Biochemistry Biochemistry最新文献_第9页

Role of Ribosomal Protein bS1 in Orthogonal mRNA Start Codon Selection.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 Epub Date: 2025-01-24 DOI: 10.1021/acs.biochem.4c00688

Kristina V Boyko, Rebecca A Bernstein, Minji Kim, Jamie H D Cate

{"title":"Role of Ribosomal Protein bS1 in Orthogonal mRNA Start Codon Selection.","authors":"Kristina V Boyko, Rebecca A Bernstein, Minji Kim, Jamie H D Cate","doi":"10.1021/acs.biochem.4c00688","DOIUrl":"10.1021/acs.biochem.4c00688","url":null,"abstract":"In many bacteria, the location of the mRNA start codon is determined by a short ribosome binding site sequence that base pairs with the 3'-end of 16S rRNA (rRNA) in the 30S subunit. Many groups have changed these short sequences, termed the Shine-Dalgarno (SD) sequence in the mRNA and the anti-Shine-Dalgarno (ASD) sequence in 16S rRNA, to create \"orthogonal\" ribosomes to enable the synthesis of orthogonal polymers in the presence of the endogenous translation machinery. However, orthogonal ribosomes are prone to SD-independent translation. Ribosomal protein bS1, which binds to the 30S ribosomal subunit, is thought to promote translation initiation by shuttling the mRNA to the ribosome. Thus, a better understanding of how the SD and bS1 contribute to start codon selection could help efforts to improve the orthogonality of ribosomes. Here, we engineered the Escherichia coli ribosome to prevent binding of bS1 to the 30S subunit and separate the activity of bS1 binding to the ribosome from the role of the mRNA SD sequence in start codon selection. We find that ribosomes lacking bS1 are slightly less active than wild-type ribosomes in vitro. Furthermore, orthogonal 30S subunits lacking bS1 do not have an improved orthogonality. Our findings suggest that mRNA features outside the SD sequence and independent of binding of bS1 to the ribosome likely contribute to start codon selection and the lack of orthogonality of present orthogonal ribosomes.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"710-718"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11800381/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143031515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Peptidisc-Assisted Hydrophobic Clustering Toward the Production of Multimeric and Multispecific Nanobody Proteins. 多肽辅助疏水聚类生产多聚体和多特异性纳米体蛋白。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 Epub Date: 2025-01-14 DOI: 10.1021/acs.biochem.4c00793

Yilun Chen, Franck Duong van Hoa

{"title":"Peptidisc-Assisted Hydrophobic Clustering Toward the Production of Multimeric and Multispecific Nanobody Proteins.","authors":"Yilun Chen, Franck Duong van Hoa","doi":"10.1021/acs.biochem.4c00793","DOIUrl":"10.1021/acs.biochem.4c00793","url":null,"abstract":"Multimerization is a powerful engineering strategy for enhancing protein structural stability, diversity and functional performance. Typical methods for clustering proteins include tandem linking, fusion to self-assembly domains and cross-linking. Here we present a novel approach that leverages the Peptidisc membrane mimetic to stabilize hydrophobic-driven protein clusters. We apply the method to nanobodies (Nbs), effective substitutes to traditional antibodies due to their production efficiency, cost-effectiveness and lower immunogenicity, and we demonstrate the formation of multimeric assemblies termed \"polybodies\" (Pbs). Starting with Nbs directed against the green fluorescent protein (GFP), we produce Pbs that display an increased affinity for GFP due to the avidity effect. The benefit of this increased avidity in affinity-based assays is demonstrated with Pbs directed against the human serum albumin. Using the same autoassembly principle, we produce bispecific and auto-fluorescent Pbs, validating our method as a versatile engineering strategy to generate multispecific and multifunctional protein entities. Peptidisc-assisted hydrophobic clustering thus expand the protein engineering toolbox to broaden the scope of protein multimerization in life sciences.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"655-665"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142982258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Computational Investigation of the Role of Metal Center Identity in Cytochrome P450 Enzyme Model Reactivity. 金属中心同一性对细胞色素P450酶模型反应性影响的计算研究。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 Epub Date: 2025-01-21 DOI: 10.1021/acs.biochem.4c00594

