# Presentation: 187999

## Revisiting the central pathway in Desulfovibrio vulgaris Hildenborough using GC-MS and FT-ICR mass spectrometry

- Yinjie Tang,  Keasling Group, Lawrence Berkeley Lab
- Oct 8th, 2006

**Pyr**

**Oxa**

**Citr**

**Suc**

**Fum**

**Oxo**

**TCA**

- An environmental important anaerobe

- Mass spectrometer

- Pathways

## ?

## Objectives 

**Genome *****Desulfovibrio vulgaris***** Hildenborough is recently sequenced, which raised questions**
- Complete amino acids and pentose phosphate pathway?
- Atypical citrate synthase?
- Acetate oxidation?
- α-ketoglutarate dehydrogenase vs ferredoxin-dependent α-ketoglutarate synthase (TCA cycle might be able to function oxidatively)?
**Develop GC/MS and FT-ICR methods to investigate metabolic pathways and quantify metabolic fluxes**
- GC/MS can only measure general isotopomer pattern.
- NMR method is not very sensitive and very slow.
- FT can locate labeled C13 position in the targeted molecule.

- Heidelberg et al., 2004, Nature biotechnology, 22(5), pp554-559

## Annotated pathway map and experimental design(Answer first question: DvH can grow in the completely minimal L-Lactate medium, indicating they have a complete biosynthesis pathway!)

- Batch culture with 1-C13 labeled lactate in the anaerobic hood

**Glutamate**

**Oxaloacetate**

**Citrate**

**Isocitrate**

******-Ketoglutarate**

**Succinate**

**Malate**

**Acetyl-CoA****           ****Acetate**
**	****	**
**	****          ****Lipid**

**Aspartate**

**Ala**

- EMP and PP pathway

- C-C

** ****C-C-****C****OOH**

- PEP

- Lactate

**PYR**

- CO2

**Phe**

- TCA cycle

## Method and materials

- 1. Acetate and Lactate are measured enzymatically.
- 2. GC/MS are used to analyze the isotopomer distribution
-      -Lactate, pyruvate and succinate were derivatized by BSTFA.
-      -Amino acids were derivatized by TBDMS.
- 3. Total biomass, protein, fatty acid, amino acid composition, RNA, DNA and carbohydrate are also measured
- 4. An flux model was developed to evaluate the fluxes through central pathway.

## Results: Growth kinetics

| Growth condition | Yx/c
g/mole | YP/C 
g protein /mole | YSO4/Lac
mol/mol | t1/2 hr* | μmax hr-1 | Ks
mM | End product, mol/mol lactate |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Minimal lactate medium | 3.1 ± 0.9 | 0.93 ± 0.27 | 0.55 
± 0.09 | 8.2     ±2.0 | 0.14 ±0.01 | 28 ±2 | acetate: 0.88± 0.06
alcohol: <0.002 |

**Growth curve of D. vulgaris in minimal L-lactate medium. ◊: OD600; ■: protein concentration; solid line: Model fitting.**

**Lactate and sulfate consumption, acetate production curves in minimal L-lactate medium.  ◊: lactate; □: acetate; ▲: sulfate; solid line: model fitting**

**Parameters of growth kinetics in L- lactate medium.**

## GC-MS measurement of protein hydrolysate from DvH. 

- GC-MS spectrum of glutamate of Desulfovibrio *vulgaris *lysate.

- GC-MS measurement of protein hydrolysate from DvH.

## Result: fragment mass distribution of C13 labeled metabolites

|  | Fragment | M0 | M1 | M2 | M3 | C13 enriched position  |
| --- | --- | --- | --- | --- | --- | --- |
| Glycine | No loss | 0.18 | 0.82 |  |  | carboxyl group |
| Serine  | No loss | 0.18 | 0.80 | 0.01 |  | carboxyl group |
|  | Loss of COOH | 0.98 | 0.02 | 0 |  |  |
| Alanine | No loss | 0.17 | 0.83 |  |  | carboxyl group |
|  | Loss of COOH | 0.97 | 0.03 |  |  |  |
| Glutamate | No loss | 0.21 | 0.76 | 0.03 |  | carboxyl group |
|  | Loss of COOH | 0.23 | 0.77 | 0 |  |  |
| Asparate | No loss | 0.05 | 0.27 | 0.68 |  | α or / and γ carboxyl group |
|  | Loss of COOH | 0.20 | 0.80 | 0 |  |  |
| Methionine | No loss | 0.04 | 0.24 | 0.65 | 0.01 | carboxyl group |
|  | Loss of COOH | 0.18 | 0.75 | 0.03 | 0.04 |  |

## Table continued.   

| Histidine | No loss | 0.20 | 0.58 | 0.08 | 0.13 | The carboxyl group is NOT enriched. |
| --- | --- | --- | --- | --- | --- | --- |
|  | Loss of COOH | 0.17 | 0.62 | 0.09 | 0.11 |  |
| Phenylalanine | No loss | 0.03 | 0.12 | 0.35 | 0.49 | The carboxyl group |
|  | Loss of COOH | 0.08 | 0.32 | 0.56 | 0.03 |  |
| *Lactate | No loss | 0.12 | 0.86 | 0.02 |  | carboxyl group |
|  | Loss of COOH | 0.97 | 0.02 | 0.01 |  |  |
| *Pyruvate | No loss | 0.10 | 0.87 | 0.03 |  | carboxyl group |
|  | Loss of COOH | 0.95 | 0.03 | 0.01 |  |  |
| *Succinate | No loss | 0.11 | 0.22 | 0.68 |  | α or / and γ carboxyl group |

- Table continued.

