificity can be
attributed to specific secondary structural insertions
14

15. 15
Expression, purification and
biochemical characterization of
AnAEst

16. 16
Biochemical characterization of AnAEst
Activity Substrate Method Activity
Protease Casein
Casein
Gelatin
Zymography
Colorimetry
Zymography
-
-
-
Lipase Olive oil
Sesame oil
Tributyrin
p-Nitrophenyl palmitate
Titrimetry
Spectrophotometry
-
-
-
-
Arylesterase α-Naphthyl acetate
α-Naphthyl propionate
α-Naphthyl butyrate
α-Naphthyl valerate
β-Naphthyl acetate
p-Nitrophenyl acetate
p-Nitrophenyl butyrate
p-Nitrophenyl caprate
p-Nitrophenyl laurate
Paraoxon
Phenyl acetate
4-methyl umbelliferyl acetate
Resorufin acetate
Methyl benzoate
Spectrophotometry
HPLC
+
+
-
-
-
+
-
-
-
+
+
+
+
-
Esterase Ethyl acetate, n-butyl acetate,
isopropyl acetate
Titrimetry
HPLC
-
Lysophospholipase Egg yolk phosphocholine
1-myristoyl-sn-glycero-3
phosphocholine
TLC -
Thioesterase Phenyl thioacetate
Acetyl CoA
Spectrophotometry +
-
Enantiospecificity 2-ethoxyethanol ester of Ibuprofen
Acetyl ester of (R) (+) α-methyl-2-
naphthalene methanol
HPLC -
-
AnAEst is an arylesterase hydrolysing specifically
aryl esters of short chain fatty acid

17. Regular biochemical characterization
 AnAEst is an arylesterase which hydrolyses small chain
fatty acid aryl esters
 It exhibits an optimal activity at pH 7.5 and in a broad
temperature range 25-45 °C
 Among all the divalent cations Cu+2 and Fe+2 shows
inhibitory effect of the esterase activity
What are the active site residues to be considered for
mutational and kinetic studies ?
17
Bakshy K, Gummadi SN, Manoj N, Biochim Biophys Acta. 2009, 2:324-334

18. 18
Selection, generation and purification of active site
mutants
L86
R54
S17
PDB ID 1z8h
WT S17A R54G L86A M
kDa
80
66
56
40
29
25
20
17
14
SDS-PAGE analysis of purified AnAEst
and its mutants
Wild-type and mutants were purified
under similar conditions
Selection of AnAEst mutations
The following residues were selected and
mutated by site-directed mutagenesis:
S17 nucleophile Ala
R54 oxyanion Gly
L86 active site Ala

19. 19
WT S17A R54G L86A
Zymogram showing the activities of
AnAEst and its mutants
•Arylesterase zymogram : 1-NA, Fast blue B
•Native PAGE for basic proteins under
neutral conditions
Altered specific activity of R54G mutant with
increased activity against phenyl esters
Standard assay condition: 50 mM sodium phosphate (pH 7.5); 1
mM substrate; 2 µg purified enzyme; at 25 °C. Results displayed
are mean of three individual experiments
Determination of activity profile of active site mutants
Spectrophotometric assays using various
substrates
Bakshy K, Gummadi SN, Manoj N, Biochim Biophys Acta. 2009, 2:324-334.

20. 20
Substrate Kinetic parameters Wild-type R54G L86A
α-naphthyl
acetate
Km (mM)
kcat (x103min-1)
kcat /Km (x103mM-1min-1)
0.28±0.01
1.32
4.71
0.61±0.02
0.96
1.61
0.28±0.05
0.18
0.64
α-naphthyl
propionate
Km (mM)
kcat (x103min-1)
kcat /Km (x103mM-1min-1)
0.71±0.05
0.36
0.51
2.06±0.67
0.36
0.17
0.24±0.02
0.05
0.21
p-nitrophenyl
acetate
Km (mM)
kcat (x103min-1)
kcat /Km (x103mM-1min-1)
2.44±0.31
6.36
2.60
6.35±0.50
26.50
4.17
3.70±0.46
1.44
0.39
Phenyl
thioacetate
Km (mM)
kcat (x103min-1)
kcat /Km (x103mM-1min-1)
3.30±0.42
6.14
1.86
6.46±0.51
29.30
4.53
2.14±0.16
1.35
0.63
Results displayed are mean of three individual experiments
Standard assay condition: 50 mM phosphate pH 7.5; varied [substrate]; 2 µg purified
enzyme; at 25 °C.
Kinetic parameters of AnAEst and its active site mutants
Wild-type shows highest affinity and catalytic efficiency to 1-NA
R54G shows highest affinity to 1-NA whereas highest catalytic efficiency to PTA
L86A shows highest affinity to 1-NP whereas highest catalytic efficiency to 1-NA

