1. Enzymatic Mechanism of Oxalate Production in the TCA
and Glyoxylate Pathways using Antrodia radiculosa
Katie M. Jenkins
Department of Forest Products, Mississippi State University
USDA FS, Forest Products Laboratory, Madison, WI

2. Objective
 Gain insight into the
biosynthesis of
oxalate in order to
understand the
mechanism utilized
by varying isolates of
Antrodia radiculosa
2

3. Background
 Copper-tolerant fungi produce and accumulate
HIGH levels of oxalate
 Oxalate biosynthesis has been linked to the
Glyoxylate and Tricarboxylic Acid (TCA)
Pathways
 Oxalateproduction relies on twelve specific
enzymes
 Two major oxalate producing enzymes:
 GlyoxylateDehydrogenase
 Oxaloacetase
Munir, et al., 2001. PNAS 98(20): 11126-11130. 3

4. 4

5. Methods
Antrodia radiculosa
 Treatment: Isolates
 Untreated (Controls) FP-90848-T
 1.2% Copper Citrate L-11659-SP
 Timeline: TFFH 294
 2 weeks L-9414-SP
 4 weeks
 6 weeks
 8 weeks
 Oxalate Analysis:
 Trinity Biotech Reagents
 560nm
5

6. Methods
 Enzyme Analysis Enzyme Wavelength
 Prepping the sample Fumarase 240nm
 Rasped into saw dust Malate Synthase 412nm
 ~ 0.2g Oxaloacetase 255nm
 Frozen in liquid N2 Isocitrate Lyase 324nm
 Stored at -80°C Glyoxylate Dehydrogenase 550nm
 Determining Activity
 Spectrophotometrically
 Biotech Epoch
 96-well plates
 UV
 VIS
6

7. 7
RESULTS – OXALATE
Oxalate Concentration
1.4
1.2
1.0
Concentration (mg/mL)
L-11659-SP Copper Citrate
0.8 L-11659-SP Untreated
FP-90848-T Copper Citrate
FP-90848-T Untreated
L-9414-SP Copper Citrate
0.6 L-9414-SP Untreated
TFFH 294 Copper Citrate
TFFH 294 Untreated
0.4
0.2
0.0
2 Weeks 4 Weeks 6 Weeks 8 Weeks

8. 8
RESULTS – ISOCITRATE LYASE ACTIVITY
ISOCITRATE LYASE ACTIVITY: TFFH 294
1.1
1.0
0.9
0.8
Change in Absorbance/Minute
0.7
Copper Citrate Week 2
0.6 Untreated Week 2
Copper Citrate Week 4
0.5 Untreated Week 4
Copper Citrate Week 6
Untreated Week 6
0.4
Copper Citrate Week 8
Untreated Week 8
0.3
0.2
0.1
0.0
0 10 20 30 40 50 60
Enzyme Filtrate (ul)

9. 9
RESULTS – GLYOXYLATE DEHYDROGENASE ACTIVITY
GLYOXYLATE DEHYDROGENASE ACTIVITY: TFFH 294
0.35
0.30
Change in Absorbance/Minute
0.25
Copper Citrate Week 2
0.20
Untreated Week 2
Copper Citrate Week 4
Untreated Week 4
0.15 Copper Citrate Week 6
Untreated Week 6
Copper Citrate Week 8
Untreated Week 8
0.10
0.05
0.00
0 10 20 30 40 50 60
Enzyme Filtrate (ul)

10. 10
RESULTS – FUMARASE ACTIVITY
FUMARASE ACTIVITY: TFFH 294
0.5
0.4
Change in Absorbance/Minute
0.3
Copper Citrate Week 2
Untreated Week 2
Copper Citrate Week 4
0.2 Untreated Week 4
Copper Citrate Week 6
Untreated Week 6
Copper Citrate Week 8
Untreated Week 8
0.1
0
0 10 20 30 40 50 60
-0.1
Enzyme Filtrate (ul)

11. 11
RESULTS – MALATE SYNTHASE ACTIVITY
MALATE SYNTHASE ACTIVITY: TFFH 294
0.16
0.14
0.12
Change in Absorbance/Minute
0.10
Copper Citrate Week 2
Untreated Week 2
0.08 Copper Citrate Week 4
Untreated Week 4
Copper Citrate Week 6
0.06 Untreated Week 6
Copper Citrate Week 8
Untreated Week 8
0.04
0.02
0.00
0 10 20 30 40 50 60
Enzyme Filtrate (ul)

12. Discussion - Oxalate
 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

13. Discussion – Enzymatic Analysis
 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
13

14. Conclusion
 Theoretically, oxalate production relies on
Isocitrate Lyase and Glyoxylate Dehydrogenase
14

15. 15
15

16. Future Objectives
 Evaluate Protein Concentration
 Gene expression analysis
 Copper Influence
 Citrate
16

17. Special Thanks
 Forest Products Laboratory - Madison, WI
 Carol Clausen
 Rick Green
 Mississippi State University
 Dr. Susan Diehl
 Dr. Ken Willeford
 Juliet Tang
17