Vyshnavi Vennelakanti, Mugyeom Jeon, Heather J Kulik

{"title":"Computational Investigation of the Role of Metal Center Identity in Cytochrome P450 Enzyme Model Reactivity.","authors":"Vyshnavi Vennelakanti, Mugyeom Jeon, Heather J Kulik","doi":"10.1021/acs.biochem.4c00594","DOIUrl":"10.1021/acs.biochem.4c00594","url":null,"abstract":"Mononuclear Fe enzymes such as heme-containing cytochrome P450 enzymes catalyze a variety of C-H activation reactions under ambient conditions, and they represent an attractive platform for engineering reactivity through changes to the native enzyme. Using density functional theory, we study both native Fe and non-native group 8 (Ru, Os) and group 9 (Ir) metal centers in an active site model of P450. We quantify how changing the metal changes spin state preferences throughout the catalytic cycle. Our calculations reveal an intermediate-spin ground state for all Fe intermediates while the heavier metals prefer low-spin ground states across most intermediates in the reaction cycle. We also study the rate-determining hydrogen atom transfer (HAT) step and the subsequent rebound step. We observe comparable HAT barriers for Fe and Ru, a much higher barrier for Os, and the lowest HAT barrier for Ir. Rebound steps are barrierless for all metals, and the rebound intermediate for Fe is most significantly stabilized. Examination of ground spin states of all intermediates in the reaction cycle reveals spin-allowed pathways for the group 8 metals and spin-forbidden energetics for the group 9 Ir with potential two-state reactivity. Our work highlights the differences between the group 8 metals and the group 9 Ir, and it suggests that engineered P450 enzymes with Ru in particular result in improved enzyme reactivity toward C-H hydroxylation.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"678-691"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142996268","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Mechanisms of Peptide Agonist Dissociation and Deactivation of Adhesion G-Protein-Coupled Receptors

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 DOI: 10.1021/acs.biochem.4c0053110.1021/acs.biochem.4c00531

Keya Joshi, and , Yinglong Miao*,

{"title":"Mechanisms of Peptide Agonist Dissociation and Deactivation of Adhesion G-Protein-Coupled Receptors","authors":"Keya Joshi,  and , Yinglong Miao*, ","doi":"10.1021/acs.biochem.4c0053110.1021/acs.biochem.4c00531","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00531https://doi.org/10.1021/acs.biochem.4c00531","url":null,"abstract":"Adhesion G protein–coupled receptors (ADGRs) belong to Class B2 of GPCRs and are involved in a wide array of important physiological processes. ADGRs contain a GPCR autoproteolysis-inducing domain that is proximal to the receptor N-terminus and undergoes autoproteolysis during the biosynthesis to generate two fragments: the N-terminal fragment (NTF) and the C-terminal fragment (CTF). Dissociation of NTF reveals a tethered agonist to activate the CTF of ADGRs for G protein signaling. Synthetic peptides that mimic the tethered agonist can also activate ADGRs. However, mechanisms of peptide agonist dissociation and the deactivation of ADGRs remain poorly understood. In this study, we have performed all-atom enhanced sampling simulations using a novel protein–protein interaction Gaussian-accelerated molecular dynamics (PPI-GaMD) method on the ADGRG2-IP15 and ADGRG1-P7 complexes. The PPI-GaMD simulations captured the dissociation of the IP15 and P7 peptide agonists from their target receptors. We were able to identify important low-energy conformations of ADGRG2 and ADGRG1 in the active, intermediate, and inactive states, as well as different states of the peptide agonists IP15 and P7 during dissociation. Therefore, our PPI-GaMD simulations have revealed dynamic mechanisms of peptide agonist dissociation and deactivation of ADGRG1 and ADGRG2, which will facilitate the rational design of peptide regulators of the two receptors and other ADGRs.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 4","pages":"871–878 871–878"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428548","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Roles of Loop Region in Folding Kinetics and Transcription Inhibition of DNA G-Quadruplexes. 环区在DNA g -四联体折叠动力学和转录抑制中的作用。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 Epub Date: 2024-12-02 DOI: 10.1021/acs.biochem.4c00601

