Microbiology II Microbial physiology II - uni-due.de · The Entner‐Doudoroff(ED) pathway • 1...

Microbiology IIMicrobial physiology II –Some principles and mechanisms in the the central carbon metabolism

Christopher Bräsen

Lecture Plan

17.10. 2017 Mikrobielle Physiologie I ‐ Energetik Bräsen

24.10. 2017 Mikrobielle Physiologie II – Einige Prinzipien und Mechanismen im zentralen Kohlenstoffmetabolismus

Bräsen

31.10. 2017 Keine Vorlesung Bräsen

07.11. 2017 Mikrobielle Physiologie III – Nitrat‐Atmung Bräsen

14.11. 2017 Mikrobielle Physiologie IV – Acetogenese und der Acetyl‐CoA/Kohlenmonoxid Dehydrogenase‐Weg

Bräsen

21.11. 2017 Mikrobielle Physiologie V – Anaerobe Nahrungskette und Methanogenese Bräsen

28.11. 2017 Mikrobielle Physiologie VI – Sulfate Reduktion Bräsen

05.12. 2017 Antibiotika (Penicillium notatum) Meckenstock

12.12. 2017 Mikroorganismen in der Umwelt (Geobacter metallireducens) Meckenstock

19.12. 2017 Mikrobielles Wachstum (Elusimicrobium minutum) Meckenstock

09.01. 2018 Mikrobielle Fortbewegung (Thioploca) Meckenstock

16.01. 2018 Viren (T4) Meckenstock

23.01. 2018 Geschichte der Mikrobiologie Meckenstock

30.01. 2018 Wrap up/Ausweichtermin Meckenstock/Bräsen

Questions 1

• In which two parts can the energy metabolism be devided?• How much energy is required to synthesize ATP from ADP and Pi

considering cellular concentrations of reactants?• Which are the two basal mechanisms of ATP synthesis and what are their

characteristics? • How much energy is approximately gained from one H+ flowing back into

the cell (e.g. E. coli)?• How can ATP yields be estimated if the energy yielding catabolic reaction

is known (overall efficiency of energy metabolism ~60%)?• What are energy rich intermediates? Give three examples.• How can ΔG values be calculated from Redoxpotentials, give the

equation?• Name the sites of SSP in glycolysis? Which enzyme catalyses the only

oxidation reaction in this pathway?• What is the frequently used cosubstrate in dehydrogenase catalyzed

reactions?

Example: Aerobic Glucose oxidation

Glucose = S1red. 6 O2 = S2ox.

6 CO2 = P1ox. 12 H2O = P2red.

ATP ATP

12 x 2[H]

Oxidative part Reductive part

ATP synthesis viaSubstrate levelphosphorylation(SSP)

ATP synthesis viaElectron transportphosphorylation(ETP)

Glucose (red.) + 6 O2 (ox.) → 6 CO2 (ox.) + 6H2O (red.)

Most energy yielding catabolic rections are redox reactions

Electron donating half reaction Electron accepting half reaction

Example: Aerobic Glucose oxidation

Glucose = S1red. 6 O2 = S2ox.

6 CO2 = P1ox. 12 H2O = P2red.

ATP ATP

12 x 2[H]

Oxidative part Reductive part

ATP synthesis viaSubstrate levelphosphorylation(SSP)

ATP synthesis viaElectron transportphosphorylation(ETP)

Glucose (red.) + 6 O2 (ox.) + 6 H2O → 6 CO2 (ox.) + 12 H2O (red.)

Most energy yielding catabolic rections are redox reactions

Electron donating half reaction Electron accepting half reaction

6 H2O

Aerobic glucose degradation

Glucose

6 O2

4 CO2

12 H2O

~34 ATP(ETP)

24 [H]

Oxidative part

Reductive part

Transport

Embden‐Meyerhof pathwayEntner‐Doudoroff pathway

Pyruvatedehydrogenasecomplex

Citric acid cycle

Glucose

2 Acteyl‐CoA

2 Pyruvate

4 [H]

4 [H]

16 [H]

2 CO2

1‐2 ATP(SSP)

