GO:0050353 trimethyllysine dioxygenase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0050353 (trimethyllysine dioxygenase activity) catalyzes the hydroxylation of N6,N6,N6-trimethyl-L-lysine to 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, the first committed step of carnitine biosynthesis.
• The reaction is a 2-oxoglutarate (alpha-ketoglutarate, alphaKG)-dependent dioxygenation that consumes O2 and produces CO2 and succinate.
• The human enzyme is TMLHE (trimethyllysine hydroxylase, epsilon), a non-heme Fe(II)/alphaKG-dependent oxygenase localized to mitochondria.
• Loss of TMLHE function blocks carnitine synthesis and has been linked to autism spectrum disorder and carnitine deficiency.
• alphaKG-mediated carnitine synthesis supports DNA repair and histone acetylation, linking this activity to chromatin regulation and genome stability.
• TMLHE is a tractable target for CRISPR knockout, point-mutation, knock-in, and overexpression models to dissect carnitine-dependent and chromatin-dependent phenotypes.
Description
GO:0050353, trimethyllysine dioxygenase activity, is a molecular function that catalyzes the conversion of N6,N6,N6-trimethyl-L-lysine (TML) to 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, the first committed step in the carnitine biosynthesis pathway. This reaction is a 2-oxoglutarate (alphaKG)-dependent dioxygenation that requires molecular oxygen and produces carbon dioxide and succinate. The human enzyme responsible for this activity is TMLHE (trimethyllysine hydroxylase, epsilon), a non-heme Fe(II)/alphaKG-dependent oxygenase. Because carnitine is essential for mitochondrial fatty acid beta-oxidation, this activity sits at the interface of amino acid catabolism, energy metabolism, and epigenetic regulation. Researchers study GO:0050353 to understand how metabolic flux through carnitine synthesis influences chromatin modifications, DNA repair, and neurodevelopmental outcomes.
trimethyllysine dioxygenase activity At A Glance
| GO ID | GO:0050353 |
|---|---|
| GO term | trimethyllysine dioxygenase activity |
| Ontology | molecular_function |
| Synonym | TMLD activity; TML hydroxylase activity; trimethyllysine,2-oxoglutarate dioxygenase activity |
| Major function | Catalyzes the first committed step of carnitine biosynthesis: hydroxylation of N6,N6,N6-trimethyl-L-lysine |
| Cofactors | Non-heme Fe(II) and 2-oxoglutarate (alphaKG) |
| Substrates | N6,N6,N6-trimethyl-L-lysine, 2-oxoglutarate, O2 |
| Products | 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, CO2, succinate |
| Human gene | TMLHE (trimethyllysine hydroxylase, epsilon) |
What Is GO:0050353?
In plain terms, trimethyllysine dioxygenase activity is the enzyme activity that adds a hydroxyl group to trimethyllysine using oxygen, while converting 2-oxoglutarate to succinate and releasing carbon dioxide. This is the first step in making carnitine, a molecule required for fatty acid transport into mitochondria.
Why Is trimethyllysine dioxygenase activity Important in Cell Biology?
GO:0050353 is important because it gates the entire carnitine biosynthesis pathway, and carnitine is indispensable for mitochondrial long-chain fatty acid oxidation and energy homeostasis. Beyond bioenergetics, recent work shows that alphaKG-mediated carnitine synthesis supports DNA repair and histone acetylation, connecting this enzymatic activity to chromatin regulation and genome stability. Clinically, loss of TMLHE function causes carnitine deficiency and has been associated with autism spectrum disorder, making this activity a focus for neurodevelopmental and metabolic research.
• It is the rate-limiting first step of carnitine biosynthesis, controlling flux into the pathway.
• Carnitine is required for mitochondrial fatty acid beta-oxidation and energy production.
• The reaction consumes alphaKG and O2, linking it to the TCA cycle and oxygen sensing.
• alphaKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation.
• TMLHE deficiency causes carnitine deficiency and has been linked to autism spectrum disorder.
