Carnitine O-octanoyltransferase (CROT or COT) is a member of the transferase family, more specifically a carnitine acyltransferase, a type of enzyme which catalyzes the transfer of acyl groups from acyl-CoAs to carnitine, generating CoA and an acyl-carnitine. (EC2.3.1.137) Specifically, CROT catalyzes the chemical reaction:
This reaction is easily reversible, and does not require any energy input, as both fatty acyl-CoAs and fatty acylcarnitines are considered chemically “activated” forms of fatty acyl groups.[2]
Nomenclature
The systematic name of this enzyme is octanoyl-CoA:L-carnitine O-octanoyltransferase. Other names in common use include:
As of late 2007, 4 structures have been solved for this class of enzymes, with PDB accession codes 1XL7, 1XL8, 1XMC, and 1XMD.
CROT in complex with carnitine
Another representation of the CROT active site pocket in complex with carnitine
The significant catalytic residue within all carnitine acyltransferases, including CROT, has been found to be a histidine residue, confirmed by site-directed mutagenesis studies. In CROT, this residue is at position 327. This residue, including the rest of the active site, is found at the interface between the N and C domains. The active site stabilizes carnitine by an intricate network of hydrogen-bonding residues, along with a key water molecule. The longer acyl chain is stabilized by hydrophobic residues arrayed in an approximately-cylindrical fashion.[3]
As may be expected from members of the same enzymatic family, there is strong similarity between the structures of carnitine acetyltransferase (CRAT) and CROT, as these enzymes have 36% sequence homology.[3] A key difference between these enzymes that may explain their selectivities between short and medium-chain acyl-CoAs hinges on a glycine residue which is present in the acyl binding site in CROT, Gly-553. In CRAT, however, the residue in the same position in the acyl binding site is a methionine residue, Met-564.[3] These residues has been shown to serve as a substrate “gatekeeper” in both CRAT and CROT. M564G CRAT mutants have been shown to accept a wider variety of acyl-CoA substrates.[4] Similarly, G553M CROT mutants show marked inactivity with octanoyl-CoA, while maintaining activity with short-chain acyl-CoAs.[3]
Function
One function of CROT is to supply acetyl-CoA to glucose-starved cells. In the absence of carnitine acetyltransferase (CRAT), acyltransferases such as CROT can catalyze the acetyl group transfer from acetylcarnitine to coenzyme A. Rescue experiments with CROT gene knockout cells have shown that peroxisomal CROT can mediate acetyl-CoA production under glucose-limited conditions. The peroxisome can then export these products into the cytosol.[5]
Localization
Though CROT is distributed on both sides of microsomal vesicles, it has also been found that the bulk of CROT activity in murine liver is in the cytoplasm face of the vesicles and endoplasmic reticulum. CROT may play a role in converting peroxisomal medium-chain acylcarnitine derivatives to medium-chain acyl-CoA derivatives. These can then feed into a variety of biosynthetic pathways for elongations and other modifications.[6]
In addition, CROT is inhibited by trypsin in a dose-dependent manner. A maximum of 60% inhibition was observed in purified CROT, similar to what was seen with carnitine palmitoyltransferase (CPT). CROT activity also appears to be inhibited to the same extent in both permeable and sealed microsomal membranes.[6]
CROT is thought to be peroxisomally-located. It was found that administration of di(2-ethylhexyl)phthalate (DEHP), a peroxisomal proliferator, to Wistar rats led to an increase in the expression of CROT by a factor of 14.1. This was as a result of increased translation of CROT mRNA, along with decreased degradation by a factor of 1.5.[7]
CROT activity has also been reported in mouse liver, kidney, adipocyte, mammary gland, skeletal muscle, and heart tissues. It was found that CROT activity in the kidney was mostly overt, while in the liver and heart it was mainly latent. Interestingly, the trend for a related enzyme, carnitine palmitoyl transferase (CPT), the opposite trend was found.[8]
Substrates
Canonical CROT reaction
Other acyl-CoAs which CROT can take as substrates
While CROT's canonical substrate is octanoyl-CoA, CROT is also known to be able to catalyze the deacylation of numerous acyl-CoAs, such as acetyl-CoA, propionyl-CoA, butyryl-CoA, and hexanoyl-CoA.[5] CROT can also take branched-chain fatty acyl-CoAs as substrates, such as 4,8-dimethylnonanoyl-CoA, which is derived from the metabolism of pristanic acid in the peroxisome.[9]
Regulation
Because CROT activity has a role in beta-oxidation of fatty acids and ketone body synthesis, it is an important point of regulation. One known inhibitor of CROT is malonyl-CoA, which inhibits CROT non-linearly. Complex kinetic behavior is observed when malonyl-CoA is incubated with purified CROT.