21. 21
Enzyme Accessible
surface area
(Å2)
Cavity
volume
(Å3)
Cavity
length
(Å)
WT
R54G
L86A
21.9
21.9
32.2
3.3
3.3
8.8
27.5
27.5
38.8
Active site dimensions of AnAEst and its mutants
Rationale for different substrate specificities of mutants
 Different binding modes of
phenyl and naphthyl esters
 Location of R54 and salt
bridge formation with E92
 Conversion of Michaelis
complex to tetrahedral
complex could involve
movement of amide protons of
R54 during oxyanion formation
PDB ID 1z8h
L86
R54
S17
E92
1NA
Y128
D179
H182
F18
N87
R54 and L86 are important in substrate binding and catalysis
Bakshy K, Gummadi SN, Manoj N, Biochim Biophys Acta. 2009, 2:324-334.

22. Biophysical characterization of
AnAEst
pH and thermal stability
22

23. 23
Results displayed are mean of three individual experiments
Thermal deactivation of AnAEst
 Process of deactivation is irreversible
 The enzyme follows first order deactivation kinetics
Enzyme was incubated for different time periods at different combinations of pH and temperature
whose residual activity was measured at standard assay conditions
Standard assay condition: 50 mM sodium phosphate (pH 7.5); substrate-0.6mM 1-NA; 2 µg purified enzyme;
at 25 °C.
tk
t
d
eEE 
 )( 0
DE dk


24. 24
Low pH, low temp. - 80-100 % residual activity
High pH, low temp. – 25-75 % residual activity
At 50 °C, as pH increases residual activity
decreases from 75-25 %
At 60 °C, 2 % activity remains at all pH within
2 hrs
The lines are fitted to first order deactivation
kinetic equation with R2>0.9
Thermal deactivation of AnAEst

25. 25
 The deactivation rate constant (kd) can be
obtained from the slope of the plot
ln(Et/E0) vs Time
 Half-life was calculated from the Eq.
below:
 Half-life of the enzyme decreases with
increase in pH and temperature.
 Maximum half-life was observed at 30 °C
and pH 5.5 indicating its maximum stability
at these conditions
Optimum conditions of activity and stability for AnAEst are different
Optimum activity conditions : pH 7.5, 25-45 °C
Optimum stability conditions : pH 5.5, 25-45 °C
dk
t
693.0
2/1 
Thermal deactivation of AnAEst

26. 26
DSC was performed to monitor the structural stability or thermal unfolding of
AnAEst, but the protein tends to aggregate beyond 70 °C
Transition peak, Tp at pH 5.5 and 7.5 are 64.5 and 60.2 °C respectively
Effect of pH on Molar heat capacity of wild-type AnAEst

27. 27
Structural stability : CD spectra
•The residual secondary structures correspond to the residual activity of
the protein
•Near UV-CD spectra showed presence of tertiary structures at all
conditions
•Complete loss in secondary structures was not observed so what is
happening to the microenvironment of the aromatic residues ?
pH 5.5

28. 28
Decrease in intrinsic Trp fluorescence along with a red shift
indicates exposure of Trp to polar solvent
Structural stability : Trp emission spectra
Enzyme was incubated for different time periods at different combinations of pH and temperature whose
residual fluorescence was measured at pH 7.5 and 25 °C with excitation wavelength of 290 nm

29. 29
Structural stability : fourth derivative spectra
 To determine the microenvironment of
other aromatic residues such as Tyr and Phe
 UV absorption spectra of the incubated
protein was recorded which was converted to
4th derivative spectra
Peak at 260 nm – Phe
275 nm – Tyr
292 nm - Trp
 Decrease in peak intensity was observed with increase in temperature
at 260 nm, 275 nm and 292 nm
 Microenvironment of the aromatic residues becomes more polar
 This indicates opening up of the enzyme structure

30. 30
Protein dynamics-Tryptophan quenching
 Slope of the plot F0/F vs quencher concentration gives Ksv, Stern-Volmer constant
 Linear plots – static/dynamic quenching; positive deviation from linearity – static and
dynamic quenching
 Modified Stern-Volmer equation for positive deviation from linearity:
 QK
F
F
sv10
0
1 [ ]app
F
K Q
F
     0 1
1
[ ]
app D S D S
F
K K K K K Q
F Q
 
     
 