Minori Nakata, Naoki Kosaka, Keiko Kawauchi, Daisuke Miyoshi

{"title":"Roles of Loop Region in Folding Kinetics and Transcription Inhibition of DNA G-Quadruplexes.","authors":"Minori Nakata, Naoki Kosaka, Keiko Kawauchi, Daisuke Miyoshi","doi":"10.1021/acs.biochem.4c00601","DOIUrl":"10.1021/acs.biochem.4c00601","url":null,"abstract":"Targeting G-quadruplexes, which have distinctive structures, to regulate biological reactions in cells has attracted interest due to the many disease-related genes that possess G-quadruplex-forming sequences. To achieve regulation of gene expression using G-quadruplexes, their folding kinetics and time scales should be well understood. However, the G-quadruplex folding kinetics is highly dependent on its nucleotide sequence as well as its surrounding environment, and thus a general folding mechanism is difficult to propose. Moreover, the effects of G-quadruplex folding kinetics on biological functions such as transcription inhibition are not represented yet. Here, we investigated the folding kinetics and mechanism of G-quadruplexes by focusing on the loop region. Kinetic analyses showed that the hairpin structure in the second loop region significantly accelerated G4 folding, suggesting that it served as a nucleation site for the subsequent folding process. The hairpin in the second loop adopted an intermediate state, an antiparallel G4 structure, in the folding process. Moreover, T7 polymerase assay demonstrated that faster G4 folding resulted in more efficient transcription inhibition. These findings demonstrate the importance of hairpin in the G4 folding kinetics and mechanism and a new strategy for developing G4-targeting small molecules.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"609-619"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142764537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Identification of the Polymerizing Glycosyltransferase Required for the Addition of d-Glucuronic Acid to the Capsular Polysaccharide of Campylobacter jejuni.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 Epub Date: 2025-01-24 DOI: 10.1021/acs.biochem.4c00703

Dao Feng Xiang, Alexander S Riegert, Tamari Narindoshvili, Frank M Raushel

{"title":"Identification of the Polymerizing Glycosyltransferase Required for the Addition of d-Glucuronic Acid to the Capsular Polysaccharide of Campylobacter jejuni.","authors":"Dao Feng Xiang, Alexander S Riegert, Tamari Narindoshvili, Frank M Raushel","doi":"10.1021/acs.biochem.4c00703","DOIUrl":"10.1021/acs.biochem.4c00703","url":null,"abstract":"Campylobacter jejuni is the leading cause of food poisoning in Europe and North America. The exterior surface of this bacterium is encased by a capsular polysaccharide that is attached to a diacyl glycerol phosphate anchor via a poly-Kdo (3-deoxy-d-manno-oct-2-ulosinic acid) linker. In the HS:2 serotype of C. jejuni NCTC 11168, the repeating trisaccharide consists of d-ribose, N-acetyl-d-glucosamine, and d-glucuronate. Here, we show that the N-terminal domain of Cj1432 (residues 1-356) is responsible for the reaction of the C2 hydroxyl group from the terminal d-ribose moiety of the growing polysaccharide chain with UDP-d-glucuronate as the donor substrate. This discovery represents the first biochemical identification and functional characterization of a glycosyltransferase responsible for the polymerization of the capsular polysaccharide of C. jejuni. The product of the reaction catalyzed by the N-terminal domain of Cj1432 is the substrate for the reaction catalyzed by the C-terminal domain of Cj1438 (residues 453-776). This enzyme catalyzes amide bond formation using the C6 carboxylate of the terminal d-glucuronate moiety and (S)-serinol phosphate as substrates. It is also shown that Cj1435 catalyzes the hydrolysis of phosphate from the product catalyzed by the C-terminal domain of Cj1438. These results demonstrate that amide decoration of the d-glucuronate moiety occurs after the incorporation of this sugar into the growing polysaccharide chain.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"581-590"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11800397/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143031500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An Active-Site Bro̷nsted Acid-Base Catalyst Destabilizes Mandelate Racemase and Related Subgroup Enzymes: Implications for Catalysis. 活性位点Bro -嵌套的酸碱催化剂破坏了曼德尔酸外消旋酶和相关亚群酶的稳定性：对催化的影响。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 Epub Date: 2025-01-21 DOI: 10.1021/acs.biochem.4c00572

