session 28 ic2011 jenkins

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

13

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

17