2 ATP(SSP)

10 NADH → 100 H+

2 UQH2 → 12 H+

Respiratory chain

Σ 4 ATP (SLP) + 10 NADH + 2 UQH2

Σ ~38 ATP

Transport

ΔG = ΔµH+ = F ΔΨ – 2.3 RT ΔpH (i‐o)

ΔG = ΔµH+ = ~18 kJ/mol→ provides the energy to transport a substrate against a gradientof 1000; 50 kJ/mol = 1 ATP → concentration gradient 108

ΔG FΔΨ 2.3RTlgH out

H in

In case of a non electrogenic transport: ΔG 2.3RTlgS out

S in5.7

kJmol lg

S out

S in

A concentration gradient of 0.1 (10fold higher intracellular conc. of e.g. a growthsubstrate)

→ 5.7 kJ/mol has to be invested to transportthe substrate against this gradient of 10fold

ΔG 5.7kJmol lg

110 = ‐5.7 kJ/mol x ‐1

100fold → 11.4 kJ/mol1000fold → 17.1 kJ/mol

Sugar transport in Bacteria

H+out

Glucoseout

H+in

Glucosein

Glucoseout Glucosein

Maltoseout MaltoseinATP

ADP + Pi

K

K

F

GE

BA

HPr E1

CGlucoseout Glucose‐6‐P

PEP (=1 ATP)

Pyruvate

Membraneout + in ‐

Glucose‐6‐P

Glucose‐6‐P

= 1/3 ATP

ATP ADP

ATP ADP

Facilitated Diffusion

H+ Symport

PTS System

~P

ABC Transport

Rare in bacteria, e.g. glucose uptakeZymomonas mobilis, glycerol E. coli

Many aerobic bacteria

Many anaerobic, facultative bacteria

Widespread in all domains of life for a variety of substrates;

e.g. Maltose transporter in E. coli


Glucose

6 O2

4 CO2

12 H2O

~34 ATP(ETP)

24 [H]

Oxidative part

Reductive part

Transport



Citric acid cycle

Glucose

2 Acteyl‐CoA

2 Pyruvate

4 [H]

4 [H]

16 [H]

2 CO2

1‐2 ATP(SSP)

2 ATP(SSP)

10 NADH → 100 H+

2 UQH2 → 12 H+

Respiratory chain


Σ ~38 ATP

Glycolysis(Embden‐Meyerhof‐Parnas (EMP) patwhay)

Glycolysis (EMP patwhay)


Bar‐Even et al., 2012

Constraints:• preventing loss of carbon and energy→

Phosphorylation reduces permeability• C‐C bond cleavage is easier in β position

to carbonyl group• Avoiding toxic intermediates

Aims:• building blocks• Max. ATP gain (as much as possible)


1. Permeability

2. Aldol cleavage, C‐C bond cleavage iseasier in β position to carbonyl group

3. Permeability, both final C3 cleavageproducts are phosphorylated

5. building blocks, DHAP is a precursore.g. for lipid synthesis

4. Aldol cleavage, finally yields twoidentical C3 sugar phosphates (GAP)


6. Phosphate anhydride formation, NAD avoids toxic compound formation

7. Substrate level phosphorylation; 3PG isbuilding block in AA synthesis (Ser, Gly, Cys)

8.+9. Enables phosphate activation, andSLP in 10.; PEP is building block in AA synthesis (Tyr, Trp)

10. Substrate level phosphorylation; pyruvate is building block in AA synthesis(Ala, Leu, Ile, Lys, Val)

A second glycolytic route: The Entner‐Doudoroff (ED) pathway

• 1 ATP spent for Glucose phosphorylation• 2 ATP gained from the oxidation from one

molecule of GAP to pyruvate• Net yield 1 ATP and 2 NAD(P)H + H+

• 2‐keto‐3‐deoxy‐6‐phosphogluconate (KDPG) isthe key intermediate

• GAP is further oxidized by the same reactionsas in the EMP

The Entner‐Doudoroff (ED) pathway


• 1 ATP spent for Glucose phosphorylation• 2 ATP gained from the oxidation from one

molecule of GAP to pyruvate• Net yield 1 ATP and 2 NAD(P)H + H+

Avi Flamholz et al. PNAS 2013;110:10039-10044



• ED utilizers are in most cases aerobes (this does not mean that all aerobes areED utilizers)

