A Noncatalytic Cysteine Residue Modulates Cobalamin Reactivity in the Human B12 Processing Enzyme CblC
Anna J Esser1, Santiago Sastre2,3,4, Thien-Ly Julia Dinh5
1Laboratory of Clinical Biochemistry and Metabolism, Department of General Pediatrics, Adolescent Medicine and Neonatology, Faculty of Medicine, Medical Center, University of Freiburg, Freiburg im Breisgau 79106, Germany.
Biochemistry
|January 25, 2025
Summary
The CblC enzyme processes vitamin B12. A specific cysteine residue (Cys149) in human CblC appears to fine-tune its reactivity, impacting electron transfer and cobalamin stability, especially in oxygen-rich environments.
Area of Science:
- Biochemistry
- Enzymology
- Vitamin B12 Metabolism
Background:
- Human CblC is crucial for processing dietary vitamin B12, involving ligand removal and cobalt reduction.
- CblC has five cysteine residues, with the function of Cys149 being previously unknown.
- Cys149 is conserved across mammals, suggesting a potentially important role in CblC activity.
Purpose of the Study:
- To investigate the role of the conserved Cys149 residue in human CblC function.
- To determine how Cys149 influences the catalytic activity and reaction mechanism of CblC.
- To compare the activity of wild-type CblC with engineered variants lacking or altered at Cys149.
Main Methods:
- Engineered CblC variants (Cys149Ser and Cys149Ala) were created to study the role of Cys149.
- Glutathione-driven dealkylation of methylcobalamin (MeCbl) was used to measure reaction rates.
- End-point determination of oxidized glutathione (GSSG) assessed electron transfer uncoupling.
- Cobalamin species (aquacobalamin, cob(II)alamin) were monitored over time, particularly under aerobic conditions.
Main Results:
- Mutating Cys149 to Ser or Ala resulted in faster observed dealkylation rates of MeCbl compared to wild-type CblC.
- Mutants exhibited significantly uncoupled electron transfer, leading to increased oxidized glutathione formation.
- In the presence of oxygen, mutants showed conversion of aquacobalamin to cob(II)alamin, unlike wild-type CblC.
- The observed faster dealkylation rates in mutants came at the cost of a slower rate constant.
Conclusions:
- Cys149 in human CblC tunes catalytic activity by minimizing uncoupled electron transfer and stabilizing cob(II)alamin under aerobic conditions.
- The presence of Cys149 leads to a slower, more controlled reaction, potentially advantageous in environments with high oxygen or lower cobalamin turnover needs.
- The findings suggest an evolutionary adaptation in CblC function related to oxygen availability and metabolic demands, drawing parallels with CblC from C. elegans.
Related Concept Videos
01:27Cofactors and Coenzymes
80.9K
Enzymes require additional components for proper function. There are two such classes of molecules: cofactors and coenzymes. Cofactors are metallic ions and coenzymes are non-protein organic molecules. Both of these types of helper molecule can be tightly bound to the enzyme or bound only when the substrate binds.
80.9K
02:04Covalently Linked Protein Regulators
6.8K
Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein....
These groups modify specific amino acids in a protein....
6.8K
01:19Allosteric Proteins-ATCase
5.7K
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
5.7K
01:17Phase II Reactions: Methylation Reactions
123
Methylation is a phase II biotransformation process involving the attachment of a methyl group to a substrate. Enzymes known as methyltransferases orchestrate this reaction.
The mechanism of methylation unfolds in two stages. The first stage sees a methyltransferase enzyme facilitating the transfer of a methyl group from S-adenosylmethionine (SAM) to the substrate, forming S-adenosylhomocysteine (SAH). The second stage involves further metabolism of SAH into homocysteine, which can be recycled...
The mechanism of methylation unfolds in two stages. The first stage sees a methyltransferase enzyme facilitating the transfer of a methyl group from S-adenosylmethionine (SAM) to the substrate, forming S-adenosylhomocysteine (SAH). The second stage involves further metabolism of SAH into homocysteine, which can be recycled...
123
02:23RNA Editing
8.9K
RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
8.9K
00:49Ligand Binding and Linkage
4.7K
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
4.7K