In
the Alber lab, we explore the innumerable ways in which microbes are
able to metabolize an amazing variety of carbon sources.
Using the well-studied Rhodobacter
sphaeroides as a model organism, we work to uncover
properties of unknown enzymatic pathways for carbon assimilation.
The premise of our work hinges on the assertion that all
cells must be able to synthesize precursor metabolites (e.g.
oxaloacetate, pyruvate, alpha-ketoglutarate, acetyl-CoA, etc.) from
carbon substrates, a process known as anaplerosis. The recent
realization of the immense diversity of anaplerotic reaction sequences
drives us to pursue yet undiscovered pathways of carbon assimilation
while also seeking a better understanding of currently known pathways.
Foremost, it must be understood that organisms able to grow on substrates that are metabolized solely via acetyl-CoA, meaning their only carbon source is acetyl-CoA, absolutely
require
a unique series of anaplerotic reactions.
To understand why anaplerotic reactions are required, one must examine
the relationship between acetyl-CoA and the tricarboxylic acid (TCA)
cycle (Figure 1). Figure 1 illustrates that acetyl-CoA is the
sole source of carbon input for the TCA cycle while intermediates of
the cycle are removed for biosynthesis of amino acids.
Acetyl-CoA
will enter the TCA cycle if a cell lacks an alternative or ancillary
pathway of acetyl-CoA assimilation. In this case, the cell
will
quickly
deplete the intermediates of the TCA cycle as they are removed for cell
biosynthesis. Acetyl-CoA is only a two carbon molecule and is
the only input of carbon into the cycle. As two C1
units are released as CO2
for each acetyl-CoA that enters the cycle, the
net assimilation of carbon is zero. Therefore, intermediates
from
the cycle are lost as precursor metabolites in biosynthetic pathways
and cannot be replaced. Ultimately, the cell needs a method
to
synthesize additional precursor metabolites from acetyl-CoA such that
oxaloacetate is available for
the first step of continuing this essential cycle.
This connundrum was immediately apparent to Kornberg and Krebbs in 1957 when th
ey
identified the enzyme activities for isocitrate lyase and malate
synthase in
Pseudomonas
and Escherichia coli
(Kornberg and Krebs 1957). Together, these enzymes
provided a method for incorporating an additional acetyl-CoA unit while
bypassing
the CO2 releasing steps of the TCA cycle. They
had seemingly solved the question of acetyl-CoA assimilation.
With the newly
understood "glyoxylate bypass," as they termed the pathway, an organism
could assimilate carbon during growth solely on substrates that are
metabolized via acetyl-CoA. However, they shortly realized a
new complexity. Some organisms, including R. sphaeroides, do
not exhibit isocitrate lyase activity in cell extract but were fully
capable of
assimilating acetyl-CoA (Kornberg and Lascelles 1960). For
nearly half a century, a hole loomed in the knowledge of
central metabolism. No researcher was able to identify the
pathway that made it possible for these isocitrate lyase-negative
organisms to assimilate acetyl-CoA until 2007. In 2007, a unique series of enzymatic activites was discovered in R. sphaeroides cell extract that
would make it possible for the organism to assimilate acetyl-CoA (Erb et al.
2007). This series of reactions has come to be known
as the ethylmalonyl-CoA pathway (Figure 3).
At its essence, the ethylmalonyl-CoA pathway incorporates three
acetyl-CoA
units and two CO2 equivalents and produces succinyl-CoA
and malate, two anaplerotic
intermediates. The first step is the condensation of two
acetyl-CoA molecules to
form
acetoactyl-CoA. After a series of hydrations, carboxylations,
and carbon rearrangments, the pathway passes through its
namesake, ethylmalonyl-CoA, and ends up at ß-methylmalonyl-CoA which is
cleaved to form two branches in the pathway. One branch leads
to malate by incorporation of a third acetyl-CoA unit while the other
branch proceeds through a set of reactions known from
propionate metabolism to form succinyl-CoA. All of this is
done without any loss of carbon to
carbon dioxide. In fact there are actually two carbon
dioxide units
that are co-assimilated.
With the enzymatic reaction series elucidated, our next task is to study the regulation of the ethylmalonyl-CoA pathway. We are currently seeking to identify the level of regulation that is primarily used by R. sphaeroides to control the expression of this pathway and which genetic elements and gene products might be involved in regulating the pathway.
1.
