The Entner–Doudoroff Pathway
The Entner–Doudoroff pathway is an alternate series of reactions that catabolize glucose to pyruvate.
Distinguish between the Entner-Doudoroff pathway and glycolysis
- Glycolysis is the metabolic pathway that converts glucose into pyruvate and hydrogen ions.
- The Entner-Doudoroff pathway was first reported in 1952 by Michael Doudoroff and Nathan Entner.
- There are a few bacteria that substitute classic glycolysis with the Entner-Doudoroff pathway.
- glycolysis: The metabolic pathway that converts glucose into pyruvate and hydrogen ions.
- ATP: Adenosine-5′-triphosphate (ATP) is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the “molecular unit of currency” of intracellular energy transfer. ATP transports chemical energy within cells for metabolism.
The Entner–Doudoroff pathway describes an alternate series of reactions that catabolize glucose to pyruvate using a set of enzymes different from those used in either glycolysis or the pentose phosphate pathway. Glycolysis (from glycose, an older term for glucose + -lysis degradation) is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+. The free energy released in this process is used to form the high-energy compounds ATP (adenosine triphosphate) and NADH (reduced nicotinamide adenine dinucleotide). Most bacteria use glycolysis and the pentose phosphate pathway. This pathway was first reported in 1952 by Michael Doudoroff and Nathan Entner.
Distinct features of the Entner–Doudoroff pathway are that it occurs only in prokaryotes and it uses 6-phosphogluconate dehydratase and 2-keto-3-deoxyphosphogluconate aldolase to create pyruvate from glucose. The Entner–Doudoroff pathway also has a net yield of 1 ATP for every glucose molecule processed, as well as 1 NADH and 1 NADPH. By comparison, glycolysis has a net yield of 2 ATP and 2 NADH for every one glucose molecule processed.
There are a few bacteria that substitute classic glycolysis with the Entner-Doudoroff pathway. They may lack enzymes essential for glycolysis, such as phosphofructokinase-1. This pathway is generally found in Pseudomonas, Rhizobium, Azotobacter, Agrobacterium, and a few other Gram-negative genera. Very few Gram-positive bacteria have this pathway, with Enterococcus faecalis being a rare exception. Most organisms that use the pathway are aerobes due to the low ATP yield per glucose such as Pseudomonas, a genus of Gram-negative bacteria, and Azotobacter, a genus of Gram-negative bacteria.
Aerobic Hydrocarbon Oxidation
Microbes can utilize hydrocarbons via oxidation as an energy source.
Discuss the advantages of organisms that can undergo aerobic hydrocarbon oxidation
- Microbes in aerobic conditions can use hydrocarbons via oxidation of the hydrocarbon. This leads to byproducts such as water, alcohol, and peroxide.
- Many hydrocarbons are environmentally damaging, thus the break down of hydrocarbons by microbes is of special interest.
- HUM bugs can function as biosurfactants to facilitate the emulsification of hydrocarbons.
- hydrocarbon: A compound consisting only of carbon and hydrogen atoms.
- biosurfactant: Surface-active substances synthesized by living cells.
- bioremediation: The use of biological organisms, usually microorganisms, to remove contaminants, especially from polluted water.
Microbes can use many different carbon sources for energy. The best known and perhaps most common example is glucose. Microbes can utilize hydrocarbons via a stepwise oxidation of a hydrocarbon by oxygen produces water and, successively, an alcohol, an aldehyde or a ketone, a carboxylic acid, and then a peroxide. Note the presence of oxygen, thus defining this as aerobic hydrocarbon oxidation. There are examples of anaerobic hydrocarbon oxidation, which will not be discussed here. This is of special interest as many of the environment pollutants released by human industry are often hydrocarbon based. One of the best examples is oil spills. Understanding how microbes digest hydrocarbons has started the field of microbial biodegradation, a type of bioremediation. The goal of this is to find ways of using microbes to degrade hydrocarbon spills or waste into less dangerous byproducts such as alcohol.
