1. Enterics Emphasize novel pyruvate enzymes
Example of free radicals involved in C-C bond cleavage.
Gram negative bacteria that ferment sugars to acids and gas. All use glycolysis
Mixed acid group:
Escherichia, Salmonella, Shigella, Proteus.
Make lactic, acetic, succinic, formic acids
Butanediol fermentors:
Enterobacter, Serratia, Erwinia
Mixed acid at neutral pH
Make 2,3-butanediol at low pH

2. Ways to cleave a C-C bond
Carbonyl beta to another oxygen-containing molecule
Alpha decarboxylation: use TPP to stabilize carbanion
Rearrangements: use free radical mechanism, free radical provided by 5-deoxyadenosyl on B12
Other free radicals can be formed on proteins: glycyl and tyrosyl free radicals
See how E. coli cleaves pyruvate by free radical mechanism

3. Fermentation Productsol i
E. ae r
ogenes F or m
ate
2.4 17 A c
etate
36.5
0.5 La c
tate
79.5
2.9 Su c c i ni c
10.7 -
Ethano l
49.8
69.5
Butand i ol
0.3
66.4 C O 88
172 2
35.4 H 75 2

4. Mixed acid fermentation
Glucose to pyruvate by glycolysis
New enzymes
Pyruvate metabolism by pyruvate-formate lyase
Pyruvate + CoA --> acetyl-CoA + formate (HCOOH)
No TPP (glycine free radical),
no NADH made
Still get high energy intermediate, but don’t have to recycle NADH

5. Mechanism of Pyruvate-formate lyase
Free radical cleavage of C-C bond
Transfer stable free radical on glycine to
one of the sulfurs in cysteine at the active site.

6. Mixed Acid fermentation
Succinate formation
PEP carboxylase (heterotrophic CO2 fixation)
PEP + CO2 --> oxaloacetate + Pi
ATP synthesis by electron transport
Formate metabolism
Formate: hydrogen lyase
Formate --> CO2 + H2
2 enzymes involved, formate dehydrogenase and hydrogenase
Note: Enterobacter group also does mixed acid fermentation at neutral pH

7. Mixed Acid Fermentation
Glucose +
xNAD
Glycolysis
xNADH
(See previous notes
xADP
for complete pathway P i CO 2
Not balanced!)
x ATP
Oxaoloacetate
PEP
malate
dehydrogenase
PEP carboxylase
NADH
ADP
NAD +
ATP
Malate
Pyruvate
Lactate
CoA
fumarase
NADH
NAD+ CO 2 H H O
Pyruvate-formate lyase 2
Fumarate
Formate
Acety-CoA
ADP
NADH P i
Formate-hydrogen
lyase
ATP +
NAD
CoA
Acetyl-P
fumarate
reductase +
CoA +
ADP
NAD
Acetaldehyde
ATP
Succinate
Acetate
NADH
NAD +
Ethanol

8. Butandiol fermentation
Switch to solvent production in low pH
New enzymes:
Alpha-acetolactate synthase
2 pyruvate --> acetolactate + CO2
TPP as cofactor
Butandiol fermentation
Reduce acetolactate to acetoin and butandiol.

9. ] [ 2,3-Butanediol Fermentation Glucose Glycolysis See previous
handout for reactions.
(not balanced)
xNAD +
xNADH
xADP
xATP a
-Acetolactate Synthase
PEP CO
ADP [ ] 2
ATP CH 3 C
TPP
Pyruvate
Lactate OH
CoA
Pyruvate-formate lyase
Pyruvate CO 2
Acety-CoA +
Formate
NADH OH O H
CoA +
NAD + 2 a
-Acetolactate CH 3 C C CH 3
Acetaldehyde
COOH
NADH CO a
-Acetolactate
decarboxylase 2
NAD + O
Acetoin OH
Ethanol CH 3 CH C CH 3
2,3-Butanediol
dehydrogenase
NADH
NAD + OH OH
2,3-Butanediol CH 3 CH CH CH 3

10. The problem of food and water pollution
“..its waters returning, Back to the springs, like the rain, Shall fill them full of refreshment, that which the fountain sends forth returns again to the fountain”
All the water on the planet is recycled.
Risks of fecal contamination differentiation between fecal and non-fecal enterics is critical
Shanks, O. C. et. al. (2006) Competitive Methagenomic DNA Hybridization identifies host-specific
microbial genetic markers in cow fecal samples. AEM V 72 N6 p – 4060.
Simpson, J. M. et. al. (2004). Assessment of equine fecal contamination: the search for alternative
bacterial source-tracking targets. FEMS Microbiol. Ecol. V 47 p
Dick, L. K., et. al. (2005). Host distributions of uncultivated fecal Bacteriodes bacteria reveal
genetic markers for fecal source identification. AEM V71 N6 p

11. Differentiation of Enterics
Differentiation based on metabolic characterization
Enzyme analysis
Intermediate analysis
Mixed acid
Gas: E. coli, Salmonella
No gas: Shigella, S. typhi
Butanediol (acetoin)
Gas: Enterobacter
No gas: Erwinia, Serratia.