Himank Kumar, Oliver P Kuehm, Sarah A E Aboushawareb, Atieh Rafiei, Nicole M Easton, Stephen L Bearne

{"title":"An Active-Site Bro̷nsted Acid-Base Catalyst Destabilizes Mandelate Racemase and Related Subgroup Enzymes: Implications for Catalysis.","authors":"Himank Kumar, Oliver P Kuehm, Sarah A E Aboushawareb, Atieh Rafiei, Nicole M Easton, Stephen L Bearne","doi":"10.1021/acs.biochem.4c00572","DOIUrl":"10.1021/acs.biochem.4c00572","url":null,"abstract":"Enzymes of the enolase superfamily (ENS) are mechanistically diverse, yet share a common partial reaction, i.e., the metal-assisted, Bro̷nsted base-catalyzed abstraction of the α-proton from a carboxylate substrate to form an enol(ate) intermediate. Although the catalytic machinery responsible for the initial deprotonation reaction has been conserved, divergent evolution has led to numerous ENS members that catalyze different overall reactions. Using differential scanning calorimetry, we examined the contribution of the Bro̷nsted acid-base catalysts to the thermostability (Tm) of four members of the mandelate racemase (MR)-subgroup of the ENS: MR, d-tartrate dehydratase, l-talarate/galactarate dehydratase, and l-fuconate dehydratase. Each enzyme contains an active-site Lys (part of a KxK motif) and His, which act as Bro̷nsted acid-base catalysts. The KxK → KxM substitutions increased the thermostability in all four enzymes with the effect being most prominent for MR (ΔTm = +8.6 °C). The KxK → MxK substitutions decreased the thermostability in all four enzymes, and the His → Asn substitution had a significant stabilizing effect only on MR. Thus, the active sites of MR-subgroup enzymes are destabilized by the Lys Bro̷nsted acid-base catalyst, suggesting that the destabilization energy may be used to drive a conformational change of the enzyme to yield a catalytically competent protonation state upon substrate binding.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"666-677"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142996264","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Inhibition of EphB4 Receptor Signaling by Ephrin-B2-Competitive and Non-Competitive DARPins Prevents Angiogenesis. ephrin - b2竞争性和非竞争性DARPins抑制EphB4受体信号传导可阻止血管生成。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 Epub Date: 2025-01-17 DOI: 10.1021/acs.biochem.4c00431

Weronika Trun, Amaury Fernández-Montalván, Yong-Jiang Cao, Bernard Haendler, Dieter Zopf

引用次数: 0

Yeast Eukaryotic Initiation Factor 4B Remodels the MRNA Entry Site on the Small Ribosomal Subunit.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 Epub Date: 2025-01-23 DOI: 10.1021/acs.biochem.4c00489

