Enterics

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 Products P r odu c ts E. c ol 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 - 69.5 Ethano l 49.8 Butand i ol 0.3 66.4 172 C O 88 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. CO 2 H 2 NADH + NAD Mixed Acid Fermentation Glucose + xNAD Glycolysis (See previous notes xNADH for complete pathway xADP P i CO Not balanced!) 2 x ATP Oxaoloacetate PEP PEP carboxylase ADP malate dehydrogenase NADH ATP + NAD Pyruvate Lactate Malate CoA NAD+ NADH fumarase Pyruvate-formate lyase H O 2 Formate Fumarate Acety-CoA ADP NADH P i Formate-hydrogen lyase + CoA ATP NAD Acetyl-P + fumarate reductase + CoA NAD ADP Acetaldehyde ATP Succinate Acetate 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. Glycolysis See previous handout for reactions. (not balanced) a -Acetolactate Synthase a -Acetolactate decarboxylase 2,3-Butanediol Fermentation Glucose + xNAD xNADH xADP xATP PEP ADP CO 2 ] [ ATP CH C TPP 3 Pyruvate Lactate OH CoA Pyruvate-formate lyase Pyruvate CO 2 + Acety-CoA Formate NADH H OH O 2 + + CoA NAD a -Acetolactate CH CH C C 3 3 Acetaldehyde NADH COOH CO + 2 NAD O OH Acetoin Ethanol CH CH C CH 3 3 NADH 2,3-Butanediol dehydrogenase + NAD OH OH 2,3-Butanediol CH CH CH CH 3 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. 4054 – 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. 65-75. Dick, L. K., et. al. (2005). Host distributions of uncultivated fecal Bacteriodes bacteria reveal genetic markers for fecal source identification. AEM V71 N6 p. 3184- 3191.

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: 507-529, 2000; Conway, FEMS Micro. Rev. 103: 1-28, 1992.

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

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

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

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.