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Rinkoo D. Gupta, Ph.D.Faculty of Life Sciences and Biotechnology
South Asian UniversityNew Delhi
Metal-Based Enzyme promiscuity of Human Senescence Marker Protein 30
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What is Enzyme Promiscuity?
One Enzyme
One Substra
te
One Reactio
n
“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
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PON1 SMP30Only ~38% sequence similarity
Physiological function:Lactonase: Hydrolysis of oxidized lipids
Secondary Function: OP hydrolase
Physiological function:Lactonase: Vitamin C biosynthesis
Secondary Function: OP hydrolase
Gupta RD et.al. (2011) Nature Chem
Biol.
Designed for nerve agent hydrolysis, ~86000 fold higher catalytic activity
Metal binding proteins
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SMP30SMP30 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”.
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Vitamin C biosynthesis by Lactonase activityLactones are cyclic esters, a condensation product of an –OH group and –COOH group in the same molecule
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
What is a physiological function of SMP30 in primates?
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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 stress-
0.1-1 uM 1000 uMStress
What is the role of SMP30 during resting conditions?
Does it bind to other divalent cations and perform different functions?
During stress condition, SMP30 appears to regulate Ca2+-pumping activity via interactions with calmodulin and thus protects the cells from apoptosis.
Chakraborti and Bahnson, 2010,
Biochemistry
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Organophosphate Hydrolysis OPs are esters of phosphoric acid.R1 and R2 = generally alkyl and/or alkoxy groupsX = leaving group for e.g. -F and -CN
Scott and Bahnson, 2011, Biomol concepts
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Metal dependent activities of SMP30
In resting condition---
Lactonase/OP hydrolase
During stress—Ca2+
homeostasis ?
What is the role of different divalent cations in lactonase and OP hydrolase activities of SMP30?
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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.
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Bacterial expression of SMP30
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Purification of SMP30 by affinity chromatography
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Esterase activity of huSMP30
0 1 2 3 4 5 6 7 8 9 100
0.05
0.1
0.15
0.2
0.25
0.3
ContHusmp NA
Time (mins)Ab
sorb
ance
( a
t 32
0 nm
)
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0
0.5
1
1.5
2
2.5
3
3.5
4
experimentcontrol
Ca Co Mn Ni Mg 00.5
11.5
22.5
33.5
44.5
Experimentcontrol
00.5
11.5
22.5
33.5
44.5
experimentcontrol
Ca Co Mn Ni Mg 0
0.51
1.52
2.53
3.54
4.5
experiment
control
Ca Co Mn Ni Mg
Phenyl Acetate
Napthyl Butyrate Demeton S
Thiobutyrolacton
Activity performed in the presence of different divalent cations
Ca Co Mn Ni Mg
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AcknowledgmentsProf. Rajiv K. Saxena, Dean, FLSBSouth Asian University
Financial Supports:South Asian University, New DelhiDST, India
My students:Abhishek Singh RathoreRoshan K. DuttaDrishti AgarwalRohan AroraPallavi PrasadGeetika Sharma
Alka SinghBhanuja TripathiGhulam Reza HazareParmanand PandeyShishir
Life Supports:Shobhna & Shubham
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THANK YOU