- Note: GC/MS provided general information on labeled pattern, but can not tell which position the carbon is labeled at.

- M0     M1       M2     M3

## FT-ICR technology for isotopomer analysis

- FT-ICR** **MS of DvH* *hydrolysate.                         b) Relative abundance of amino acids in cell
-                                                                                      hydrolysate (leucine/isoleucine is the base).

## Mass spectrum for glutamic acid  fragmentation

- Dissociation pathway of glutamic acid

- 1. FT-ICR can not tell leucine and isoleucine.
- 2. FT-ICR is not sensitive to glycine (Low molecular weight).

## Discussion 1: Atypical citrate synthase

- Citrate reacts asymmetrically because aconitase has the active site that is asymmetric: the enzyme have a specific three binding points to citrate (Alexander Ogston, 1948).

- CH314CO-CoA

- Labeled acetate

- Oxaloacetate

- +

- CH214COOH

-      CH2

-     O=CCOOH

- CO2

- Aconitase

- -ketoglutarate

-        Isocitrate

## FT-ICR detected unexpected labeled pattern in glutamate: suggesting stereospecificity of (R)-citrate synthase in some anaerobic sulfate-reducing and photosynthetic bacteria:

- FT-ICR detected unexpected labeled pattern in glutamate: suggesting stereospecificity of *(R)-*citrate synthase in some anaerobic sulfate-reducing and photosynthetic bacteria:
- Including: *Clostridium kluyveri, Clostridium acidi-urici, Clostridium cylindrosporum *

- First discovered by Gottschalk, 1966-1967

## Discussion 2: A complete TCA cycle?

- no α-ketoglutarate dehydrogenase,
-                 α-ketoglutarate               succinyl-CoA        (a broken TCA loop)
- ferredoxin-dependent α-ketoglutarate synthase (a close TCA cycle)
-   α-ketoglutarate + ferredoxinox + CoA  succinyl-CoA + CO2 + ferredoxinoxred

- X

## X

- Oxa (C-C-C-COOH)

- Suc (C-C-C-COOH)

- AcetylCoA
- (C-C-CoA)

- Citrate C-C-C-C-COOH
-                    C

- Oxo (C-C-C-C-COOH)

- TCA cycle

- CO2

- Succinate is the key!

- CO2

- Asp

- Glu

- TCA cycle is broken in DvH metabolism *(unlike shewanella oneidensis MR1).*
- Activity of ferredoxin-dependent α-ketoglutarate synthase is disproven.

- M0=0.05;
- M1=0.27;
- M2=0.68;
- M3=0;

- M0=0.10;  M1=0.87;  M2=0.03; M3=0

- M0=0.11; M1=0.22; M2=0.68

- M0=0.21; M1=0.76; M2=0.03; M3=0

- CO2

## Discussion 3: New acetyl-CoA oxidation route is confirmed 

- Leucine	  No loss                           0.69      0.29        0.02
-                 Loss of COOH               (0.97)    (0.02)      (0.01)

- Carboxyl group is labeled!!!

- (CH3)2CHCH2  _**C**__(NH__2__) __12/13__**C**__OOH_

- 12**CH**3           12/13**C**O-CoA

**C****H****3****X**

- 12/13**[CO]**

-   HCOOH            CO2

- 12/13**CO****2**

- 12**C**H3-12**C**O-13**C**OOH

- 13CO2

- 1-C13 Lactate

- Acetyl-CoA

- Leucine

- Bound carbon monoxide

- Bound methanol

- Lactate/Acetate oxidation to CO2 via a novel pathway not involving reactions of the citric acid cycle (Schauder et al., 1986)

- [H]

- NADH+ATP

- NADH

- All enzymes are annotated: carbon monoxide dehydrogenase,  formate dehydrogenase
- methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase

## Discussion 4: Flux distribution in D. vulgaris

- Discussion 4: Flux distribution in *D. vulgaris*
- A dotted arrow indicates that the reaction has zero flux.
- An ‘x’ indicates the absence of an annotated gene for the step.
- The data in brackets are the exchange coefficients

**X**

**X**

**X**

## Summary of this project

- 1. Develop a method using mass spectrometer (GC/MS and FT-ICR) to efficiently investigate the metabolic pathway using 13C isotopic labeling.
- Traditional techniques: NMR, GC-MS, LC-MS
- Limitations:
- NMR—Not sensitive (can’t work for certain labeling pattern).
- GC-MS---derivatization (measure total mass distribution).
- LC-MS– reproducible separation is not always possible (few examples)
- 2. Revisit the *(R)-*citrate synthase using in vivo tracer experiment.
- 3. Disprove activity of ferredoxin-dependent α-ketoglutarate synthase.
- 4.Confirm the new pathway for acetly-CoA oxidization and incomplete PP pathway
- 5. Calculate flux distribution through anaerobic pathway in DvH.

## Acknowledgement 

- Jay
- Terry Hazen (Richard, Rick and Romy)
- Francesco
- Aindrila
- Judy Wall
- Jeannie