Pub Date : 2025-02-06DOI: 10.1016/j.abb.2025.110330
Heesoo Jeong, Nathaniel M Vacanti
Glucose provides substrate for the predominant anaplerotic pathway which involves the activity of pyruvate carboxylase (PC). PC-mediated anaplerosis has been extensively studied as a metabolic regulator in glycolytic cells during tumorigenesis and metastasis. Herein, inaccuracies in established methods to measure relative intracellular flux through PC are highlighted and a compartmentalized condensed metabolic network (CCMN) is used to resolve the total malate pool into relative contributions from PC and other sources by metabolic flux analysis (MFA) with [U-13C6]glucose tracing. Performance of the CCMN method is evaluated in breast cancer cell lines that are exposed to small molecules targeting metabolism. Across conditions and cell lines, the CCMN approach yields results nearest to an accepted gold-standard methodology, using [3-13C]glucose, or even exposes the gold standard's limitations. The CCMN method does not require a separate experiment with a much more costly and generally less informative metabolic tracer, such as [3-13C]glucose, and in some cases, may outperform its application.
{"title":"A relative metabolic flux analysis model of glucose anaplerosis.","authors":"Heesoo Jeong, Nathaniel M Vacanti","doi":"10.1016/j.abb.2025.110330","DOIUrl":"https://doi.org/10.1016/j.abb.2025.110330","url":null,"abstract":"<p><p>Glucose provides substrate for the predominant anaplerotic pathway which involves the activity of pyruvate carboxylase (PC). PC-mediated anaplerosis has been extensively studied as a metabolic regulator in glycolytic cells during tumorigenesis and metastasis. Herein, inaccuracies in established methods to measure relative intracellular flux through PC are highlighted and a compartmentalized condensed metabolic network (CCMN) is used to resolve the total malate pool into relative contributions from PC and other sources by metabolic flux analysis (MFA) with [U-<sup>13</sup>C<sub>6</sub>]glucose tracing. Performance of the CCMN method is evaluated in breast cancer cell lines that are exposed to small molecules targeting metabolism. Across conditions and cell lines, the CCMN approach yields results nearest to an accepted gold-standard methodology, using [3-<sup>13</sup>C]glucose, or even exposes the gold standard's limitations. The CCMN method does not require a separate experiment with a much more costly and generally less informative metabolic tracer, such as [3-<sup>13</sup>C]glucose, and in some cases, may outperform its application.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110330"},"PeriodicalIF":3.8,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143373757","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}
Pub Date : 2025-02-05DOI: 10.1016/j.abb.2025.110327
Lara Console , Maria Tolomeo , Luciana Travo , Deborah Giudice , Alessia Nisco , Maria Barile , Cesare Indiveri
Riboflavin, the FMN and FAD precursor, is a crucial vitamin in cell metabolism. Its adsorption and tissue distribution are mediated by tree membrane transporters namely RFVT1-3. Mutations of their genes are associated with Riboflavin Transporter Deficiency. Moreover, derangements of the level of these transporters have been found in several human cancers. To obtain a suitable experimental tool for studying the function of the single proteins, for testing the effect of pathological mutations and for validating predicted ligands as candidate drugs, we have set up a proteoliposome system harbouring the functional RFVT1 or RFVT3. RFVT proteins have been produced in E. coli and purified to the homogeneity by affinity chromatography. The purified proteins show an apparent molecular mass of 45.6 or 48.4 kDa, which are very close to the theoretical mass of RFVT1 or RFVT3, respectively. The purified transporters have been reconstituted into proteoliposomes using a methodology previously pointed out for RFVT2. The transport of riboflavin shows cooperative kinetics with K0.5 values of 0.86 or 1.13 μM and Hill coefficients of 1.19 or 1.3 for RFVT1 or RFVT3, respectively. The K0.5 data of both the transporters are similar the Km reported in intact cell studies. The transporters are inhibited by the riboflavin analogues FMN and lumiflavin in agreement with the molecular docking simulations.