Hydrocarbon utilizing microorganisms, mostly Cladosporium resinae and Pseudomonas aeruginosa, colloquially known as “HUM bugs,” are commonly present in jet fuel. They live in the water-fuel interface of the water droplets, form dark black/brown/green, gel-like mats, and cause microbial corrosion to plastic and rubber parts of the aircraft fuel system by consuming them, and to the metal parts by the means of their acidic metabolic products. They are also incorrectly called algae due to their appearance. FSII, which is added to the fuel, acts as a growth retardant for them. There are about 250 kinds of bacteria that can live in jet fuel, but fewer than a dozen are meaningfully harmful.
Biosurfactants are surface-active substances synthesized by living cells. Interest in microbial surfactants has been steadily increasing in recent years due to their diversity, environmentally friendly nature, possibility of large-scale production, selectivity, performance under extreme conditions, and potential applications in environmental protection. Biosurfactants enhance the emulsification of hydrocarbons, have the potential to solubilize hydrocarbon contaminants, and increase their availability for microbial degradation. The use of chemicals for the treatment of a hydrocarbon polluted site may contaminate the environment with their by-products, whereas biological treatment may efficiently destroy pollutants, while being biodegradable themselves. Therefore, biosurfactant-producing microorganisms may play an important role in the accelerated bioremediation of hydrocarbon-contaminated sites. These compounds can also be used in enhanced oil recovery and may be considered for other potential applications in environmental protection. Other applications include herbicides and pesticides formulations, detergents, healthcare and cosmetics, pulp and paper, coal, textiles, ceramic processing and food industries, uranium ore-processing, and mechanical dewatering of peat. Several microorganisms are known to synthesize surface-active agents; most of them are bacteria and yeasts. When grown on hydrocarbon substrate as the carbon source, these microorganisms synthesize a wide range of chemicals with surface activity, such as glycolipid, phospholipid, and others. These chemicals are synthesized to emulsify the hydrocarbon substrate and facilitate its transport into the cells. In some bacterial species such as Pseudomonas aeruginosa, biosurfactants are also involved in a group motility behavior called swarming motility.
The Pentose Phosphate Shunt
The pentose phosphate pathway (PPP) converts glucose-6-phosphate into NADPH and pentoses (5-carbon sugars).
Outline the two major phases of the pentose phosphate shunt: oxidative and non-oxidative phases
- There are two distinct phases in the pathway: the oxidative phase and the non-oxidative phase.
- In the oxidative phase, two molecules of NADP+ are reduced to NADPH, utilizing the energy from the conversion of glucose-6-phosphate into ribulose-5-phosphate. These NADPH molecules can then be used as an energy source in elsewhere in the cell.
- The non-oxidative phase generates 5-carbon sugars, which can be used in the synthesis of nucleotides, nucleic acids, and amino acids.
- The pentose phosphate pathway is an alternative to glycolysis.
- glycolysis: The cellular degradation of the simple sugar glucose to yield pyruvic acid and ATP as an energy source.
- NADPH: Nicotinamide adenine dinucleotide phosphate (NADP) carrying electrons and bonded with a hydrogen (H) ion; the reduced form of NADP+.
- oxidative stress: Damage caused to cells or tissue by reactive oxygen species.
The pentose phosphate pathway (PPP; also called the phosphogluconate pathway and the hexose monophosphate shunt) is a process that breaks down glucose-6-phosphate into NADPH and pentoses (5-carbon sugars) for use in downstream biological processes.
There are two distinct phases in the pathway: the oxidative phase and the non-oxidative phase. The first is the oxidative phase in which glucose-6-phosphate is converted to ribulose-5-phosphate. During this process two molecules of NADP+ are reduced to NADPH. The overall reaction for this process is:
Glucose 6-phosphate + 2 NADP+ + H2O → ribulose-5-phosphate + 2 NADPH + 2 H+ + CO2
The second phase of this pathway is the non-oxidative synthesis of 5-carbon sugars. Depending on the body’s state, ribulose-5-phosphate can reversibly isomerize to ribose-5-phosphate. Ribulose-5-phosphate can alternatively undergo a series of isomerizations as well as transaldolations and transketolations that result in the production of other pentose phosphates including fructose-6-phosphate, erythrose-4-phosphate, and glyceraldehyde-3-phosphate (both intermediates in glycolysis). These compounds are used in a variety of different biological processes including production of nucleotides and nucleic acids (ribose-5-phosphate), as well as synthesis of aromatic amino acids (erythrose-4-phosphate).
