Escherichia, Salmonella, Shigella, Proteus. Make lactic, acetic, succinic, formic acids

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Stanley Gilbert
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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 B 12 ther 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 Products E. col i E. aerogenes For m ate Acetate La ctate Su ccini c Ethanol Butandiol C H

4 Mixed acid fermentation Glucose to pyruvate by glycolysis New enzymes Pyruvate metabolism by pyruvate-formate lyase Pyruvate + CoA --> acetyl-coa + formate (HCH) 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. cys418 C CH 3 S C cys419 cys418 HS S C C - CH 3 cys418 C CH 3 S HS cys419 CoA-S HS - cys419 CoA cys418 C CH 3 cys418 C S S cys419 HS C HC - cys419 S cys418 S HS cys419 CH 3 CH 3 -C-S-CoA

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

7 Mixed Acid Fermentation xaoloacetate NADH malate dehydrogenase ATP fumarase Malate Fumarate ADP fumarate reductase Succinate NAD+ H 2 NADH NAD + P i PEP carboxylase CoA C2 CoA Pyruvate-formate lyase P i Acetyl-P ADP ATP Acetate Glucose PEP Pyruvate Acety-CoA xnad + xnadh xadp x ATP ADP ATP NADH NAD + Formate Acetaldehyde Ethanol Lactate Formate-hydrogen lyase CoA + NAD + NADH NAD + C2 H 2

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

9 2,3-Butanediol Fermentation Glucose xnad + Glycolysis xnadh See previous xadp handout for reactions. xatp (not balanced) α -Acetolactate Synthase PEP C ADP [ ] 2 ATP CH 3 C TPP Pyruvate Lactate H CoA Pyruvate-formate lyase Pyruvate C 2 Acety-CoA + Formate NADH H CoA + NAD + H 2 α -Acetolactate CH 3 C C CH 3 Acetaldehyde CH NADH α C -Acetolactate 2 decarboxylase NAD+ H Acetoin Ethanol 2,3-Butanediol dehydrogenase 2,3-Butanediol CH 3 NADH NAD + CH CH 3 C H CH CH 3 H 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 Henry Wadsworth Longfellow All the water on the planet is recycled. Risks of fecal contamination differentiation between fecal and non-fecal enterics is critical Shanks,. C. et. al. (2006) Competitive Methagenomic DNA Hybridization identifies host-specific microbial genetic markers in cow fecal samples. AEM V 72 N6 p 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 pathway Glycolytic pathway Glucose 2 pyruvates Net 2 ATP 2 NADH's made Pyruvate decarboxylase 2 C 2 2 acetaldehyde H 3 C C H 2 NADH 2 NAD + xidative reactions: 3-phosphoglyceraldehyde + P i + NAD + -> 1,3-bisphosphoglycerate + NADH Reductive reactions: acetaldehyde + NADH -> ethanol + NAD + Substrate-level phosphorylation: PEP + ADP -> pyruvate + ATP 1,3-bisphosphoglycerate + ADP -> 3 phosphoglycerate + ATP Net ATP use 2 ATP make 4 ATP net of 2 ATP 2 ethanol

15 Pyruvate decarboxylase Pyruvate decarboxylase Pyruvate -> acetaldehyde (CH 3 CH) + C 2 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.

Gram negative, motile, small rods, anaerobic to microaerophilic Usually make

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 ften more versatile than yeast in substrates used rganism of choice for bulk ethanol production (gasohol)

17 Zymomonas Uses a new pathway for glucose metabolism called Entner-Doudoroff xidation 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 6PG dehydratase 3-phosphoglyceraldehyde NAD P + i NADH 1,3-bisphosphoglycerate ADP ATP 3-phosphoglycerate 2-phosphoglycerate Glucose ATP ADP Glucose-6-P NADP + NADPH 6-Phosphogluconate H 2 2-Keto-3-deoxy-6-phosphogluconate H 2 KD6PG aldolase pyruvate pyruvate ATP ADP phosphoenol pyruvate Pyruvate decarboxylase Alcohol dehydrogenase ATP summary used 1 made 2 net = 1 Reoxidation of NAD(P)H 2 pyruvate 2 acetaldehyde 2 ethanol 2 C 2 2 NAD(P)H 2 NAD(P) +

19 Key enzymes and intermediates H CH HC H CH HC H HC H H 2 6-Phosphogluconate dehydratase H CH C CH HC H HC H 2-keto-3-deoxy- 6-phosphoglucon a te (K D6PG) H 2 C P H 2 C P CH KD 6PG aldolase HC C HC H CH 3 Pyruvate H 2 C P 3-phosphoglyce raldehyde

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.

Respiration. Organisms can be classified based on how they obtain energy: Autotrophs

Respiration. Organisms can be classified based on how they obtain energy: Autotrophs Respiration rganisms can be classified based on how they obtain energy: Autotrophs Able to produce their own organic molecules through photosynthesis Heterotrophs Live on organic compounds produced by