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

Enterics

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 ProductsProducts E. col i E. aerogenes

Formate 2.4 17

Acetate 36.5 0.5

La ctate 79.5 2.9

Su ccinic 10.7 -

Ethanol 49.8 69.5

Butandiol 0.3 66.4

CO2 88 172

H2 75 35.4

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

CoA

C CCH3

O O

O-

C CCH3

•O O

O-

Scys418

CO

O•

HS

cys419

HS

cys419

C

CH3

Scys418

O

C

CH3

Scys418

OS•

cys419

HS

cys419

S•cys418

HCOO-

CoA-S•C

CH3

Scys418

OHS

cys419

S•cys418

HS

cys419

O

CH3-C-S-CoA

Mechanism of Pyruvate-formate lyaseFree radical cleavage of C-C bondTransfer 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

NADH NAD+

Glucose

PEP

Glycolysis(See previous notesfor complete pathwayNot balanced!)

Oxaoloacetate

Fumarate

Malate

Succinate

H 2O

P i

Pyruvate

xNADH

xNAD+

Acety-CoA Formate

Ethanol

Acetyl-P

Acetate

Acetaldehyde

CO2

Lactate

CO2

H2

CoA

CoAP i

ATPADP

NADHNAD+

CoA

+ NAD

+

ATPADP

x ATP

xADP

NAD+

NAD+

NADH

NADHADP

ATP

Pyruvate-formate lyase

PEP carboxylasemalate dehydrogenase

fumarase

fumaratereductase

Formate-hydrogenlyase

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

PEP

CO2

GlycolysisSee previoushandout for reactions.(not balanced)

Pyruvate

xNADHxNAD +

Acety-CoA Formate

Ethanol

Acetaldehyde

H2

Lactate

+

CoA

NADHNAD+

CoANADH

+ NAD +

ATPADP

xATP

xADP

C

OH

TPPCH 3

CO2

-Acetolactate

Pyruvate

Acetoin

2,3-Butanediol

NADH

NAD +

-Acetolactate Synthase

CO2

CH 3 C

COOH

C

OH O

CH 3

CHCH 3 C

OH O

CH 3

OH

CHCH 3 CH

OH

CH3

[ ]Pyruvate-formate lyase

2,3-Butanediol dehydrogenase

-Acetolactatedecarboxylase

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-specificmicrobial 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 alternativebacterial source-tracking targets. FEMS Microbiol. Ecol. V 47 p. 65-75.

Dick, L. K., et. al. (2005). Host distributions of uncultivated fecal Bacteriodes bacteria revealgenetic 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

Glucose

2 pyruvates

Glycolyticpathway

Net 2 ATP

2 NADH's made

2 acetaldehyde H3C CH

O

2 CO2

Pyruvate decarboxylase

2 ethanol

2 NADH

2 NAD+

Oxidative reactions:3-phosphoglyceraldehyde + Pi + NAD+ -> 1,3-bisphosphoglycerate + NADH

Reductive reactions:acetaldehyde + NADH -> ethanol + NAD+

Substrate-level phosphorylation:PEP + ADP -> pyruvate + ATP1,3-bisphosphoglycerate + ADP -> 3 phosphoglycerate + ATP

Net ATP use 2 ATP make 4 ATP net of 2 ATP

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.

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.

6PG dehydratase

KD6PG aldolase

Glucose

Glucose-6-P

1,3-bisphosphoglycerate

3-phosphoglycerate

phosphoenol pyruvate

pyruvate

ATPADP

3-phosphoglyceraldehyde

2-phosphoglycerate

H2O

NAD+

NADH

ATP

ATP

ADP

ADP

Pi

6-Phosphogluconate

H2O

2-Keto-3-deoxy-6-phosphogluconate

pyruvate

NADP+

NADPH

Reoxidation of NAD(P)H

2 pyruvate

2 acetaldehyde

2 ethanol

2 NAD(P)+

2 NAD(P)H

2 CO2

Pyruvatedecarboxylase

Alcoholdehydrogenase

ATP summary used 1 made 2 net = 1

Key enzymes and intermediates

COOH

HC

CH

HC

HC

H2C

OH

HO

OH

OH

O P

COOH

C

CH

HC

HC

H2C

HO

OH

OH

O P

H2OO

6-Phospho-gluconatedehydratase

COOH

C

CH3

O

Pyruvate

HC

HC

H2C O P

OH

O

KD6PG aldolase

2-keto-3-deoxy-6-phosphoglucon a te(KD6PG)

3-phosphoglyceraldehyde

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.