• The enzyme is a non-heme Fe(II)/alphaKG-dependent oxygenase, a druggable enzyme class.
• It provides a metabolic entry point for studying chromatin-modifying metabolite availability.
• Ascorbate (vitamin C) supports carnitine biosynthesis, implicating redox status in pathway output.
• Vitamin D metabolites can regulate lipid beta-oxidation and carnitine biosynthesis.
• It is a tractable target for CRISPR-based metabolic and epigenetic screens.
What Happens During trimethyllysine dioxygenase activity?
Substrate recognition and binding
In simple terms: The enzyme grabs trimethyllysine and alphaKG so they sit next to each other in the active site.
TMLHE binds N6,N6,N6-trimethyl-L-lysine and 2-oxoglutarate in a non-heme Fe(II) active site, positioning the substrates for oxidative chemistry. The active site architecture of human trimethyllysine hydroxylase has been investigated to define residues that coordinate the metal and recognize the trimethyllysine side chain.
Oxidative decarboxylation of 2-oxoglutarate
In simple terms: Oxygen is split, and one part is used to convert alphaKG into succinate and CO2.
In the canonical alphaKG-dependent dioxygenase mechanism, O2 is activated at the Fe(II) center, one oxygen atom is incorporated into succinate, and the other is transferred to the substrate; CO2 is released from the decarboxylation of 2-oxoglutarate. This step is shared with other Fe(II)/alphaKG oxygenases such as JMJD3.
Hydroxylation of trimethyllysine
In simple terms: The enzyme adds an OH group to trimethyllysine, making 3-hydroxy-trimethyllysine.
The activated oxygen species hydroxylates N6,N6,N6-trimethyl-L-lysine at the 3-position to yield 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, the product of GO:0050353. This hydroxylated intermediate is subsequently cleaved to glycine and 4-trimethylaminobutyraldehyde in the next steps of carnitine biosynthesis.
Coupling to carnitine biosynthesis and downstream metabolism
In simple terms: The product made by this enzyme is the raw material for making carnitine.
The 3-hydroxy-trimethyllysine produced by TMLHE is converted through subsequent enzymatic steps to carnitine, which is required for mitochondrial fatty acid import and beta-oxidation. Ascorbate (vitamin C) supports carnitine biosynthesis, and vitamin D metabolites can regulate lipid beta-oxidation and carnitine biosynthesis, indicating that pathway output is sensitive to nutritional and hormonal status.
Metabolic and epigenetic consequences
In simple terms: This enzyme's activity affects not only energy metabolism but also how DNA is repaired and how genes are turned on.
alphaKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation, linking GO:0050353 to chromatin state and DNA repair. Because alphaKG is a co-substrate for many chromatin-modifying enzymes, changes in TMLHE activity can influence the availability of metabolites that regulate histone acetylation and gene expression.
Key Genes Involved in GO:0050353 trimethyllysine dioxygenase activity
The following genes and proteins are directly or functionally connected to trimethyllysine dioxygenase activity (GO:0050353) and its downstream carnitine biosynthesis and chromatin-related processes.