• Some facultative anaerobes (e.g. E. coli) utilize this pathway for gluconatedegradation

• Widespread in strict aerobes like Pseudomonads

• Sugar transport as H+ symport• Exception Zymomonas mobilis


• 1 ATP spent for Glucose phosphorylation

• 2 ATP gained from the oxidation fromone molecule of GAP to pyruvate

• Net yield 1 ATP and 2 NAD(P)H + H+

• Widespread in strict aerobes like Pseudomonads

• Sugar transport as H+ symport• Exception Zymomonas mobilis


Glucose

6 O2

4 CO2

12 H2O

~34 ATP(ETP)

24 [H]

Oxidative part

Reductive part

Transport



Citric acid cycle

Glucose

2 Acteyl‐CoA

2 Pyruvate

4 [H]

4 [H]

16 [H]

2 CO2

1‐2 ATP(SSP)

2 ATP(SSP)

10 NADH → 100 H+

2 UQH2 → 12 H+

Respiratory chain


Σ ~38 ATP

Pyruvate dehydrogenase complex

• 3 Enzymes • 5 Coenzymes• Irreversible!


Glucose

6 O2

4 CO2

12 H2O

~34 ATP(ETP)

24 [H]

Oxidative part

Reductive part

Transport



Citric acid cycle

Glucose

2 Acteyl‐CoA

2 Pyruvate

4 [H]

4 [H]

16 [H]

2 CO2

1‐2 ATP(SSP)

2 ATP(SSP)

10 NADH → 100 H+

2 UQH2 → 12 H+

Respiratory chain


Σ ~38 ATP

The Citric acid cycle

• Acetyl‐CoA oxidation to CO2 andgeneration of reducing power → respiratory chain/energy via ETP (3 NADH + 1 UQH2 per acetyl‐CoA)

• 1 ATP/acetyl‐CoA via SSP:Enzyme + Succinyl‐CoA + Pi Enzyme(Succinyl~P) + HS‐CoA

Enzyme(Succinyl~P) Enzyme‐His246α~P + Succinate

Enzyme‐His246α~P + ADP E + ATP

• One C2 compound enters the cyleand two CO2 are formed→ the C2 compound is thus completelyoxidized

CH3CO‐S‐CoA + 3 H2O 2 CO2+ 8[H] + HS‐CoA


• The first step the aldolkondensationof acetyl‐CoA and oxaloacetate isirreversibel/highly exergonic ΔG0‘ = ‐32 kJ/mol

• Due to thioester hydrolysis• Not coupled to energy formation

CH3CO‐S‐CoA + 3 H2O + ADP + Pi 2 CO2+ ATP + 8[H] + HS‐CoA


‐190 mV

ΔG0‘ = ‐ n F ΔE0‘ΔE0‘ =(E0‘[Akzeptor]‐ E0‘[Donor])

ΔG0‘ = ‐ n F (‐0.320 V – (‐0,19 V))= ‐200 kJ/mol V * (‐0.13 V)= + 26 kJ/mol

• Reaction is endergonic• Equilibrium lies far on the side

of malate

At which ratio ofoxaloacetate/malate is the reductionof NAD+ feasable?


ΔG0‘ = ‐ n F ΔE0‘ΔE0‘ =(E0‘[Akzeptor]‐ E0‘[Donor])

ΔG0‘ = ‐ n F (‐0.320 V – (‐0,19 V))= ‐200 kJ/mol V * (‐0.13 V)= + 26 kJ/mol

• Reaction is endergonic• Equilibrium lies far on the side

of malate

At which ratio ofoxaloacetate/malate is the reductionof NAD+ feasable?

E‘ E0‘ ..

E‘ V E0‘ , lg ..

‐0,32 V 0,19 V , lg ..

-0,32 0,19 /0.03 lg ..

-0,32 0,19 /0.03 lg ..

-4.3 lg ..

~ 10-4 ..


