THE GLUCONEOGENESIS PATHWAY

E. coli employs the gluconeogenesis pathway to synthesize the six carbon sugar glucose-6-phosphate plus three other important cell building blocks, fructose-6-phosphate, PEP and 3-phosphoglycerate. Other six carbon sugars needed for cell synthesis are derived from the pathway end product.

The overall gluconeogenesis pathway reaction is:

2 pyruvate + phosphate + 4 ATP + 2 NADH + 2H+ → glucose-6-phosphate + 4 ADP + 4 Pi + 2NAD+

Key Concepts

  • E. coli cells synthesize glucose -6-phosphate from lower molecular weight carbon compounds when this six carbon sugar is unavailable for uptake from the cell surroundings.
  • The gluconeogenesis pathway converts two molecules of pyruvate into glucose-6- phosphate by using many of the enzymes common to the glycolysis pathway.
  • Two reactions are not shared and are unique to this anabolic pathway.
  • Synthesis of glucose -6-phosphate requires input of energy in the form of four molecules of ATP and two molecules of NADH + H+.
  • Several gluconeogenesis pathway intermediates are used as precursors for other biosynthetic pathways.
  • The pathway end product, glucose -6-phosphate, can be converted into other six carbon sugars needed for cell biosynthesis.

Operation of the Gluconeogenesis Pathway

E. coli employs the gluconeogenesis pathway to synthesize the six carbon sugar, glucose-6-phospage when glucose and/or other related sugars are unavailable for uptake from the cell surroundings.

Starting from pyruvate the synthesis of glucose-6-phosphate mirrors the reverse of the glycolysis pathway. Here, seven of the gluconeogenesis pathway enzymes are shared with the glycolysis pathway but operate in the reverse direction. Two enzymes are unique.

The operation of the gluconeogenesis pathway is energy intensive; energy in the form of 4 ATP and the reducing power of 2 NADH are used to drive this anabolic (i.e., biosynthetic) pathway.

The operation of the gluconeogenesis and glycolysis pathways is coordinated to supply biosynthetic intermediates and/or cell energy. This depends on the nutritional conditions of cell growth. If glucose is unavailable, the cell must synthesize glucose-6-phosphate to make various cell macromolecules. When glucose is freely available from the cell surroundings it is preferentially transported, broken down and converted into the needed cell intermediates.

Note that four carbon compounds, malate and oxaloacetate as well as other, smaller carbon compounds including lactate and glycerol, can serve as precursors for the gluconeogenesis pathway. These precursors are either converted to pyruvate or enter the gluconeogenesis pathway at a later stage.

The gluconeogenesis pathway end product, glucose-6-phosphate is used to form other six carbon or five carbon sugars needed for cell envelope synthesis. These include precursors for the peptidoglycan layer located in the periplasmic space, the LPS molecules present in the outer membrane, and several other sugars located in other cell compartments.

Details of the Gluconeogenesis Pathway

I. The gluconeogenesis pathway reactions starting with pyruvate.

The nine enzymes comprising the gluconeogenesis pathway produce glucose-6-phosphate from the three carbon precursor molecule, pyruvate. Two of the enzymes are unique to this pathway while the other seven are shared with the glycolysis pathway that operates in the reverse direction in order to break down glucose.

In the first pathway reaction the high energy molecule ATP is used to donate a phosphate to form phosphoenolpyruvate (PEP). Phosphoenolpyruvate synthetase is unique to the pathway and a different enzyme operates in the reverse direction (pyruvate kinase) in the glycolysis pathway.

pyruvate + ATP + H2O ↔ phosphoenolpyruvate + AMP + phosphate +2H+

This second pathway enzyme adds a water molecule into the substrate to prepare it for the subsequent pathway reaction. This same enzyme participates in the glycolysis pathway where operates in the reverse direction.

phosphoenolpyruvate + H2O → 2-phospho-D-glycerate

This mutase enzyme relocates the phosphate on the three carbon backbone of 2-phospho-D-glycerate to give 3-phospho-D-glycerate. E. coli has two isoenzmes encoded by gpmM and gpmA genes that can perform this reaction and both also perform the reverse reaction in the glycolysis pathway.

2-phospho-D-glycerate ↔ 3-phospho-D-glycerate

This energy consuming reaction employs ATP to donate a second phosphate molecule onto the three carbon precursor molecule. The same Pgk kinase operates in the reverse direction in the glycolysis pathway where it harvests energy by the process called substrate level phosphorylation (link to SLP in portal).

3-phospho-D-glycerate + ATP ↔ 1,3-bisphospho-D-glycerate + ADP

In this next energy consuming reaction, two electrons are introduced into the three carbon substrate to reduce it to glyceraldehyde-3-phosphate. At the same time, a phosphate group is removed to give Pi (inorganic phosphate). The same GapA dehydrogenase operates in the reverse direction in the glycolysis pathway.

1,3-bisphospho-D-glycerate + NADH + H+ ↔ D-glyceraldehyde 3-phosphate + NAD+ + Pi

The TpiA isomerase rapidly inter-converts glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. This reaction provides needed intermediates for other cell biosynthetic pathways.

D-glyceraldehyde 3-phosphate ↔ dihydroxyacetone phosphate

This aldolase enzyme combines two triose phosphate molecules to form the six carbon sugar, fructose-1,6-bisphosphate. E. coli has two enzymes called FbaA and FbaB that perform this reaction and both operate in the reversible direction in the glycolysis pathway.

dihydroxyacetone phosphate + D-glyceraldehyde ↔ 3-phosphate fructose-1,6-bisphosphate

This phosphatase enzyme is the second unique pathway enzyme. It removes a phosphate and a water molecule to form fructose-6-phosphate. The energy released is lost as heat. Note that this enzyme is not shared with the glycolysis pathway. Three more fructose-1,6-bisphosphatase have been identified in E. coli, GlpX, YggF and YbhA. The physiological role of these enzymes is not clear as their genes can be deleted without impacting E. coli to grow on gluconeogenic substrates.

fructose-1,6-bisphosphate + H2O → D-fructose 6-phosphate + Pi

In this final pathway reaction, the Pgi enzyme isomerizes the six carbon molecule to form glucose-6-phosphate. It also participates in the glycolysis pathway.

D-fructose 6-phosphate ↔ D-glucose 6-phosphate

II. Conversion of glucose-6-phosphate to other six carbon sugars

The formation of glucose-6-phosphate provides a starting point to make a number of other sugar phosphate molecules destined for incorporation into growing cells. For example, the amino sugars NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid) are needed in large amounts to form the peptidoglycan layer in the cell envelope. The outer leaflet of the outer membrane contains large amounts of the molecule called LPS (lipopolysaccharide). Lastly, some E. coli strains make exo-polysaccharide (EPS) that coats the cell surface and/or is needed to make bio-films for attachment to surfaces.

Summary

  • The gluconeogenesis pathway requires considerable energy input to convert pyruvate into glucose-6-phosphate.
  • The pathway consumes four molecules of ATP and 2 molecules of NADH + H+ for every two molecules of the precursor, pyruvate.
  • Two enzymes of the gluconeogenesis pathway are unique while the other enzymes are shared with glycolysis pathway.
  • The demand for the pathway intermediates and end product depends on the cell growth conditions (i.e., what precursors are available for uptake from the cell surroundings).

Credits:

Authored by Robert Gunsalus and Imke Schröder
©The Escherichia coli Student Portal

This project acknowledges support from:
NIH Grant Award GM077678 to SRI, International
Peter Karp and coworkers at EcoCyc.org
The UCLA Department of MIMG