| Gene | Major Role | Research Relevance |
|---|---|---|
| TMLHE | Encodes trimethyllysine hydroxylase, the enzyme with GO:0050353 activity | Core target for knockout, point-mutation, and overexpression studies of carnitine biosynthesis |
| TMLHE (active site variants) | Residues coordinating Fe(II) and recognizing trimethyllysine | Point-mutation models to dissect catalytic mechanism and substrate specificity |
| SLC25A20 | Carnitine-acylcarnitine translocase for mitochondrial fatty acid import | Downstream readout of carnitine availability in TMLHE models |
| CPT1A | Carnitine palmitoyltransferase 1, rate-limiting for fatty acid oxidation | Functional readout of carnitine-dependent beta-oxidation |
| CPT2 | Carnitine palmitoyltransferase 2, inner membrane beta-oxidation enzyme | Model to test carnitine-dependent mitochondrial metabolism |
| SLC22A5 | Carnitine transporter (OCTN2) controlling cellular carnitine uptake | Knockout/overexpression to separate transport from synthesis |
| JMJD3 (KDM6B) | Fe(II)/alphaKG-dependent histone demethylase, mechanistic paralog | Comparative model for alphaKG-dependent dioxygenase chemistry |
| KDM6A (UTX) | Fe(II)/alphaKG-dependent histone demethylase | Comparative model for alphaKG-dependent chromatin regulation |
| TET2 | Fe(II)/alphaKG-dependent DNA demethylase | Model to test competition for alphaKG with TMLHE |
| HIF1A | Oxygen-sensing transcription factor affected by alphaKG availability | Readout of metabolic oxygen/alphaKG crosstalk |
| VDR | Vitamin D receptor regulating lipid beta-oxidation and carnitine biosynthesis | Model to test hormonal regulation of TMLHE pathway |
| SLC23A2 | Vitamin C transporter supporting ascorbate-dependent carnitine biosynthesis | Model to test redox cofactor dependence |
| GULO | Vitamin C synthesis enzyme in mice, relevant to ascorbate-dependent carnitine biosynthesis | Mouse model for ascorbate-carnitine interaction |
| PPARGC1A (PGC-1alpha) | Transcriptional coactivator of mitochondrial and fatty acid oxidation genes | Model to test transcriptional coupling to carnitine biosynthesis |
| ACADVL | Very-long-chain acyl-CoA dehydrogenase in beta-oxidation | Downstream readout of carnitine-dependent fatty acid oxidation |
| HADHA | Mitochondrial trifunctional protein subunit in beta-oxidation | Model to test carnitine-dependent energy metabolism |
| SIRT1 | NAD+-dependent deacetylase linked to metabolic and chromatin regulation | Model to test acetylation crosstalk with carnitine synthesis |
How Is trimethyllysine dioxygenase activity Regulated?
TMLHE expression and GO:0050353 activity are regulated at multiple levels. The pathway is sensitive to nutritional status, as ascorbate (vitamin C) supports carnitine biosynthesis, and vitamin D metabolites can regulate lipid beta-oxidation and carnitine biosynthesis. Because the enzyme consumes alphaKG and O2, its activity is also coupled to mitochondrial metabolic state and oxygen availability. At the chromatin level, alphaKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation, indicating that changes in TMLHE activity can feed back on epigenetic regulation.
trimethyllysine dioxygenase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| TMLHE | Carnitine deficiency and autism spectrum disorder association | TMLHE knockout and point-mutation cell models |
| TMLHE | Impaired mitochondrial fatty acid oxidation | Knockout with palmitate oxidation and acylcarnitine profiling |
| TMLHE | Homologous recombination and DNA repair defects | Knockout with DNA damage sensitivity assays |
| TMLHE | Histone acetylation and chromatin regulation | Knockout with histone acetylation profiling |
| SLC22A5 | Carnitine transport deficiency | Knockout/overexpression to separate transport from synthesis |
Carnitine deficiency and metabolic disease
Loss of TMLHE function impairs carnitine biosynthesis, and carnitine deficiency compromises mitochondrial long-chain fatty acid oxidation, leading to energy failure and metabolic decompensation. Because GO:0050353 is the first committed step of carnitine biosynthesis, TMLHE variants are directly relevant to primary carnitine deficiency and related metabolic phenotypes.
Autism spectrum disorder and neurodevelopment
TMLHE deficiency has been associated with autism spectrum disorder, suggesting that carnitine biosynthesis and GO:0050353 activity influence neurodevelopment. The mechanistic link may involve mitochondrial energy metabolism and epigenetic regulation in the developing brain.
Cancer and DNA repair
alphaKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation, linking GO:0050353 to DNA repair capacity and genome stability. This raises the possibility that TMLHE activity modulates sensitivity to DNA-damaging agents in cancer cells.