{"title":"Production of the recombinant human riboflavin transporters SLC52A1, 3 and functional assay in proteoliposomes","authors":"Lara Console , Maria Tolomeo , Luciana Travo , Deborah Giudice , Alessia Nisco , Maria Barile , Cesare Indiveri","doi":"10.1016/j.abb.2025.110327","DOIUrl":"10.1016/j.abb.2025.110327","url":null,"abstract":"<div><div>Riboflavin, the FMN and FAD precursor, is a crucial vitamin in cell metabolism. Its adsorption and tissue distribution are mediated by tree membrane transporters namely RFVT1-3. Mutations of their genes are associated with Riboflavin Transporter Deficiency. Moreover, derangements of the level of these transporters have been found in several human cancers. To obtain a suitable experimental tool for studying the function of the single proteins, for testing the effect of pathological mutations and for validating predicted ligands as candidate drugs, we have set up a proteoliposome system harbouring the functional RFVT1 or RFVT3. RFVT proteins have been produced in <em>E. coli</em> and purified to the homogeneity by affinity chromatography. The purified proteins show an apparent molecular mass of 45.6 or 48.4 kDa, which are very close to the theoretical mass of RFVT1 or RFVT3, respectively. The purified transporters have been reconstituted into proteoliposomes using a methodology previously pointed out for RFVT2. The transport of riboflavin shows cooperative kinetics with K<sub>0.5</sub> values of 0.86 or 1.13 μM and Hill coefficients of 1.19 or 1.3 for RFVT1 or RFVT3, respectively. The K<sub>0.5</sub> data of both the transporters are similar the Km reported in intact cell studies. The transporters are inhibited by the riboflavin analogues FMN and lumiflavin in agreement with the molecular docking simulations.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"766 ","pages":"Article 110327"},"PeriodicalIF":3.8,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143363423","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}
Pub Date : 2025-02-05DOI: 10.1016/j.abb.2025.110326
Pablo Gonzalez-Jabalera, Andres Jäschke
Flavin adenine dinucleotide (FAD), a versatile metabolic cofactor, is emerging as an important non-canonical RNA cap across various life domains. This review explores FAD's dual role as a coenzyme and an RNA modifier, focusing on its incorporation as a 5′ cap structure during transcription initiation and its subsequent implications for RNA metabolism and cellular functions. A comprehensive view of the mechanisms underlying FAD capping and decapping is presented, highlighting key enzymes that play a role in these processes. FAD-capped RNA is shown to play critical roles in viral replication, as demonstrated in the Hepatitis C virus, where FAD capping supports cellular immune evasion. Analytical techniques, including mass spectrometry and innovative sequencing methodologies, have advanced our understanding of the flavin cap, enabling its identification and quantification in different biological systems. This review underscores the significance of FAD-RNA capping as a novel regulatory mechanism, proposes innovative methodologies for its study, and emphasizes its potential therapeutic applications in viral and cellular biology.