Glucose-6-phosphate dehydrogenase is the rate-controlling enzyme in this pathway. It is allosterically stimulated by NADP+. NADPH-utilizing pathways, such as fatty acid synthesis, generate NADP+, which stimulates glucose-6-phosphate dehydrogenase to produce more NADPH. In mammals, the PPP occurs exclusively in the cytoplasm; it is found to be most active in the liver, mammary gland, and adrenal cortex. The ratio of NADPH:NADP+ is normally about 100:1 in liver cytosol, making the cytosol a highly-reducing environment.
The PPP is one of the three main ways the body creates molecules with reducing power, accounting for approximately 60% of NADPH production in humans. While the PPP does involve oxidation of glucose, its primary role is anabolic rather than catabolic, using the energy stored in NADPH to synthesize large, complex molecules from small precursors.
Additionally, NADPH can be used by cells to prevent oxidative stress. NADPH reduces glutathione via glutathione reductase, which converts reactive H2O2 into H2O by glutathione peroxidase. For example, erythrocytes generate a large amount of NADPH through the pentose phosphate pathway to use in the reduction of glutathione.
Organic Acid Metabolism
Microbes can harness energy and carbon derived from organic acids by using a variety of dedicated metabolic enzymes.
Give examples of types of organic acid metabolism that are used by microorganisms for a sole source of energy
- Some microbes are capable of utilizing organic acids such as fatty acids, amino acids, or straight-chain unsaturated acids (e.g., lactate) as a sole source of energy.
- Metabolism of the organic acid formate is important in methylotrophic organisms. It is vital in the catabolism of C1 compounds (such as methanol).
- Many bacteria are capable of utilizing fatty acids as sole energy and carbon sources through the cyclic β-oxidation pathway, which ultimately yields acetyl-CoA.
- fatty acid: Any of a class of aliphatic carboxylic acids, of general formula CnH2n+1COOH, that occur combined with glycerol as animal or vegetable oils and fats. Only those with an even number of carbon atoms are normally found in natural fats.
- acyl: Any of class of organic radicals, RCO-, formed by the removal of a hydroxyl group from a carboxylic acid.
- acetyl CoA: Acetyl coenzyme A or acetyl-CoA is an important molecule in metabolism, used in many biochemical reactions. Its main function is to convey the carbon atoms within the acetyl group to the citric acid cycle (Krebs cycle) to be oxidized for energy production.
Organic Acid Metabolism
A great many organisms generate organic acids (such as lactate) as a byproduct of fermentation. Some microbes are capable of utilizing such compounds as a sole source of energy.
The most commonly metabolized organic acids are the carboxylic acids, which are organic acids containing at least one carboxyl (-COOH) group. The general formula of a carboxylic acid is R-COOH, where R is a monovalent functional group. Many types of carboxylic acids can be metabolized by microbes, including:
- Fatty acids (carboxylic acids with long acyl tails)
- Amino acids (the building blocks of proteins)
- Straight-chained, saturated acids (e.g., formate, acetate, and palmitate)
Formate metabolism is important in methylotrophic organisms. It is vital in the catabolism of C1 compounds such as methanol (see the “Methylotrophy and Methanotrophy” atom for more information on C1 compound utilization). Methylotrophic microbes convert single-carbon compounds to formaldehyde, which is oxidized to formate by formaldehyde dehydrogenase. Degradation of formate is then catalyzed by formate dehydrogenase (FDH), which oxidizes formate to ultimately yield CO2. It permits the donation of electrons to a second substrate (such as NAD+) in the process. This is a critical late step in the hydrocarbon utilization pathway. The ability to metabolize formate is also critical in bacterial anaerobic metabolism, in which case formate is also oxidized by an FDH enzyme but the electrons are donated to cytochromes (proteins involved in electron transport).