Chromatin regulation and epigenetic disease
Because TMLHE consumes alphaKG, a co-substrate for Fe(II)/alphaKG-dependent chromatin enzymes such as JMJD3 and TET2, changes in GO:0050353 activity can influence histone and DNA methylation landscapes. This connects the enzyme to epigenetic regulation in development and disease.
From trimethyllysine dioxygenase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is TMLHE required for carnitine biosynthesis? | TMLHE knockout cell line |
| Which active-site residues are required for GO:0050353 activity? | TMLHE point-mutation knock-in |
| Does TMLHE activity support homologous recombination? | TMLHE knockout with DNA repair reporter |
| Does TMLHE activity influence histone acetylation? | TMLHE knockout with acetylation profiling |
| Can TMLHE overexpression increase carnitine and beta-oxidation? | TMLHE overexpression cell model |
| How does TMLHE activity affect mitochondrial metabolism? | TMLHE knockout with Seahorse and acylcarnitine analysis |
How to Study the trimethyllysine dioxygenase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzyme activity assay | Conversion of trimethyllysine to 3-hydroxy-trimethyllysine | Direct measurement of GO:0050353 activity |
| Metabolomics | Carnitine, acylcarnitine, and trimethyllysine levels | Pathway flux and carnitine deficiency |
| Seahorse respirometry | Mitochondrial fatty acid oxidation capacity | Functional consequence of TMLHE loss |
| Histone acetylation profiling | Chromatin acetylation state | Epigenetic readout of alphaKG-mediated carnitine synthesis |
| Homologous recombination reporter | DNA repair efficiency | Genome stability assessment |
| CRISPR knockout | Loss-of-function phenotype | Causal testing of TMLHE in metabolism and chromatin |
| CRISPR point mutation | Specific residue function | Active-site mechanism dissection |
| Overexpression | Gain-of-function phenotype | Testing sufficiency of TMLHE for carnitine synthesis |
Enzymatic activity assays
GO:0050353 activity can be measured by monitoring the conversion of N6,N6,N6-trimethyl-L-lysine to 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, or by coupling to 2-oxoglutarate consumption and succinate production. Active-site studies of human trimethyllysine hydroxylase provide a framework for designing such assays.
Metabolomics and carnitine profiling
Because TMLHE controls carnitine biosynthesis, metabolomic profiling of carnitine, acylcarnitines, and trimethyllysine is a direct readout of pathway activity. Ascorbate and vitamin D status can be varied to test nutritional regulation.
Chromatin and DNA repair assays
Histone acetylation profiling and homologous recombination reporters can test whether alphaKG-mediated carnitine synthesis supports DNA repair and chromatin state. Comparative analysis with JMJD3 and TET2 can distinguish shared alphaKG-dependent mechanisms.
Genetic and CRISPR screens
CRISPR knockout, point-mutation, knock-in, and overexpression models can be combined with metabolic and chromatin readouts to define the causal role of TMLHE and GO:0050353 in cell physiology.
How CRISPR Can Be Used to Study GO:0050353 trimethyllysine dioxygenase activity
Knockout
CRISPR knockout of TMLHE eliminates GO:0050353 activity, providing a clean model to test carnitine dependence, mitochondrial fatty acid oxidation, histone acetylation, and DNA repair. Knockout cells can be rescued with carnitine or 3-hydroxy-trimethyllysine to distinguish substrate from downstream effects.
Point Mutation
Point mutations in TMLHE active-site residues can be introduced to dissect the Fe(II)/alphaKG-dependent catalytic mechanism of GO:0050353. Such models help separate catalytic activity from protein stability or interaction effects.
Knock-in
Knock-in of tagged TMLHE allows localization, interaction, and stability studies while preserving endogenous regulation of GO:0050353. Tagged knock-in can also support proteomic analysis of TMLHE complexes.
Overexpression
Overexpression of TMLHE can test whether increased GO:0050353 activity is sufficient to raise carnitine levels, enhance beta-oxidation, and alter chromatin acetylation. This is useful for gain-of-function studies in metabolic and epigenetic contexts.
