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Transcript
Rinkoo D. Gupta, Ph.D.
Faculty of Life Sciences and Biotechnology
South Asian University
New Delhi
What is Enzyme Promiscuity?
“The term ‘promiscuous’ is used to describe enzymes that catalyze
more than one reaction”
“Enzyme promiscuity is an ability to catalyze secondary reactions that
are physiologically irrelevant”
 Substrate Promiscuity
 Catalytic Promiscuity
 Alternative Cofactors
Copley S.D. (2015), Trends in Biochemical Sciences
PON1
Metal binding proteins
SMP30
Only ~38% sequence
similarity
Physiological function:
Lactonase: Hydrolysis of oxidized
lipids
Physiological function:
Lactonase: Vitamin C biosynthesis
Secondary Function: OP hydrolase
Secondary Function: OP hydrolase
Designed for nerve agent hydrolysis,
~86000 fold higher catalytic activity
Gupta RD et.al. (2011) Nature Chem Biol.
SMP30
 SMP30 was reported as a marker protein for ageing where
concentration decreases with the age.
 It is positioned on the X-chromosome, consists of seven
exons and six introns and has an open reading frame of 897
bp which encodes for 299 amino acids long protein.
 70-90% of amino acid sequence identity was observed among
vertebrates, which is evocative of an essential biological
function.
 It is also reported as Ca2+-binding protein involved in the
regulation of free cellular Ca2+ , hence also named as
“Regucalcin”.
Vitamin C biosynthesis by Lactonase activity
Lactones are cyclic esters, a condensation product of an –OH group and
–COOH group in the same molecule
What is a physiological function of SMP30 in primates?
The final 3 steps of the biosynthesis of L-ascorbate from D-glucuronate. In
mammals lacking the ability to synthesize ascorbate, gulonolactone oxidase
(GULO) is non-functional and highly mutated.
Scott and Bahnson, 2011, Biomol concepts
Role of SMP30 in Ca2+ binding
• Metalloenzyme, known to bind with Ca2+ and play an
important role in Ca2+ homeostasis.
• Cellular Ca2+ level goes high during stress0.1-1 uM
Stress
1000 uM
During stress condition, SMP30 appears to regulate Ca2+-pumping
activity via interactions with calmodulin and thus protects the cells
from apoptosis.
What is the role of SMP30 during resting conditions?
Does it bind to other divalent cations and perform
different functions?
Chakraborti and Bahnson, 2010,
Biochemistry
Organophosphate Hydrolysis
OPs are esters of phosphoric acid.
R1 and R2 = generally alkyl and/or alkoxy
groups
X = leaving group for e.g. -F and -CN
Scott and Bahnson, 2011, Biomol concepts
Metal dependent activities of SMP30
What is the role of different divalent cations in
lactonase and OP hydrolase activities of SMP30?
In resting condition-Lactonase/OP
hydrolase
During stress—
Ca2+ homeostasis ?
Cloning of huSMP30 and
MoSMP30
Cloning of mouse SMP30 ORF in
pJET1.2/blunt cloning vector.
Cloning of mouse SMP30 ORF
in Bacterial expression pET28(a)
vector.
Bacterial expression of SMP30
Purification of SMP30 by affinity
chromatography
Esterase activity of huSMP30
Control
(without
IPTG)
Experimental
(With IPTG)
Esterase
activity
CaCl2
0.001
0.003
+
CoCl2
0.09
0.185
++++
MnCl2
0.002
0.003
+
MgCl2
0
CdCl2
0
0
0.3
Absorbance ( at 320 nm )
Metals
0.25
0.2
0.15
Cont
Husmp NA
0.1
0.05
-0
0
--
0
1
2
3
4
5
6
Time (mins)
7
8
9 10
Activity performed in the presence of different
divalent cations
4
5
experiment
3.5
Experiment
4
control
3
control
3
2.5
2
2
1.5
1
1
0.5
0
0
Ca
Co
Mn
Ni
Mg
Ca
Co
Mn
Ni
Mg
Phenyl Acetate
Thiobutyrolacton
5
5
experiment
experiment
4
4
control
control
3
3
2
2
1
1
0
Ca
Co
Mn
Ni
Napthyl Butyrate
Mg
0
Ca
Co
Mn
Demeton S
Ni
Mg
Acknowledgments
Prof. Rajiv K. Saxena, Dean,
FLSB
South Asian University
Financial Supports:
South Asian University, New Delhi
DST, India
Life Supports:
Shobhna & Shubham
My students:
Abhishek Singh Rathore
Roshan K. Dutta
Drishti Agarwal
Rohan Arora
Pallavi Prasad
Geetika Sharma
Alka Singh
Bhanuja Tripathi
Ghulam Reza Hazare
Parmanand Pandey
Shishir