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Welcome!
Welcome!
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 anoxygenic phototrophic bacterium 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.
The Ethylmalonyl-CoA Pathway
The Ethylmalonyl-CoA Pathway
Growth on substrates that are exclusively metabolized via acetyl-CoA requires one of many specialized pathways for anaplerosis. In order 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 necessarily removed for biosynthetic purposes. In the absence of an ancillary pathway for anaplerosis, acetyl-CoA, a two-carbon molecule, enters the citric acid cycle as the sole input of carbon. The cycle outputs two carbons as CO2 while intermediates of the cycle are simultaneously withdrawn as biosynthetic presursors. Therefore, intermediates required for maintaining the cycle are removed from without any net carbon input, collapsing the cycle. Ultimately, the cell needs a method to synthesize additional intermediates from acetyl-CoA to continue production of the biosynthetic precursors within the cycle.
The necessity for anaplerotic reactions was immediately apparent to Kornberg and Krebbs in 1957 when they identified the enzyme activities for isocitrate lyase and malate synthase in Pseudomonas and Escherichia coli (Kornberg and Krebbs, 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 Lacelles, 1960).
For nearly half a century, a hole loomed in the knowledge of central carbon 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 citric acid cycle 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, which is converted to beta-methylmalonyl-CoA. The latter is then 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.
3-Hydroxypropionate Assimilation
3-Hydroxypropionate Assimilation
The C3 compound 3-hydroxypropionate (CH2OH-CH2-COO−) is increasingly being recognized as an important intermediate or end product of carbon metabolism in a variety of organisms. So far, there are at least five known metabolic processes involving 3-hydroxypropionate.
- Propionyl-CoA metabolism in plants
- Propionyl-CoA is derived from the breakdown of chlorophyll, odd-chain fatty acids, or amino acids like isoleucine and is oxidized to 3-hydroxypropionate which is then further oxidized to acetyl-CoA (Giovanelli and Stumpf, 1958; Lucas et al., 2007; Rendina and Coon, 1957). Some animals and algae may also metabolize propionate via a similar route (Callely and Lloyd 1964; Hanarnkar et al., 1985; Lloyd et al., 1968).
- Some autotrophic CO2 fixation pathways:
- In bacteria and archaea, the reductive conversion of acetyl-CoA and CO2 to propionyl-CoA via 3-hydroxypropionate is part of two CO2 fixation pathways; however, different enzymes are used in either pathway to catalyze the common steps in the conversion of acetyl-CoA and CO2 to propionyl-CoA (Berg et al., 2007; Berg et al., 2002; Herter et al. 2001; Talarico et al., 1988). For example, the reductive conversion of 3-hydroxypropionate to propionyl-CoA is catalyzed by a fusion protein, named propionyl-CoA synthase, in Chloroflexus aurantiacus (3-hydroxypropionate bi-cycle), whereas Metallosphaera sedula (hydroxypropionate/4-hydroxybutyrate cycle) requires three separate enzymes to catalyze the same reaction sequence (Alber and Fuchs, 2002; Teufel et al., 2009).
- Dimethylsulfonopropionate (DMSP) metabolism.
- 3-Hydroxypropionate is an intermediate in the metabolism of the secondary metabolite DMSP by microorganisms (Ansede et al., 1999; Todd et al., 2007). DMSP is synthesized by marine algae and some land plants, and there are currently three different mechanisms known for the initial step of DMSP degradation: demethylation to methylmercaptopropionate (Talarico et al., 1988, Teufel et al., 2009), cleavage by a DMSP lyase into dimethylsulfide and acrylate (Ansede et al., 1999; Hanarnkar et al., 1985; Wagner and Stadtman, 1962), and the cleavage of DMSP into 3-hydroxypropionate and dimethylsulfide by an unusual CoA-transferase (Todd et al., 2007; Todd et al., 2010). Acrylate or 3-hydroxypropionate generated from the cleavage of DMSP may be further metabolized to acetyl-CoA and CO2; in the case of acrylate, this proceeds via 3-hydroxypropionate (Ansede, 1999; Ansede 2001; Todd et al., 2007). However, some bacteria use DMSP solely as a sulfur source and may therefore release 3-hydroxypropionate or acrylate as an end product (Gonzalez et al., 1999).
- Uracil degradation:
- 3-Hydroxypropionate has been identified as the end product of two different pathways for uracil degradation in bacteria like Escherichia coli as well as in the yeast Saccharomyces kluyveri (Andersen et al, 2008; Loh et al., 2006, Osterman, 2006).
- Anaerobic metabolism of glycerol:
- There have been reports of 3-hydroxypropionate formation by the fermentation of glycerol by lactic acid bacteria (Luo et al. 2011; Talarico et al., 1988) and the anaerobic oxidation of glycerol by a sulfate-reducing bacterium (Qatibi et al., 1998).
Considering its abundance, 3-hydroxypropionate is likely to play an important role in the overall carbon cycle as an end product or intermediate in the carbon metabolism of a variety of compounds. However, its use as a carbon source in bacteria is poorly understood.
Rhodobacter sphaeroides likely encounters 3-hydroxypropionate in its environment, as this compound is released from other organisms. In addition, we have demonstrated that R. sphaeroides is able to use 3-hydroxypropionate as a sole carbon source (Schneider, 2011). If 3-hydroxypropionate were exclusively oxidized to acetyl-CoA and CO2, a functional ethylmalonyl-CoA pathway for acetyl-CoA assimilation would be essential to convert acetyl-CoA for anaplerosis (see above).
However, inactivation of the gene (ccr) for crotonyl-CoA carboxylase, a neccessary enzyme of the ethylmalonyl-CoA pathway, did not affect growth during 3-hydroxypropionate-dependent growth. Instead, acuI, encoding acrylyl-CoA reductase (Asao and Alber, 2013), was shown to be essential for growth with 3-hydroxypropionate and for the reductive conversion of 3-hydroxypropionate to propionyl-CoA in cell extracts. Upon production of propionyl-CoA, the cells would be able to generate succinyl-CoA from which all of the cell's precursor metabolites can be manufactured (See Figure 4), obviating the need for the ethylmalonyl-CoA pathway.
Results regarding R. sphaeroides growth on 3-hydroxypropionate has prompted us to further investigate the functional role of acrylyl-CoA reductase (AcuI). After working toward characterizing AcuI (Asao and Alber, 2013), it became remarkably curious that the presence of ccr in trans was able to partially restore growth on 3-hydroxypropionate in an acuI null mutant. Additionally, biochemical characterization of AcuI has demonstrated that it forms a new class of acrylyl-CoA reductases (Asao and Alber, 2013), and its amino acid sequence shows similarity to Ccr. In an effort to satisfy our curiousity, we are developing an understanding of the functional characteristics that distinguish AcuI (enoyl-CoA reductase) from Ccr (enoyl-CoA carboxylase/reductase).
References
References
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