{"title":"Flavin adenine dinucleotide (FAD) as a non-canonical RNA cap: Mechanisms, functions, and emerging insights","authors":"Pablo Gonzalez-Jabalera, Andres Jäschke","doi":"10.1016/j.abb.2025.110326","DOIUrl":"10.1016/j.abb.2025.110326","url":null,"abstract":"<div><div>Flavin adenine dinucleotide (FAD), a versatile metabolic cofactor, is emerging as an important non-canonical RNA cap across various life domains. This review explores FAD's dual role as a coenzyme and an RNA modifier, focusing on its incorporation as a 5′ cap structure during transcription initiation and its subsequent implications for RNA metabolism and cellular functions. A comprehensive view of the mechanisms underlying FAD capping and decapping is presented, highlighting key enzymes that play a role in these processes. FAD-capped RNA is shown to play critical roles in viral replication, as demonstrated in the Hepatitis C virus, where FAD capping supports cellular immune evasion. Analytical techniques, including mass spectrometry and innovative sequencing methodologies, have advanced our understanding of the flavin cap, enabling its identification and quantification in different biological systems. This review underscores the significance of FAD-RNA capping as a novel regulatory mechanism, proposes innovative methodologies for its study, and emphasizes its potential therapeutic applications in viral and cellular biology.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"766 ","pages":"Article 110326"},"PeriodicalIF":3.8,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143373442","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}
Pub Date : 2025-02-01DOI: 10.1016/j.abb.2024.110272
Eduardo Vagner Rodrigues da Silva , Caroline Torres , Hariel Nemamiah Escolarique Ribeiro , Camila Rolemberg Santana Travaglini Berti de Correia , Taissa de Oliveira de Castro , Giovanna da Costa Mancin , Mayla Gabriela Zanchetta Venancio , Munira Muhammad Abdel Baqui , Felipe Roberti Teixeira , Marcelo Damário Gomes
UBC13 is an orthologue of Homo sapiens ubiquitin-conjugation E2 enzymes described in Leishmania mexicana, a null mutant lacking this gene cannot be produced, suggesting essential functions in this parasite. Leishmania infantum is an etiological agent of visceral leishmaniasis, the most severe type of disease that is potentially fatal if untreated. The ubiquitination process has been targeted for leishmanicidal compounds, indicating its essential function in parasite homeostasis. Therefore, the molecular characterization of the ubiquitination process may provide a better understanding of the molecular and cellular basis of leishmaniasis. Here, we characterized the gene LINF_350017900 in Leishmania infantum, which was named LinfUBC13, an E2 orthologue of UBC13 in Leishmania mexicana and the UBE2D family in Homo sapiens, sharing 72–74 % identity with UBE2D1, UBE2D2, and UBE2D3. LinfUbc13 contains conserved catalytic residues, including Cys86 and the HPN motif, which are essential for ubiquitin-conjugating activity. Structural analysis revealed a high similarity between LinfUbc13 and human UBE2D proteins, with a root-mean-square deviation (RMSD) of 0.4 Å, suggesting conserved functions. Recombinant LinfUbc13 was expressed and shown to accept ubiquitin from E1, forming a thioester intermediate. Functional assays demonstrated that LinfUbc13 transfers ubiquitin to p53 through human HDM2 E3 ligase, confirming its role in ubiquitination. Subcellular localization showed that LinfUbc13 was distributed throughout the parasite cytoplasm. These findings highlight the conserved nature of the ubiquitin-proteasome system between Leishmania infantum and Homo sapiens, showing that LinfUbc13 is an E2 enzyme that plays a crucial role in parasitic development.