How EDITGENE Supports trimethyllysine dioxygenase activity Research
Researchers studying trimethyllysine dioxygenase activity-related genes often need to determine whether a candidate gene is causally involved in carnitine biosynthesis, mitochondrial metabolism, or chromatin regulation. EDITGENE provides publication-ready CRISPR models and bioinformatics support to test these hypotheses rigorously.
Contact EDITGENE today to design your custom CRISPR model for trimethyllysine dioxygenase activity research.
Frequently Asked Questions About trimethyllysine dioxygenase activity
What is trimethyllysine dioxygenase activity?
It is the enzyme activity (GO:0050353) that converts N6,N6,N6-trimethyl-L-lysine to 3-hydroxy-N6,N6,N6-trimethyl-L-lysine using 2-oxoglutarate and O2, producing CO2 and succinate.
What gene encodes trimethyllysine dioxygenase activity in humans?
The human gene is TMLHE, which encodes trimethyllysine hydroxylase, epsilon.
What is the first step of carnitine biosynthesis?
The first committed step is the hydroxylation of trimethyllysine catalyzed by GO:0050353.
What cofactors are required for trimethyllysine dioxygenase activity?
The enzyme requires non-heme Fe(II) and 2-oxoglutarate (alphaKG) as co-substrates.
How is trimethyllysine dioxygenase activity linked to DNA repair?
alphaKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation, connecting GO:0050353 to DNA repair.
What diseases are associated with TMLHE deficiency?
TMLHE deficiency causes carnitine deficiency and has been associated with autism spectrum disorder.
Does vitamin C affect carnitine biosynthesis?
Ascorbate (vitamin C) supports carnitine biosynthesis, indicating a redox cofactor requirement.
Can vitamin D regulate carnitine biosynthesis?
Vitamin D metabolites can regulate lipid beta-oxidation and carnitine biosynthesis.
How can I study trimethyllysine dioxygenase activity in the lab?
You can use enzyme activity assays, metabolomics, Seahorse respirometry, histone acetylation profiling, and CRISPR knockout or overexpression models.
What CRISPR models are available for TMLHE research?
Knockout, point-mutation, knock-in, tagged knock-in, and overexpression models can be generated to study GO:0050353 function.
Conclusion
GO:0050353, trimethyllysine dioxygenase activity, is the first committed step of carnitine biosynthesis and a metabolic node connecting mitochondrial fatty acid oxidation, alphaKG availability, histone acetylation, and DNA repair. The human enzyme TMLHE is a non-heme Fe(II)/alphaKG-dependent oxygenase whose active site and regulation are increasingly well defined. Because loss of TMLHE function causes carnitine deficiency and has been linked to autism spectrum disorder, this activity is relevant to metabolic, neurodevelopmental, and cancer research. CRISPR-based knockout, point-mutation, knock-in, and overexpression models provide powerful tools to dissect the causal roles of GO:0050353 in health and disease.
References
- 1. Uboveja A et al.. 2026. αKG-mediated carnitine synthesis drives DNA repair via histone acetylation.. Nature 655(8123):790-800 PMID: 42203879
- 2. Rebouche CJ. 1991. Ascorbic acid and carnitine biosynthesis.. Am J Clin Nutr 54(6 Suppl):1147S-1152S PMID: 1962562
- 3. Zhang X et al.. 2019. JMJD3 in the regulation of human diseases.. Protein Cell 10(12):864-882 PMID: 31701394
- 4. Wang Y et al.. 2019. Investigating the active site of human trimethyllysine hydroxylase.. Biochem J 476(7):1109-1119 PMID: 30898847
- 5. Strijbis K et al.. 2010. Enzymology of the carnitine biosynthesis pathway.. IUBMB Life 62(5):357-62 PMID: 20306513
- 6. Mendoza A et al.. 2022. Controlled lipid β-oxidation and carnitine biosynthesis by a vitamin D metabolite.. Cell Chem Biol 29(4):660-669.e12 PMID: 34506728
- 7. Uboveja A et al.. 2024. αKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation.. bioRxiv PMID: 38370789