12. Summary Free radicals on proteins can also be used to break C-C bonds.
Enterics are a good example of reactions. They metabolize pyruvate to most of the products we discussed.
ID of enterics critical to assess water quality.

13. Alcohol fermentations
Two possibilities: yeast and Zymomonas.
Yeast
1815: Gay-Lussac found that yeast made 2 ethanols and 2 carbon dioxides from glucose
Buchner: cell-free extract, beginnings of biochemistry
Uses glycolytic pathway to make pyruvate
Difference from Streptococcus is in what happens to pyruvate
New pyruvate enzyme:
References: Flores et al. FEMS Micro. Rev. 24: , 2000; Conway, FEMS Micro. Rev. 103: 1-28, 1992.

14. Summary of the yeast pathwayG l u c o s e O x i d a t v e r c o n s : 3 - p h g l y + P N A D
> 1 , b H N e t 2 A T P G l y c o t i p a h w 2 N A D H ' s m a d e R e d u c t i v r a o n s : l h y + N A D H -
> 2 p y r u v a t e s P y r u v a t e d c b o x l s S u b s t r a e - l v p h o y i n : P E + A D
> T 1 , 3 g c 2 C O 2 a c e t l d h y H 3 C C H O N e t A T P u s 2 m a k 4 n o f 2 N A D H 2 N A D + 2 e t h a n o l

15. Pyruvate decarboxylase
Pyruvate -> acetaldehyde (CH3CHO) + CO2
Cofactor: thiamine pyrophosphate (TPP).
Thus, no oxidation/reduction and no high energy intermediate is made
The “active aldehyde” rearranges and forms acetaldehyde as one of the products
Function of TPP here is decarboxylation.

16. Zymomonas
Natural agent of alcohol fermentations in tropics, isolated from Mexican pulque.
Gram negative, motile, small rods, anaerobic to microaerophilic
Usually make more than 2 mol ethanol per mol glucose
Often more versatile than yeast in substrates used
Organism of choice for bulk ethanol production (gasohol)

17. Zymomonas
Uses a new pathway for glucose metabolism called Entner-Doudoroff
Oxidation of the number one carbon of glucose as in Leuconostoc to form 6-phosphogluconate
Followed by a dehydration to give a new intermediate: 2-keto-3-deoxy-6-phosphogluconate.

18. G l u c o s e A T P A T P s u m a r y e d 1 2 n t = A D P G l u c o s- 6 P N A D P + N A D P H 6 - P h o s p g l u c n a t e R e o x i d a t n f N A D ( P ) H
6PG dehydratase H 2 O 2 p y r u v a t e 2 - K e t o 3 d x y 6 p h s g l u c n a P y r u v a t e d c b o x l s
KD6PG aldolase 2 C O 3 - p h o s g l y c e r a d p y r u v a t e N A D + 2 a c e t l d h y P i N A D H A l c o h d e y r g n a s 2 N A D ( P ) H 1 , 3 - b i s p h o g l y c e r a t A D P 2 N A D ( P ) + p y r u v a t e A T P 2 e t h a n o l 3 - p h o s g l y c e r a t A T P H 2 O A D P 2 - p h o s g l y c e r a t p h o s e n l y r u v a t

19. Key enzymes and intermediatesC O H C O H H 2 O H C O H C O 2 - k e t o - 3 - d e o x y - 6 - p h o s p h o g l u c on a t e H O C H H O C H ( K D 6 P G ) H C O H 6 - P h o s p g l u c n a t e d y r H C O H H C O H H C O H H 2 C O P H 2 C O P K D 6 P G a l d o s e C O H O H C C O H C O H C H 3 H 2 C O P P y r u v a t e 3 - p h o s g l y c e r a d

20. Summary
Pyruvate decarboxylase: uses TPP to decarboxylate pyruvate but only makes acetaldehyde
ED pathway: only one G-3-P made, limits ATP production
ED pathway oxidizes C-1 of glucose and makes new intermediate, 2-keto-3-deoxy-6-phosphogluconate
Extra oxidative reaction in Zymomonas limits ATP production compared to yeast.