FATTY ACID METABOLISM
Many bacteria are capable of utilizing fatty acids of various tail lengths as sole energy and carbon sources. This process requires the β-oxidation pathway, a cyclic process that catalyzes the sequential shortening of fatty acid acyl chains to the final product, acetyl-CoA. The step-by-step process occurs as follows:
- Fatty acid chains are converted to enoyl-CoA (catalyzed by acyl-CoA dehydrogenase).
- Enoyl-CoA is converted to 3-hydroxyacyl-CoA (catalyzed by enoyl-CoA hydratase).
- 3-hydroxyacyl-CoA is converted to 3-ketoacyl-CoA (catalyzed by 3-hydroxyacyl-CoA dehydrogenase).
- 3-ketoacyl-CoA is thiolated (by 3-ketoacyl-CoA thiolase) to yield one molecule of acetyl-CoA and a derivative of the original input fatty acid that is now shorter by two carbons.
The fatty acid chain that is left over after the thiolation step can then reenter the β-oxidation pathway, which can cycle until the fatty acid has been completely reduced to acetyl-CoA. Acertyl-CoA is the entry molecule for the TCA cycle. The TCA cycle is the process used by all aerobic organisms to generate energy.
Biological lipids, which are broken down and utilized though β-oxidation, represent a potent energy source.
Outline the process of lipid metabolism, specifically beta-oxidation
- In addition to their role as the primary component of cell membranes, lipids can be metabolized for use as a primary energy source.
- Lipid metabolism involves the degradation of fatty acids, which are fundamental biological molecules and the building blocks of more structurally complex lipids.
- In order to be metabolized by the cell, lipids are hydrolyzed to yield free fatty acids that then converted to acetyl-CoA through the β- oxidation pathway.
- One major feature of anaerobic digestion is the production of biogas (with the most useful component being methane), which can be used in generators for electricity production and/or in boilers for heating purposes.
- carboxylic acid: Any of a class of organic compounds containing a carboxyl functional group.
- coenzyme A: A coenzyme, formed from pantothenic acid and adenosine triphosphate, that is necessary for fatty acid synthesis and metabolism.
Lipids are universal biological molecules. Not only does this broad class of compounds represent the primary structural component of biological membranes in all organisms, they also serve a number of vital roles in microorganisms. Among these, lipids can be metabolized by microbes for use as a primary energy source. Although not stated explicitly, the “Organic Acid Metabolism” atom in this module introduces the concept of lipid metabolism by describing the process of fatty acid metabolism through β-oxidation. This atom will expand on the metabolic pathway that enables degradation and utilization of lipids. Fatty acids are the building blocks of lipids. They are made of a hydrocarbon chain of variable length that terminates with a carboxylic acid group (-COOH). The fatty acid structure (see below) is one of the most fundamental categories of biological lipids. It is commonly used as a building block of more structurally complex lipids (such as phospholipids and triglycerides). When metabolized, fatty acids yield large quantities of ATP, which is why these molecules are important energy sources. Lipids are an energy and carbon source. Before complex lipids can be used to produce energy, they must first be hydrolyzed. This requires the activity of hydrolytic enzymes called lipases, which release fatty acids from derivatives such as phospholipids. These fatty acids can then enter a dedicated pathway that promotes step-wise lipid processing that ultimately yields acetyl-CoA, a critical metabolite that conveys carbon atoms to the TCA cycle (aka Krebs cycle or citric acid cycle) to be oxidized for energy production.
The metabolic process by which fatty acids and their lipidic derivatives are broken down is called β-oxidation. This process bears significant similarity to the mechanism by which fatty acids are synthesized, except in reverse. In brief, the oxidation of lipids proceeds as follows: two-carbon fragments are removed sequentially from the carboxyl end of the fatty acid after dehydrogenation, hydration, and oxidation to form a keto acid, which is then cleaved by thiolysis. The acetyl-CoA molecule liberated by this process is eventually converted into ATP through the TCA cycle.