31. 31
Protein dynamics-Tryptophan quenching
Enzyme was incubated for 1hr at different combinations of pH and temperature and titrated with the
quencher at pH 7.5 and 25 °C. ‘*’ Indicates Kapp or Ksv’
Acrylamide
 Ksv at all temperatures for pH 5.5 > 7.5 and 9.5 states indicating higher
diffusion of acrylamide through the protein matrix
 At pH 5.5, Ksv remains constant with increase in deactivation
temperature indicating nearly same extent of quenching
 Fluorescence studies indicate that the enzyme states incubated at pH
5.5 is blue shifted (~2-4 nm) in comparison with those incubated at pH 9.5
indicating buried Trp

32. 32
KI
Protein dynamics-Tryptophan quenching
Enzyme was incubated for 1hr at different combinations of pH and temperature and titrated with the
quencher at pH 7.5 and 25 °C. ‘*’ Indicates Kapp or Ksv’
• Larger Ksv values at all temperatures observed for pH 5.5 > 7.5 and 9.5
• Varying Ksv trends observed for different pH states
• At pH 5.5, Ksv remained constant with increase in deactivation temperature
but with a sharp increase at 60 °C
• This varying behavior of quenching by KI at different pH states can be
attributed to the varying charge around the microenvironment of Trp
residues
Electrostatic interactions seem to play a crucial
role in determining the structural stability of
AnAEst
What happens to the hydrophobic regions of the protein ?

33. 33
Structural stability : ANS binding spectra
 With increase in temperature, ANS binding
increases indicating increased exposure of
hydrophobic regions on the protein
 At 60 °C, ANS binding decreases with
increase in pH
 Maximum hydrophobic patches can be
observed at pH 5.5 and 60 °C
60 °C
•Size exclusion analysis of AnAEst after incubating at pH 5.5, 7.5 and 9.5
separately at 45 and 60 °C revealed that the protein exists as a dimer
•This indicates that the protein exhibits a high degree of conformational
plasticity in its core dimeric structure

34. Conclusions
• Enzyme is stable at pH 5.5 from 25-45 °C,
for 6-8 hrs and follows a first order
deactivation kinetics
• Thermal deactivation occurs as a result of
protein unfolding gradually exposing the
hydrophobic regions of the protein
• The highest thermal stability of AnAEst
exposed to pH 5.5 is mostly due to the
global conformational changes involving
unique ionic interactions
34

35. 35
Crystallization of Wt-AnAEst
 Crystals were observed in 0.1 M MOPS pH 6.8, 11 % (w/v)
PEG 4000 and isopropanol 9 & 10 % (v/v) at 21 °C.
 Crystal fine screens were set up to reproduce the
previously formed crystals of AnAEst.
 Various ratios of reservoir solution: protein was also used
(1:1, 1:2, 2:1) at the above mentioned conditions.
Crystals were observed in the fine screens after about 3
months at almost the same conditions.
 0.1 M MOPS pH 6.6, 11 % (w/v) PEG 4000 and isopropanol
9, 11 and 13 % (v/v) at 21 °C
1) 0.1 M HEPES sodium pH 6.8, 10% (v/v)
isopropanol, 11% (w/v) PEG 4000, 4°C, 25 mg/ml
protein conc.
2) 0.1 M MOPS pH 6.8, 11 % (w/v) PEG 4000 and
isopropanol 9 & 10 % (v/v) at 21 °C

36. 36
Acknowledgements
 The Department of Science and Technology, New Delhi, India.
 The Bioinformatics Infrastructure Facility at IITMadras
 The Genomics Institute of the Novartis Research Foundation, USA, for their
kind gift of the clone of AnAEst
Department of Biotechnology, IITMadras
 HOD – Prof K. B. Ramachandran
 Prof G. K. Suraish Kumar
 Supervisor: Dr. Manoj Narayanan
 Doctoral committee members
 Dr. G. Satyanarayana Naidu
 Dr. A. Gopalakrishna
 Prof D. Loganathan – Department of Chemistry, IITMadras
 Prof A. K. Mishra - Department of Chemistry, IITMadras
 Dr. V. Kesavan, Department of Biotechnology, IITMadras
 Prof K. Suguna and group – MBU, IISc, Bangalore
 Dr. R. Sankaranarayanan and group– CCMB, Hyderabad
 Prof Shekar C. Mande – CDFD, Hyderabad
 Prof M.J. Swamy and group– Hyderabad University, Hyderabad
 Friends and labmates: Sirisha, Navin, Ravi, Santosh, Harshavardhan, MJ,
Madhavi, Sai Krishna, Prashant, Prabhahar, Vidya, Vipin, Jayakumar, Abhipsa,
Shyam, Swati, Santosh, Aneesh and others
 Family

37. 37
THANK YOU