{"title":"Molecular characterization of the E2 conjugating enzyme LinfUbc13 in Leishmania infantum","authors":"Eduardo Vagner Rodrigues da Silva , Caroline Torres , Hariel Nemamiah Escolarique Ribeiro , Camila Rolemberg Santana Travaglini Berti de Correia , Taissa de Oliveira de Castro , Giovanna da Costa Mancin , Mayla Gabriela Zanchetta Venancio , Munira Muhammad Abdel Baqui , Felipe Roberti Teixeira , Marcelo Damário Gomes","doi":"10.1016/j.abb.2024.110272","DOIUrl":"10.1016/j.abb.2024.110272","url":null,"abstract":"<div><div>UBC13 is an orthologue of <em>Homo sapiens</em> ubiquitin-conjugation E2 enzymes described in <em>Leishmania mexicana</em>, a null mutant lacking this gene cannot be produced, suggesting essential functions in this parasite. <em>Leishmania infantum</em> is an etiological agent of visceral leishmaniasis, the most severe type of disease that is potentially fatal if untreated. The ubiquitination process has been targeted for leishmanicidal compounds, indicating its essential function in parasite homeostasis. Therefore, the molecular characterization of the ubiquitination process may provide a better understanding of the molecular and cellular basis of leishmaniasis. Here, we characterized the gene <em>LINF_350017900</em> in <em>Leishmania infantum</em>, which was named <em>LinfUBC13</em>, an E2 orthologue of UBC13 in <em>Leishmania mexicana</em> and the UBE2D family in <em>Homo sapiens</em>, sharing 72–74 % identity with UBE2D1, UBE2D2, and UBE2D3. LinfUbc13 contains conserved catalytic residues, including Cys86 and the HPN motif, which are essential for ubiquitin-conjugating activity. Structural analysis revealed a high similarity between LinfUbc13 and human UBE2D proteins, with a root-mean-square deviation (RMSD) of 0.4 Å, suggesting conserved functions. Recombinant LinfUbc13 was expressed and shown to accept ubiquitin from E1, forming a thioester intermediate. Functional assays demonstrated that LinfUbc13 transfers ubiquitin to p53 through human HDM2 E3 ligase, confirming its role in ubiquitination. Subcellular localization showed that LinfUbc13 was distributed throughout the parasite cytoplasm. These findings highlight the conserved nature of the ubiquitin-proteasome system between <em>Leishmania infantum</em> and <em>Homo sapiens</em>, showing that LinfUbc13 is an E2 enzyme that plays a crucial role in parasitic development.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"764 ","pages":"Article 110272"},"PeriodicalIF":3.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142845690","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}
Pub Date : 2025-02-01DOI: 10.1016/j.abb.2024.110264
Liqiang Wang , Changfeng Li , Yumei Song , ZhenKun Yan
{"title":"Retraction notice to “Inhibition of carnitine palmitoyl transferase 1A-induced fatty acid oxidation suppresses cell progression in gastric cancer” [Archiv. Biochem. Biophys. 696 (2020) 108664]","authors":"Liqiang Wang , Changfeng Li , Yumei Song , ZhenKun Yan","doi":"10.1016/j.abb.2024.110264","DOIUrl":"10.1016/j.abb.2024.110264","url":null,"abstract":"","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"764 ","pages":"Article 110264"},"PeriodicalIF":3.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142811703","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}
Pub Date : 2025-02-01DOI: 10.1016/j.abb.2024.110265
V. Pooja Rathan, K. Bhuvaneshwari, G. Nideesh Adit, S. Kavyashree, N. Thulasi, A.V.S. Geetha, K.L. Milan, K.M. Ramkumar
Diabetic nephropathy (DN) is a common complication of diabetes and a leading cause of end-stage renal disease, characterized by progressive kidney fibrosis and inflammation. The transforming growth factor-beta (TGF-β) signaling pathway plays a crucial role in the pathogenesis of diabetes nephropathy, and SMAD7 is a key negative regulator of this pathway. Recent studies have highlighted the involvement of miRNA in the progression of DN. Computational analysis identified 11 potential miRNAs such as miR-424, miR-195, miR-216a, miR-503, miR-15a-5p, miR-15b-5p, miR-665, miR-520h, miR16-5p, miR-21 and miR-32-5p which are predicted to target 3′UTR of SMAD7 mRNA. This review aims to explore the role of these miRNAs in the progression of DN. Notably, these miRNAs have shown therapeutic potential in mitigating fibrosis and inflammation by modulating SMAD7 expression in DN. Future directions can be to investigate the mechanistic pathways through which these miRNAs exert their effects, as well as optimizing delivery systems for effective clinical application. Targeting miRNAs that modulate SMAD7 expression represents a promising strategy for developing specific and effective therapies for diabetic nephropathy.