A decrease in pH can also enhance malonyl-CoA inhibition of CROT. Some studies have indicated that when the pH of assaying conditions was decreased from 7.4 to 6.8, inhibition could increase by 20-30%. Further, the Ki for malonyl-CoA in CROT decreases from 106 uM to 35 uM over this drop. This change is not seen for palmitoyl-CoA and decanoyl-CoA. However, the degree of inhibition by malonyl-CoA is similar to that observed with other short-chain acyl-CoA esters, such as glutaryl-CoA, hydroxymethylglutaryl-CoA, and methylmalonyl-CoA.
The ionization state of malonyl-CoA does not change significantly over the pH range 7.4-6.8. The change in sensitivity to inhibitors may be due to the CROT active-site His-327 residue. Malonyl-CoA is also found at a lower concentration in the cell (1-6 uM) than its Ki.Thus, its inhibition of CROT may not be physiologically significant under homeostatic conditions.[10]
Related Research Articles
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In biochemistry and metabolism, beta oxidation (also β-oxidation) is the catabolic process by which fatty acid molecules are broken down in the cytosol in prokaryotes and in the mitochondria in eukaryotes to generate acetyl-CoA. Acetyl-CoA enters the citric acid cycle, generating NADH and FADH2, which are electron carriers used in the electron transport chain. It is named as such because the beta carbon of the fatty acid chain undergoes oxidation and is converted to a carbonyl group to start the cycle all over again. Beta-oxidation is primarily facilitated by the mitochondrial trifunctional protein, an enzyme complex associated with the inner mitochondrial membrane, although very long chain fatty acids are oxidized in peroxisomes.
Numerous genetic disorders are caused by errors in fatty acid metabolism. These disorders may be described as fatty oxidation disorders or as a lipid storage disorders, and are any one of several inborn errors of metabolism that result from enzyme defects affecting the ability of the body to oxidize fatty acids in order to produce energy within muscles, liver, and other cell types.
Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin carboxylase (BC) and carboxyltransferase (CT). ACC is a multi-subunit enzyme in most prokaryotes and in the chloroplasts of most plants and algae, whereas it is a large, multi-domain enzyme in the cytoplasm of most eukaryotes. The most important function of ACC is to provide the malonyl-CoA substrate for the biosynthesis of fatty acids. The activity of ACC can be controlled at the transcriptional level as well as by small molecule modulators and covalent modification. The human genome contains the genes for two different ACCs—ACACA and ACACB.
Malonyl-CoA is a coenzyme A derivative of malonic acid.
Carnitine palmitoyltransferase II deficiency, sometimes shortened to CPT-II or CPT2, is an autosomal recessively inherited genetic metabolic disorder characterized by an enzymatic defect that prevents long-chain fatty acids from being transported into the mitochondria for utilization as an energy source. The disorder presents in one of three clinical forms: lethal neonatal, severe infantile hepatocardiomuscular and myopathic.
Carnitine O-palmitoyltransferase is a mitochondrial transferase enzyme involved in the metabolism of palmitoylcarnitine into palmitoyl-CoA. A related transferase is carnitine acyltransferase.