• The first step the aldolkondensationof acetyl‐CoA and oxaloacetate isirreversibel/highly exergonic ΔG0‘ =‐32 kJ/mol

• Due to thioester hydrolysis• Not coupled to energy formation


→ the oxaloacetate concentration has tobe kept very low (~ four orders ofmagnitude below malate) to drivemalate oxidation with NAD+

→ accomplished by citrate synthasecatalzed exergonic reaction whichwithdraw oxaloacetate and pulls theequilibrium towards malate oxidation


• Rearragement from Citrate toisocitrate from a tertiary to a secondary alcohol

• With a tertiary alcohol hydrideabstraction is not possible becaus a free hydrogen atom at the hydroxyl C is missing

+ H‐ + H+

AldehydeAlcohol

Malate→ OxaloacetateIsocitrate→ Oxalosuccinate



‐500 mV

• α‐Ketoglutarate Dehydrogenase catalyses the second irreversible reaction of the cycle

• Similar (molecular and catalytic) topyruvate DH complex




• Closed cycle only with a terminal electron acceptor more positive than+30 mV

+30 mV

Electron transferring coenzymes andprosthetic groups: Flavin nucleotides

• Flavoproteins• Isoalloxazin system• Can perform one and two electron transitions• Flavin is tightly bound in most flavoproteins = prosthetic group• Redox potential depends on the protein association: ‐400 mV – +100 mV• In succinate dehydrogenase near 0 mV (free FADH2 ‐220 mV)



‐500 mV

• α‐Ketoglutarate Dehydrogenase catalyses the second irreversible reaction of the cycle

• Similar (molecular and catalytic) topyruvate DH complex




• Closed cycle only with a terminal electron acceptor more positive than+30 mV

• In succinate dehydrogenase FAD ~0 mV (FAD covalently bound), succinateDH transfers electrons from succinatefinally to the UQ pool in themembrane

+30 mV

The citric acid cycle as a metabolic hub

Anaplerotic reactions

PEP carboxykinase in Bacteriamore for gluconeogenesis


Glucose

6 O2

4 CO2

12 H2O

~34 ATP(ETP)

24 [H]

Oxidative part

Reductive part

Transport



Citric acid cycle

Glucose

2 Acteyl‐CoA

2 Pyruvate

4 [H]

4 [H]

16 [H]

2 CO2

1‐2 ATP(SSP)

2 ATP(SSP)

10 NADH → 100 H+

2 UQH2 → 12 H+

Respiratory chain


Σ ~38 ATP

Basic principle

Fig. 9.5 Biology (6th edition, Campbell & Reece)

Beispiel Zellatmung

Respiratory chain

Fig. 5.19 Brock Biology of Microorganisms (10th edition) (Madigan et al.)

Electron transfer coenzymes

Fig. 14.22 Essential Cell Biology (2nd edition, Alberts, Bray et al.)Fig. 5.12 Cell & Molecular Biology (4th edition, Karp)

Flavin-Mononukleotid Chinon Cytochrom Eisen-Schwefel-Zentren

Je 2 H+ und e- nur e-

Electron transport chain

• Electron transport chainmitochondria vs. E. coli

0.369 V → ~70 kJ/mol

0.209 V → ~40kJ/mol

0.563 V → ~100 kJ/mol

2 e‐

Komplex I

Komplex III

Komplex IV


http://watcut.uwaterloo.ca/webnotes/Metabolism/RespiratoryChain.html


Σ 10 H+/NADH→ ~3ATPΣ 6 H+/UQH2→ ~2 ATP

Mitochondria, Paracoccus denitrificans

Fumarate/Succinate +0,03 VFAD/FADH2 ~0.0 V

Komplex I NADH/ubiquinone oxidoreductase

Komplex III Ubichinone/Cytochrome c oxidoreductase (bc1 Komplex)

Komplex IV Cytochrome c oxidase

Komplex II Succinate dehydrogenase

Electron transport chainmitochondria vs. E. coli

2

2

Σ 10 H+/NADH→ ~3ATPΣ 6 H+/UQH2→ ~2 ATP

Σ 8 H+/NADH→ ~2 ATPΣ 4 H+/UQH2→ ~1 ATP

Mitochondria, Paracoccus denitrificans

E. coli

• without cyt c and bc1‐complex• No cyt c oxidase• Chinol oxidase

Branched respiratory chains

Cyt0 low oxygen affinityCytd high oxygen affinity

→ Fine tuning of respiratory chain in responce tooxygen concentrations


Σ 10H+/2e‐→ ~3ATP

ATP Synthase

Reversibel (ATPase)F1: 5 UE (α3β3γεδ)F0: 3 UE (ab2c12)Katalytische Aktivität: β UERotor: c12,γ, εStator: a, b2, δ