{"title":"Therapeutic potential of SMAD7 targeting miRNA in the pathogenesis of diabetic nephropathy","authors":"V. Pooja Rathan, K. Bhuvaneshwari, G. Nideesh Adit, S. Kavyashree, N. Thulasi, A.V.S. Geetha, K.L. Milan, K.M. Ramkumar","doi":"10.1016/j.abb.2024.110265","DOIUrl":"10.1016/j.abb.2024.110265","url":null,"abstract":"<div><div>Diabetic nephropathy (DN) is a common complication of diabetes and a leading cause of end-stage renal disease, characterized by progressive kidney fibrosis and inflammation. The transforming growth factor-beta (TGF-β) signaling pathway plays a crucial role in the pathogenesis of diabetes nephropathy, and SMAD7 is a key negative regulator of this pathway. Recent studies have highlighted the involvement of miRNA in the progression of DN. Computational analysis identified 11 potential miRNAs such as miR-424, miR-195, miR-216a, miR-503, miR-15a-5p, miR-15b-5p, miR-665, miR-520h, miR16-5p, miR-21 and miR-32-5p which are predicted to target 3′UTR of SMAD7 mRNA. This review aims to explore the role of these miRNAs in the progression of DN. Notably, these miRNAs have shown therapeutic potential in mitigating fibrosis and inflammation by modulating SMAD7 expression in DN. Future directions can be to investigate the mechanistic pathways through which these miRNAs exert their effects, as well as optimizing delivery systems for effective clinical application. Targeting miRNAs that modulate SMAD7 expression represents a promising strategy for developing specific and effective therapies for diabetic nephropathy.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"764 ","pages":"Article 110265"},"PeriodicalIF":3.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142817070","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}
Pub Date : 2025-02-01DOI: 10.1016/j.abb.2024.110221
Atinuke Odunsi , Mariia A. Kapitonova , George Woodward , Erfan Rahmani , Farid Ghelichkhani , Jun Liu , Sharon Rozovsky
Selenoprotein K (selenok) is linked to the integrated stress response, which helps cells combat stressors and regain normal function. The selenoprotein contains numerous protein interaction hubs and post-translational modification sites and is involved in protein palmitoylation, vesicle trafficking, and the resolution of ER stress. Anchored to the endoplasmic reticulum (ER) membrane, selenok interacts with protein partners to influence their stability, localization, and trafficking, impacting various cellular functions such as calcium homeostasis, cellular migration, phagocytosis, gene expression, and immune response. Consequently, selenok expression level is linked to cancer and neurodegenerative diseases.
Because it contains the reactive amino acid selenocysteine, selenok is likely to function as an enzyme. However, highly unusual for enzymes, the protein segment containing the selenocysteine lacks a stable secondary or tertiary structure, yet it includes multiple interaction sites for protein partners and post-translational modifications. Currently, the reason(s) for the presence of the rare selenocysteine in selenok is not known. Furthermore, of selenok's numerous interaction sites, only some have been sufficiently characterized, leaving many of selenok's potential protein partners to be discovered. In this review, we explore selenok's role in various cellular pathways and its impact on human health, thereby highlighting the links between its diverse cellular functions.