Erb, T. J. et al. (2007) Synthesis of
C5-dicarboxylic acids from C2-units involving crotonyl-CoA
carboxylase/reductase: The ethymalonyl-CoA pathway. Proc. Nat.
Acad. Sci.: 25: 10631 – 10636.
(See Reference)
2. Kornberg, H. L. and Krebs, H. A. (1957) Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. Nature: 179: 988 – 991. (See Reference)
3. Kornberg, H. L. and Lascelles. (1960) The Formation of Isocitratase by the Athiorhodaceae. J. gen. Microbiol: 23: 511-217. (See Reference)
Foremost, it must be understood that organisms able to grow on substrates that are metabolized solely via acetyl-CoA, meaning their only carbon source is acetyl-CoA, absolutely
require
a unique series of anaplerotic reactions.
To understand why anaplerotic reactions are required, one must examine
the relationship between acetyl-CoA and the tricarboxylic acid (TCA)
cycle (Figure 1). Figure 1 illustrates that acetyl-CoA is the
sole source of carbon input for the TCA cycle while intermediates of
the cycle are removed for biosynthesis of amino acids.
Acetyl-CoA
will enter the TCA cycle if a cell lacks an alternative or ancillary
pathway of acetyl-CoA assimilation. In this case, the cell
will
quickly
deplete the intermediates of the TCA cycle as they are removed for cell
biosynthesis. Acetyl-CoA is only a two carbon molecule and is
the only input of carbon into the cycle. As two C1
units are released as CO2
for each acetyl-CoA that enters the cycle, the
net assimilation of carbon is zero. Therefore, intermediates
from
the cycle are lost as precursor metabolites in biosynthetic pathways
and cannot be replaced. Ultimately, the cell needs a method
to
synthesize additional precursor metabolites from acetyl-CoA such that
oxaloacetate is available for
the first step of continuing this essential cycle.This connundrum was immediately apparent to Kornberg and Krebbs in 1957 when th
ey
identified the enzyme activities for isocitrate lyase and malate
synthase in
Pseudomonas
and Escherichia coli
(Kornberg and Krebs 1957). Together, these enzymes
provided a method for incorporating an additional acetyl-CoA unit while
bypassing
the CO2 releasing steps of the TCA cycle. They
had seemingly solved the question of acetyl-CoA assimilation.
With the newly
understood "glyoxylate bypass," as they termed the pathway, an organism
could assimilate carbon during growth solely on substrates that are
metabolized via acetyl-CoA. However, they shortly realized a
new complexity. Some organisms, including R. sphaeroides, do
not exhibit isocitrate lyase activity in cell extract but were fully
capable of
assimilating acetyl-CoA (Kornberg and Lascelles 1960). For
nearly half a century, a hole loomed in the knowledge of
central metabolism. No researcher was able to identify the
pathway that made it possible for these isocitrate lyase-negative
organisms to assimilate acetyl-CoA until 2007. In 2007, a unique series of enzymatic activites was discovered in R. sphaeroides cell extract that
would make it possible for the organism to assimilate acetyl-CoA (Erb et al.
2007). This series of reactions has come to be known
as the ethylmalonyl-CoA pathway (Figure 3).
At its essence, the ethylmalonyl-CoA pathway incorporates three
acetyl-CoA
units and two CO2 equivalents and produces succinyl-CoA
and malate, two anaplerotic
intermediates. The first step is the condensation of two
acetyl-CoA molecules to
form
acetoactyl-CoA. After a series of hydrations, carboxylations,
and carbon rearrangments, the pathway passes through its
namesake, ethylmalonyl-CoA, and ends up at ß-methylmalonyl-CoA which is
cleaved to form two branches in the pathway. One branch leads
to malate by incorporation of a third acetyl-CoA unit while the other
branch proceeds through a set of reactions known from
propionate metabolism to form succinyl-CoA. All of this is
done without any loss of carbon to
carbon dioxide. In fact there are actually two carbon
dioxide units
that are co-assimilated. With the enzymatic reaction series elucidated, our next task is to study the regulation of the ethylmalonyl-CoA pathway. We are currently seeking to identify the level of regulation that is primarily used by R. sphaeroides to control the expression of this pathway and which genetic elements and gene products might be involved in regulating the pathway.
2. Kornberg, H. L. and Krebs, H. A. (1957) Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. Nature: 179: 988 – 991. (See Reference)
3. Kornberg, H. L. and Lascelles. (1960) The Formation of Isocitratase by the Athiorhodaceae. J. gen. Microbiol: 23: 511-217. (See Reference)