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

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

Fermentation Products 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 - Ethano l 49.8 69.5 Butand i ol 0.3 66.4 C O 88 172 2 35.4 H 75 2

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

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.

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

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

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.

] [ 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

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.

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.

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.

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.

Summary of the yeast pathway G 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

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.

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)

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.

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

Key enzymes and intermediates C 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

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.