{"title":"Selenoprotein K at the intersection of cellular pathways","authors":"Atinuke Odunsi , Mariia A. Kapitonova , George Woodward , Erfan Rahmani , Farid Ghelichkhani , Jun Liu , Sharon Rozovsky","doi":"10.1016/j.abb.2024.110221","DOIUrl":"10.1016/j.abb.2024.110221","url":null,"abstract":"<div><div>Selenoprotein K (selenok) is linked to the integrated stress response, which helps cells combat stressors and regain normal function. The selenoprotein contains numerous protein interaction hubs and post-translational modification sites and is involved in protein palmitoylation, vesicle trafficking, and the resolution of ER stress. Anchored to the endoplasmic reticulum (ER) membrane, selenok interacts with protein partners to influence their stability, localization, and trafficking, impacting various cellular functions such as calcium homeostasis, cellular migration, phagocytosis, gene expression, and immune response. Consequently, selenok expression level is linked to cancer and neurodegenerative diseases.</div><div>Because it contains the reactive amino acid selenocysteine, selenok is likely to function as an enzyme. However, highly unusual for enzymes, the protein segment containing the selenocysteine lacks a stable secondary or tertiary structure, yet it includes multiple interaction sites for protein partners and post-translational modifications. Currently, the reason(s) for the presence of the rare selenocysteine in selenok is not known. Furthermore, of selenok's numerous interaction sites, only some have been sufficiently characterized, leaving many of selenok's potential protein partners to be discovered. In this review, we explore selenok's role in various cellular pathways and its impact on human health, thereby highlighting the links between its diverse cellular functions.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"764 ","pages":"Article 110221"},"PeriodicalIF":3.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142685777","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}
Acetolactate synthase (ALS) is an essential enzyme involved in the biosynthesis of platform chemicals acetoin and 2,3-butanediol in several microorganisms. In this study, we investigated the catalytic differences among three bacterial ALSs involved in the ligation of two molecules of pyruvate or 2-ketobutyrate. Based on the findings, we predicted three amino acid residues in each enzyme that caused a discrepancy in accordance with the multi-sequence alignment and molecular docking experiments: I398, A402, and T480 in Bacillus subtilis ALS; V400, Y404, and S482 in Listeria seleigeri serovar 1/2b ALS; and M394, H398, and G476 in Klebsiella pneumoniae ALS. Subsequently, we mutually mutated the residues in the three ALSs. The data obtained confirmed our inference that these three residues in each enzyme are truly correlated with substrate recognition, particularly in recognizing compounds that are larger than pyruvate, such as 2-ketobutyrate, benzaldehyde, and nitrosobenzene. This study further clarifies the biochemical traits of ALSs derived from various bacteria and expands the scope of ALS research.
{"title":"Catalytic insights of acetolactate synthases from different bacteria","authors":"Yan-Fei Liang, Ze-Xin Niu, Zi-Wen Wu, Qing-Yang Zhang, Xin-Yi Zhao, Lei-Lei Chao, Heng Li, Wen-Yun Gao","doi":"10.1016/j.abb.2024.110248","DOIUrl":"10.1016/j.abb.2024.110248","url":null,"abstract":"<div><div>Acetolactate synthase (ALS) is an essential enzyme involved in the biosynthesis of platform chemicals acetoin and 2,3-butanediol in several microorganisms. In this study, we investigated the catalytic differences among three bacterial ALSs involved in the ligation of two molecules of pyruvate or 2-ketobutyrate. Based on the findings, we predicted three amino acid residues in each enzyme that caused a discrepancy in accordance with the multi-sequence alignment and molecular docking experiments: I398, A402, and T480 in <em>Bacillus subtilis</em> ALS; V400, Y404, and S482 in <em>Listeria seleigeri serovar</em> 1/2b ALS; and M394, H398, and G476 in <em>Klebsiella pneumoniae</em> ALS. Subsequently, we mutually mutated the residues in the three ALSs. The data obtained confirmed our inference that these three residues in each enzyme are truly correlated with substrate recognition, particularly in recognizing compounds that are larger than pyruvate, such as 2-ketobutyrate, benzaldehyde, and nitrosobenzene. This study further clarifies the biochemical traits of ALSs derived from various bacteria and expands the scope of ALS research.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"764 ","pages":"Article 110248"},"PeriodicalIF":3.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142765719","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}
Pub Date : 2025-02-01DOI: 10.1016/j.abb.2024.110226
Jaidriel Meg G. Cabanding , Steve S.-F. Yu , Zhi-Han Lin , Myrnel A. Fortuna , Adam Jo J. Elatico , Ricky B. Nellas
Bacterial lipases are versatile extracellular enzymes with a catalytic triad at the active site and a flexible ‘lid’ that modulates catalytic accessibility. We combined computational modeling with preliminary in vitro testing to assess the structural stability and activity of the Pseudomonas aeruginosa PAO1 lipase (PAL). We evaluated several systems consisting of the native and mutant forms of the lipase in n-hexane using molecular dynamics simulations. Structural stability was assessed by calculating the B-factor for each system. We measured the gorge radius of the catalytic channel and the RMSD of the catalytic triad to approximate enzymatic activity. Based on the correlation of these metrics, mutant forms were selected for their potential activity and stability. Selected mutant forms were expressed in E. coli BL21, mass-produced, and validated through a lipase-catalyzed esterification assay in n-hexane. Several helices outside the ‘lid’ region were found to influence lid conformational switching. Moreover, our preliminary experimental results show promise in validating our in silico predictions. Our integrated in silico and in vitro pipeline offers a promising approach for designing and producing industrially relevant lipases.