Malonyl-CoA decarboxylase, is found in bacteria and humans and has important roles in regulating fatty acid metabolism and food intake, and it is an attractive target for drug discovery. It is an enzyme associated with Malonyl-CoA decarboxylase deficiency. In humans, it is encoded by the MLYCD gene.
Acyl-CoA is a group of coenzymes that metabolize carboxylic acids. Fatty acyl-CoA's are susceptible to beta oxidation, forming, ultimately, acetyl-CoA. The acetyl-CoA enters the citric acid cycle, eventually forming several equivalents of ATP. In this way, fats are converted to ATP, the common biochemical energy carrier.
Fatty acid degradation is the process in which fatty acids are broken down into their metabolites, in the end generating acetyl-CoA, the entry molecule for the citric acid cycle, the main energy supply of living organisms, including bacteria and animals. It includes three major steps:
The long chain fatty acyl-CoA ligase is an enzyme of the ligase family that activates the oxidation of complex fatty acids. Long chain fatty acyl-CoA synthetase catalyzes the formation of fatty acyl-CoA by a two-step process proceeding through an adenylated intermediate. The enzyme catalyzes the following reaction,
Carnitine palmitoyltransferase I (CPT1) also known as carnitine acyltransferase I, CPTI, CAT1, CoA:carnitine acyl transferase (CCAT), or palmitoylCoA transferase I, is a mitochondrial enzyme responsible for the formation of acyl carnitines by catalyzing the transfer of the acyl group of a long-chain fatty acyl-CoA from coenzyme A to l-carnitine. The product is often palmitoylcarnitine, but other fatty acids may also be substrates. It is part of a family of enzymes called carnitine acyltransferases. This "preparation" allows for subsequent movement of the acyl carnitine from the cytosol into the intermembrane space of mitochondria.
Thiolases, also known as acetyl-coenzyme A acetyltransferases (ACAT), are enzymes which convert two units of acetyl-CoA to acetoacetyl CoA in the mevalonate pathway.
Pantothenate kinase (EC 2.7.1.33, PanK; CoaA) is the first enzyme in the Coenzyme A (CoA) biosynthetic pathway. It phosphorylates pantothenate (vitamin B5) to form 4'-phosphopantothenate at the expense of a molecule of adenosine triphosphate (ATP). It is the rate-limiting step in the biosynthesis of CoA.
Palmitoyl-CoA hydrolase (EC 3.1.2.2) is an enzyme in the family of hydrolases that specifically acts on thioester bonds. It catalyzes the hydrolysis of long chain fatty acyl thioesters of acyl carrier protein or coenzyme A to form free fatty acid and the corresponding thiol:
In enzymology, a [acyl-carrier-protein] S-acetyltransferase is an enzyme that catalyzes the reversible chemical reaction
Carnitine O-acetyltransferase also called carnitine acetyltransferase is an enzyme that encoded by the CRAT gene that catalyzes the chemical reaction
Fatty-acyl-CoA synthase, or more commonly known as yeast fatty acid synthase, is an enzyme complex responsible for fatty acid biosynthesis, and is of Type I Fatty Acid Synthesis (FAS). Yeast fatty acid synthase plays a pivotal role in fatty acid synthesis. It is a 2.6 MDa barrel shaped complex and is composed of two, unique multi-functional subunits: alpha and beta. Together, the alpha and beta units are arranged in an α6β6 structure. The catalytic activities of this enzyme complex involves a coordination system of enzymatic reactions between the alpha and beta subunits. The enzyme complex therefore consists of six functional centers for fatty acid synthesis.
Peroxisomal carnitine O-octanoyltransferase is an enzyme that in humans is encoded by the CROT gene.
↑ Ferdinandusse S, Mulders J, IJlst L, Denis S, Dacremont G, Waterham HR, etal. (September 1999). "Molecular cloning and expression of human carnitine octanoyltransferase: evidence for its role in the peroxisomal beta-oxidation of branched-chain fatty acids". Biochemical and Biophysical Research Communications. 263 (1): 213–218. doi:10.1006/bbrc.1999.1340. PMID10486279.
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