3/4 H+ transportiert pro ATP

Mechanism of ATP synthesis

Paul Boyer-Rotation der γ-UE zu α,β-Ring-L: loose site (ADP + Pi Bindung)-T: tight site (ATP Bildung)-O: open site (leer)

O

T

L

Question 2

• What is the frequently used cosubstrate in dehydrogenase catalyzed reactions? How areelectrons transfered?

• What are the four main steps in the oxidative part of aerobic glucose degradation? What isthe net gain of reduction equivalents and ATP?

• What are the main functions of glycolysis (EM pathway)? What is the substrate what theproduct? What happens in the preparatory phase? Name the sites of SSP in glycolysis (incl. Enzymes)? Which enzyme catalyses the only oxidation reaction in this pathway? What are theirreversible steps (incl. Enzymes)? Give the net yield of ATP and reduction equivalents.

• Name the differences between EM and ED pathway.

• Which enzymes and coenzymes take part in the pyruvate oxidation? Give the name of thecomplex.

• What is the basic function of the citric acid cycle? Which compound fit into the cycle andwhat are the products? What are the irreversible steps. Name the site of SSP in the cycle. Where is CO2 generated? What are the oxidation steps? Can electrons be transfered fromSuccinate to NAD+?

• How does ETP basically function? What is the difference in the electron transport chain ofmitochondria/Paracoccus and E. coli? What does that mean for the ATP yield?

Question 2

• Was ist das in Degydrogenase caztalysierten Reaktionen häufig verwendete Cosubstrat und wievieleElektronen überträgt es?

• In welche vier Hauptschritte kann man den aeroben Glucose‐Abbau einteilen? Was sind jeweils die (Netto)Ausbeuten an ATP und Reduktionsäquivalenten?

• Was sind die Hauptfunktionen der Glycolyse (EMP‐Weges)? Was ist das Substrat was sind die Produkte, geben Sie die Nettoausbeute an ATP und Reduktionsäquivalenten an? Was passiert in der Vorbereitungsphase? Welche Reaktionen der Glycolyse sind mit Substratstufenphosphorylierung verbunden, wie heißen die katalysierenden Enzyme? Welches Enzym katalysiert die einzige Oxidationsreaktion im EMP‐Weg? Welches sind die irreversiblen Schritte der Gylcolyse, welche Enzyme katalysieren sie?

• Benennen Sie die Unterschiede zwischen EMP‐ und ED‐Weg.

• Welche Enzyme und Coenzyme sind an der Pyruvat‐Oxidation beteiligt? Wie heißt der Enzym‐Komplex?

• Was sind die Hauptfunktionen des Citrat‐Zyklus? Welche Verbindung wird in den Zyklus eingeschleust und was sind die Produkte? Benennen Sie die irreversiblen Schritte. In welcher Reaktion wird ATP/GTP über SSP gebildet. Welches sind die CO2 generierenden Schritte? Welches sind die Oxidationsschritte? Können Elektronen von Succinat auf NAD+ übertragen werden?

• Wie funktioniert grundsätzlich die Elektronentransportphosphorylierung? Was ist der Unterschied zwischen den Elektronentransportketten der Mitochondrien bzw. von Paracoccus denitrificans und E. coli und was bedeutet das für die ATP‐Ausbeute?

Nitrate respiration („Homework“)

aGlucose + bNO3‐ + → cCO2 + dNO2

‐ +

Many organisms like e.g. E. coli and Paracoccus denitrificans grow with glucoseand NO3

‐ as electron donor and acceptor, respectively:

• How does the complete redox equation/balance look like?• How much energy is gained and how much ATP can maximally be synthetized?• Does complete glucose oxidation include a closed/complete TCA cycle?

Microbiology II Microbial physiology II - uni-due.de · The Entner‐Doudoroff(ED) pathway • 1...

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