β-oxidation can be broken down into a series of discrete steps:
- Activation: Before fatty acids can be metabolized, they must be “activated. ” This activation step involves the addition of a coenzyme A (CoA) molecule to the end of a long-chain fatty acid, after which the activated fatty acid (fatty acyl -CoA) enters the β-oxidation pathway.
- Oxidation: The initial step of β-oxidation is catalyzed by acyl-CoA dehydrogenase, which oxidizes the fatty acyl-CoA molecule to yield enoyl-CoA. As a result of this process, a trans double bond is introduced into the acyl chain.
- Hydration: In the second step, enoyl-CoA hydratase hydrates the double bond introduced in the previous step, yielding an alcohol (-C-OH).
- Oxidation: Hydroxyacyl-CoA dehydrogenase oxidizes the alcohol formed in the previous step to a carbonyl (-C=O).
- Cleavage: A thiolase then cleaves off acetyl-CoA from the oxidized molecule, which also yields an acyl-CoA that is two carbons shorter than the original molecule that entered the β-oxidation pathway.
This cycle repeats until the fatty acid has been completely reduced to acetyl-CoA, which is fed through the TCA cycle to ultimately yield cellular energy in the form of ATP.
Connecting Proteins to Glucose Metabolism
Excess amino acids are converted into molecules that can enter the pathways of glucose catabolism.
Describe the role played by proteins in glucose metabolism
- Amino acids must be deaminated before entering any of the pathways of glucose catabolism: the amino group is converted to ammonia, which is used by the liver in the synthesis of urea.
- Deaminated amino acids can be converted into pyruvate, acetyl CoA, or some components of the citric acid cycle to enter the pathways of glucose catabolism.
- Several amino acids can enter the glucose catabolism pathways at multiple locations.
- catabolism: Destructive metabolism, usually including the release of energy and breakdown of materials.
- keto acid: Any carboxylic acid that also contains a ketone group.
- deamination: The removal of an amino group from a compound.
Metabolic pathways should be thought of as porous; that is, substances enter from other pathways and intermediates leave for other pathways. These pathways are not closed systems. Many of the substrates, intermediates, and products in a particular pathway are reactants in other pathways. Proteins are a good example of this phenomenon. They can be broken down into their constituent amino acids and used at various steps of the pathway of glucose catabolism.
Proteins are hydrolyzed by a variety of enzymes in cells. Most of the time, the amino acids are recycled into the synthesis of new proteins or are used as precursors in the synthesis of other important biological molecules, such as hormones, nucleotides, or neurotransmitters. However, if there are excess amino acids, or if the body is in a state of starvation, some amino acids will be shunted into the pathways of glucose catabolism.
Each amino acid must have its amino group removed (deamination) prior to the carbon chain’s entry into these pathways. When the amino group is removed from an amino acid, it is converted into ammonia through the urea cycle. The remaining atoms of the amino acid result in a keto acid: a carbon chain with one ketone and one carboxylic acid group. In mammals, the liver synthesizes urea from two ammonia molecules and a carbon dioxide molecule. Thus, urea is the principal waste product in mammals produced from the nitrogen originating in amino acids; it leaves the body in urine. The keto acid can then enter the citric acid cycle.
When deaminated, amino acids can enter the pathways of glucose metabolism as pyruvate, acetyl CoA, or several components of the citric acid cycle. For example, deaminated asparagine and aspartate are converted into oxaloacetate and enter glucose catabolism in the citric acid cycle. Deaminated amino acids can also be converted into another intermediate molecule before entering the pathways. Several amino acids can enter glucose catabolism at multiple locations.
Methylotrophy and Methanotrophy
Methylotrophs and methanotrophs are a diverse group of microorganisms that can derive energy from the metabolism of single-carbon compounds.