Ayushi Datey, Prafful Sharma, Faisal Tarique Khaja, Huma Rahil, Tanweer Hussain

{"title":"Yeast Eukaryotic Initiation Factor 4B Remodels the MRNA Entry Site on the Small Ribosomal Subunit.","authors":"Ayushi Datey, Prafful Sharma, Faisal Tarique Khaja, Huma Rahil, Tanweer Hussain","doi":"10.1021/acs.biochem.4c00489","DOIUrl":"10.1021/acs.biochem.4c00489","url":null,"abstract":"Eukaryotic Initiation Factor 4 (eIF4) is a group of factors that activates mRNA for translation and recruit 43S preinitiation complex (PIC) to the mRNA 5' end, forming the 48S PIC. The eIF4 factors include mRNA 5' cap-binding protein eIF4E, ATP-dependent RNA helicase eIF4A, and scaffold protein eIF4G, which anchors eIF4A and eIF4E. Another eIF4 factor, eIF4B, stimulates the RNA helicase activity of eIF4A and facilitates mRNA recruitment. However, the mechanisms by which eIF4B binds the 40S ribosomal subunit and promotes mRNA recruitment remain poorly understood. Using cryo-Eletron Microscopy (cryo-EM), we obtained a map of the yeast 40S ribosomal subunit in a complex with eIF4B (40S-eIF4B complex). An extra density, tentatively assigned to yeast eIF4B, was observed near the mRNA entry channel of the 40S, contacting ribosomal proteins uS10, uS3, and eS10 as well as rRNA helix h16. Predictive modeling of the 40S-eIF4B complex suggests that the N-terminal domain of eIF4B binds near the mRNA entry channel, overlapping with the extra density observed in the 40S-eIF4B map. The partially open conformation of 40S in the 40S-eIF4B map is incompatible with eIF3j binding observed in the 48S PIC. Additionally, the extra density at the mRNA entry channel poses steric hindrance for eIF3g binding in the 48S PIC. Thus, structural insights suggest that eIF4B facilitates the release of eIF3j and the relocation of the eIF3b-g-i module during mRNA recruitment, thereby advancing our understanding of eIF4B's role in translation initiation.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"600-608"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143021297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Noncatalytic Cysteine Residue Modulates Cobalamin Reactivity in the Human B₁₂ Processing Enzyme CblC.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-02-04 Epub Date: 2025-01-25 DOI: 10.1021/acs.biochem.4c00613

Anna J Esser, Santiago Sastre, Thien-Ly Julia Dinh, Viola Tanner, Victoria Wingert, Katharina Klotz, Donald W Jacobsen, Ute Spiekerkoetter, Oliver Schilling, Ari Zeida, Rafael Radi, Luciana Hannibal

{"title":"A Noncatalytic Cysteine Residue Modulates Cobalamin Reactivity in the Human B12 Processing Enzyme CblC.","authors":"Anna J Esser, Santiago Sastre, Thien-Ly Julia Dinh, Viola Tanner, Victoria Wingert, Katharina Klotz, Donald W Jacobsen, Ute Spiekerkoetter, Oliver Schilling, Ari Zeida, Rafael Radi, Luciana Hannibal","doi":"10.1021/acs.biochem.4c00613","DOIUrl":"10.1021/acs.biochem.4c00613","url":null,"abstract":"Human CblC catalyzes the indispensable processing of dietary vitamin B12 by the removal of its β-axial ligand and an either one- or two-electron reduction of its cobalt center to yield cob(II)alamin and cob(I)alamin, respectively. Human CblC possesses five cysteine residues of an unknown function. We hypothesized that Cys149, conserved in mammals, tunes the CblC reactivity. To test this, we recreated an evolutionary early variant of CblC, namely, Cys149Ser, as well as Cys149Ala. Surprisingly, substitution of Cys149 for serine or alanine led to faster observed rates of glutathione-driven dealkylation of MeCbl compared to wild-type CblC. The reaction yielded aquacobalamin and stoichiometric formation of S-methylglutathione as the demethylation products. Determination of end-point oxidized glutathione revealed significantly uncoupled electron transfer in both mutants compared with the wild type. Long incubation times revealed the conversion of aquacobalamin to cob(II)alamin in the presence of oxygen in mutants Cys149Ser and Cys149Ala but not in wild-type CblC, all without an effect on dealkylation rates. This finding is reminiscent of the catalytic behavior of CblC from Caenorhabditis elegans, wherein Cys149 is naturally substituted by Ser, and the reaction mechanism differs from that of human CblC precisely by the unusual stabilization of cob(II)alamin in the presence of oxygen. Thus, Cys149 tunes the catalytic activity of human CblC by minimizing uncoupled electron transfer that forms GSSG. This occurs at the expense of a slower observed rate constant for the demethylation of MeCbl. This adjustment is compatible with diminished needs for intracellular turnover of cobalamins and with life under increased oxygen concentration.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"692-709"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143035351","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0