{"title":"The importance of helical structures to the overall activity and structural stability of a lipase from Pseudomonas aeruginosa PAO1 in n-hexane","authors":"Jaidriel Meg G. Cabanding , Steve S.-F. Yu , Zhi-Han Lin , Myrnel A. Fortuna , Adam Jo J. Elatico , Ricky B. Nellas","doi":"10.1016/j.abb.2024.110226","DOIUrl":"10.1016/j.abb.2024.110226","url":null,"abstract":"<div><div>Bacterial lipases are versatile extracellular enzymes with a catalytic triad at the active site and a flexible ‘lid’ that modulates catalytic accessibility. We combined computational modeling with preliminary <em>in vitro</em> testing to assess the structural stability and activity of the <em>Pseudomonas aeruginosa</em> PAO1 lipase (PAL). We evaluated several systems consisting of the native and mutant forms of the lipase in <em>n</em>-hexane using molecular dynamics simulations. Structural stability was assessed by calculating the B-factor for each system. We measured the gorge radius of the catalytic channel and the RMSD of the catalytic triad to approximate enzymatic activity. Based on the correlation of these metrics, mutant forms were selected for their potential activity and stability. Selected mutant forms were expressed in <em>E. coli</em> BL21, mass-produced, and validated through a lipase-catalyzed esterification assay in <em>n</em>-hexane. Several helices outside the ‘lid’ region were found to influence lid conformational switching. Moreover, our preliminary experimental results show promise in validating our <em>in silico</em> predictions. Our integrated <em>in silico</em> and <em>in vitro</em> pipeline offers a promising approach for designing and producing industrially relevant lipases.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"764 ","pages":"Article 110226"},"PeriodicalIF":3.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142765724","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}
Pub Date : 2025-02-01DOI: 10.1016/j.abb.2024.110280
Amanda J. Laseke , Jeremy R. Lohman , Martin St. Maurice
Pyruvate carboxylase (PC) catalyzes the carboxylation of pyruvate to oxaloacetate which serves as an important anaplerotic reaction to replenish citric acid cycle intermediates. In most organisms, the PC-catalyzed reaction is allosterically activated by acetyl-coenzyme A. It has previously been reported that vertebrate PC can catalyze the hydrolysis of acetyl-CoA, offering a potential means for the enzyme to attenuate its allosteric activation. However, in the years since this initial report, there has been no further investigation of this phenomenon. The allosteric binding site for acetyl-CoA is now well characterized, enabling more detailed studies on acetyl-CoA hydrolysis at the allosteric site. Here, we confirm that slow acetyl-CoA hydrolysis is catalyzed by a bacterial PC from Staphylococcus aureus, indicating that this phenomenon is a broad feature of PC enzymes spanning the domains of life. Surprisingly, the enzyme can hydrolyze acetyl-CoA even when the binding site for the acetyl moiety is eliminated through truncation of the biotin carboxylase domain. This suggests that an alternative site for acetyl-CoA binding and hydrolysis may be present in the carboxyltransferase domain of S. aureus PC. We conclude that PC has evolved to minimize the rate of acetyl-CoA hydrolysis at the allosteric site and update the description of PC-catalyzed acetyl-CoA hydrolysis to suggest that this reaction is unlikely to play a significant physiological, metabolic or catalytic role.
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