Distinguish between methylotrophs and methanotrophs and their energy sources
- Microbes with the ability to utilize single-carbon (C1) compounds (or multi-carbon compounds lacking carbon bonds) as the sole energy source for their growth are known as methylotrophs.
- Methanotrophs, a specific type of methylotroph, are able to metabolize methane as their only source of carbon and energy.
- Methylotrophs aerobically utilize C1 compounds by oxidizing them to yield formaldehyde, which in turn can either be used for energy or assimilated into biomass.
- methylotroph: Any organism that utilizes simple methyl compounds (such as methane or methanol) as a source of carbon and of energy.
- monooxygenase: Any oxygenase enzyme that catalyzes the incorporation of a single atom of molecular oxygen into a substrate, the other atom being reduced to water; active in the metabolism of many foreign compounds.
- methanogenesis: The generation of methane by anaerobic bacteria.
Multiple diverse microorganisms have evolved the intriguing ability to utilize single-carbon (C1) compounds (e.g. methanol or methane) or multi-carbon compounds lacking carbon bonds (e.g. dimethyl ether and dimethylamine) as the sole energy source for their growth. Microbes with this capability are known as methylotrophs.
Methylotrophs, in general, aerobically utilize C1 compounds by oxidizing them to yield formaldehyde. Formaldehyde, in turn, can either be “burned” for energy (by dissimilation to CO2) or assimilated into biomass, allowing the cell to grow using molecules like methanol as a sole carbon source. Because methanol is more abundant, more easily purified, and cheaper than sugar carbon sources (e.g. glucose), methylotrophs are particularly useful in biotechnology for the production of amino acids, vitamins, recombinant proteins, single-cell proteins, co- enzymes, and cytochromes.
Here are examples of methylotrophs:
- Methanosarcina, which can both utilize and produce methane;
- Methylococcus capsulatus, which requires methane to survive; and
- Pichia pastoris, a biotechnologically important model organism that can use methanol as a carbon and energy source.
Some methylotrophs can degrade the greenhouse gas methane. Organisms of this type are referred to as methanotrophs. Methanotrophs are able to metabolize methane as their only source of carbon and energy. Most known methanotrophs are bacteria that strictly require methane for growth (“obligate methanotrophs”). The fact that some methylotrophs can also make use of multi-carbon compounds distinguishes them from methanotrophs, which are usually fastidious methane and methanol oxidizers.
Methanotrophs occur mostly in soils. They are especially common near environments where methane is produced, such as:
- underground environments
- rice paddies
Methanotrophs are of special interest to researchers studying global warming because they prevent a potential greenhouse gas (methane), far more potent than carbon dioxide, from being released into the atmosphere. Methanophilic (“methane-loving”) bacteria, therefore, are significant in the global methane budget.
Methanotrophs oxidize methane by first initiating reduction of oxygen (O2) to water (H2O) and oxidation of methane (CH4) to a more active species, methanol (CH3OH), using oxidoreductase enzymes called methane monooxygenases (MMOs). Two types of MMO have been isolated from methanotrophs:
- soluble methane monooxygenase (sMMO), which is found in the cell cytoplasm.
- particulate methane monooxygenase (pMMO), which is found in the cell membrane.
Cells containing pMMO demonstrate higher growth capabilities and higher affinity for methane than cells that contain sMMO. Because pMMO is a membrane protein, cells that use it for methane metabolism characteristically have a system of internal membranes within which methane oxidation occurs.
As in the general case described above for methylotrophs, methanotrophs ultimately oxidize the methanol produced by MMOs to yield formaldehyde. The method of formaldehyde fixation differs between various methanotrophic organisms. This difference (along with variability in membrane structure) divides methanotrophs into several subgroups, such as the Methylococcaceae, Methylocystaceae, and Verrucomicrobiae. Although the mechanism by which it occurs is not entirely clear, it is also apparent that certain bacteria can utilize methane anaerobically by essentially running the methanogenesis pathway (normally used by methanogenic bacteria to produce methane) in reverse. This typically occurs in microbes dwelling in marine sediments where oxygen is scarce or altogether absent.