session 28 ic2011 jenkins
DESCRIPTION
TRANSCRIPT
Katie M. Jenkins
Department of Forest Products, Mississippi State University
USDA FS, Forest Products Laboratory, Madison, WI
Enzymatic Mechanism of Oxalate Production in the TCA
and Glyoxylate Pathways using Antrodia radiculosa
Objective
Gain insight into the
biosynthesis of
oxalate in order to
understand the
mechanism utilized
by varying isolates of
Antrodia radiculosa
2
Background
Copper-tolerant fungi produce and accumulate
HIGH levels of oxalate
Oxalate biosynthesis has been linked to the
Glyoxylate and Tricarboxylic Acid (TCA)
Pathways
Oxalate production relies on twelve specific
enzymes
Two major oxalate producing enzymes:
Glyoxylate Dehydrogenase
Oxaloacetase
3Munir, et al., 2001. PNAS 98(20): 11126-11130.
4
Methods
Treatment: Untreated (Controls)
1.2% Copper Citrate
Timeline: 2 weeks
4 weeks
6 weeks
8 weeks
Oxalate Analysis: Trinity Biotech Reagents
560nm
Antrodia radiculosa
Isolates
FP-90848-T
L-11659-SP
TFFH 294
L-9414-SP
5
Methods
Enzyme Analysis
Prepping the sample
Rasped into saw dust
~ 0.2g
Frozen in liquid N2
Stored at -80°C
Determining Activity
Spectrophotometrically
Biotech Epoch
96-well plates
UV
VIS
Enzyme Wavelength
Fumarase 240nm
Malate Synthase 412nm
Oxaloacetase 255nm
Isocitrate Lyase 324nm
Glyoxylate Dehydrogenase 550nm
6
7
RESULTS – OXALATE
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
2 Weeks 4 Weeks 6 Weeks 8 Weeks
Co
nc
en
tra
tio
n (
mg
/mL
)
Oxalate Concentration
L-11659-SP Copper Citrate
L-11659-SP Untreated
FP-90848-T Copper Citrate
FP-90848-T Untreated
L-9414-SP Copper Citrate
L-9414-SP Untreated
TFFH 294 Copper Citrate
TFFH 294 Untreated
8
RESULTS – ISOCITRATE LYASE ACTIVITY
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0 10 20 30 40 50 60
Ch
an
ge
in
Ab
so
rban
ce
/Min
ute
Enzyme Filtrate (ul)
ISOCITRATE LYASE ACTIVITY: TFFH 294
Copper Citrate Week 2
Untreated Week 2
Copper Citrate Week 4
Untreated Week 4
Copper Citrate Week 6
Untreated Week 6
Copper Citrate Week 8
Untreated Week 8
9RESULTS – GLYOXYLATE DEHYDROGENASE ACTIVITY
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0 10 20 30 40 50 60
Ch
an
ge
in
Ab
so
rban
ce
/Min
ute
Enzyme Filtrate (ul)
GLYOXYLATE DEHYDROGENASE ACTIVITY: TFFH 294
Copper Citrate Week 2
Untreated Week 2
Copper Citrate Week 4
Untreated Week 4
Copper Citrate Week 6
Untreated Week 6
Copper Citrate Week 8
Untreated Week 8
10
RESULTS – FUMARASE ACTIVITY
-0.1
0
0.1
0.2
0.3
0.4
0.5
0 10 20 30 40 50 60
Ch
an
ge
in
Ab
so
rban
ce
/Min
ute
Enzyme Filtrate (ul)
FUMARASE ACTIVITY: TFFH 294
Copper Citrate Week 2
Untreated Week 2
Copper Citrate Week 4
Untreated Week 4
Copper Citrate Week 6
Untreated Week 6
Copper Citrate Week 8
Untreated Week 8
11
RESULTS – MALATE SYNTHASE ACTIVITY
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0 10 20 30 40 50 60
Ch
an
ge
in
Ab
so
rban
ce
/Min
ute
Enzyme Filtrate (ul)
MALATE SYNTHASE ACTIVITY: TFFH 294
Copper Citrate Week 2
Untreated Week 2
Copper Citrate Week 4
Untreated Week 4
Copper Citrate Week 6
Untreated Week 6
Copper Citrate Week 8
Untreated Week 8
In general, oxalate concentration was highest
at Week 6 for all isolates treated with copper
citrate.
Exceptions:
L-11659-SP – Week 2
FP-90848-T – Week 8
No observable variation in oxalate production
between untreated and copper citrate treated
blocks.
Week 8
12
Discussion - Oxalate
Isocitrate Lyase and Glyoxylate Dehydrogenase activity
Specific Activity and Total Protein
Fumarase and Oxaloacetase show NO activity
Malate Synthase showing odd behavior when treated with copper citrate
Untreated samples have NO activity
Possible substrate inhibition
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Discussion – Enzymatic Analysis
Theoretically, oxalate production relies on
Isocitrate Lyase and Glyoxylate Dehydrogenase
14
Conclusion
1515
Future Objectives
Evaluate Protein Concentration
Gene expression analysis
Copper Influence
Citrate
16
Special Thanks
Forest Products Laboratory - Madison, WI
Carol Clausen
Rick Green
Mississippi State University
Dr. Susan Diehl
Dr. Ken Willeford
Juliet Tang
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