Pub Date : 2024-07-03DOI: 10.1021/acs.biochem.4c00179
Mst Sharmin Aktar, Vesna de Serrano, Reza A Ghiladi, Stefan Franzen
Dehalperoxidase (DHP) has diverse catalytic activities depending on the substrate binding conformation, pH, and dynamics in the distal pocket above the heme. According to our hypothesis, the molecular structure of the substrate and binding orientation in DHP guide enzymatic function. Enzyme kinetic studies have shown that the catalytic activity of DHP B is significantly higher than that of DHP A despite 96% sequence homology. There are more than 30 substrate-bound structures with DHP B, each providing insight into the nature of enzymatic binding at the active site. By contrast, the only X-ray crystallographic structures of small molecules in a complex with DHP A are phenols. This study is focused on investigating substrate binding in DHP A to compare with DHP B structures. Fifteen substrates were selected that were known to bind to DHP B in the crystal to test whether soaking substrates into DHP A would yield similar structures. Five of these substrates yielded X-ray crystal structures of substrate-bound DHP A, namely, 2,4-dichlorophenol (1.48 Å, PDB: 8EJN), 2,4-dibromophenol (1.52 Å, PDB: 8VSK), 4-nitrophenol (2.03 Å, PDB: 8VKC), 4-nitrocatechol (1.40 Å, PDB: 8VKD), and 4-bromo-o-cresol (1.64 Å, PDB: 8VZR). For the remaining substrates that bind to DHP B, such as cresols, 5-bromoindole, benzimidazole, 4,4-biphenol, 4.4-ethylidenebisphenol, 2,4-dimethoxyphenol, and guaiacol, the electron density maps in DHP A are not sufficient to determine the presence of the substrates, much less their orientation. In our hands, only phenols, 4-Br-o-cresol, and 4-nitrocatechol can be soaked into crystalline DHP A. None of the larger substrates were observed to bind. A minimum of seven hanging drops were selected for soaking with more than 50 crystals screened for each substrate. The five high-quality examples of direct comparison of modes of binding in DHP A and B for the same substrate provide further support for the hypothesis that the substrate-binding conformation determines the enzyme function of DHP.
脱氢过氧化物酶(DHP)具有不同的催化活性,这取决于底物的结合构象、pH 值以及血红素上方远端口袋中的动态变化。根据我们的假设,底物的分子结构和在 DHP 中的结合方向会引导酶的功能。酶动力学研究表明,尽管序列同源性高达 96%,但 DHP B 的催化活性明显高于 DHP A。DHP B 有 30 多种底物结合结构,每种结构都能让人深入了解活性位点酶结合的性质。相比之下,与 DHP A 复合物的小分子 X 射线晶体结构中只有苯酚。本研究的重点是研究 DHP A 与底物的结合,并与 DHP B 的结构进行比较。研究人员选择了已知能与晶体中的 DHP B 结合的 15 种底物,以测试将底物浸泡到 DHP A 中是否会产生类似的结构。其中五种底物产生了与底物结合的 DHP A 的 X 射线晶体结构,即 2,4-二氯苯酚(1.48 Å,PDB:8EJN)、2,4-二溴苯酚(1.52 Å,PDB:8VSK)、4-硝基苯酚(2.03 Å,PDB:8VKC)、4-硝基邻苯二酚(1.40 Å,PDB:8VKD)和 4-溴邻甲酚(1.64 Å,PDB:8VZR)。对于与 DHP B 结合的其余底物,如甲酚、5-溴吲哚、苯并咪唑、4,4-联苯酚、4.4-亚乙基双酚、2,4-二甲氧基苯酚和愈创木酚,DHP A 中的电子密度图不足以确定底物的存在,更不用说它们的方向了。在我们的研究中,只有苯酚、4-Br-邻甲酚和 4-硝基邻苯二酚可以浸入结晶的 DHP A 中。每种基质至少选择 7 个悬滴进行浸泡,筛选出 50 个以上的晶体。这五个直接比较 DHP A 和 B 与相同底物结合模式的高质量实例进一步支持了底物结合构象决定 DHP 酶功能的假设。
{"title":"Structural Comparison of Substrate Binding Sites in Dehaloperoxidase A and B.","authors":"Mst Sharmin Aktar, Vesna de Serrano, Reza A Ghiladi, Stefan Franzen","doi":"10.1021/acs.biochem.4c00179","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00179","url":null,"abstract":"<p><p>Dehalperoxidase (DHP) has diverse catalytic activities depending on the substrate binding conformation, pH, and dynamics in the distal pocket above the heme. According to our hypothesis, the molecular structure of the substrate and binding orientation in DHP guide enzymatic function. Enzyme kinetic studies have shown that the catalytic activity of DHP B is significantly higher than that of DHP A despite 96% sequence homology. There are more than 30 substrate-bound structures with DHP B, each providing insight into the nature of enzymatic binding at the active site. By contrast, the only X-ray crystallographic structures of small molecules in a complex with DHP A are phenols. This study is focused on investigating substrate binding in DHP A to compare with DHP B structures. Fifteen substrates were selected that were known to bind to DHP B in the crystal to test whether soaking substrates into DHP A would yield similar structures. Five of these substrates yielded X-ray crystal structures of substrate-bound DHP A, namely, 2,4-dichlorophenol (1.48 Å, PDB: 8EJN), 2,4-dibromophenol (1.52 Å, PDB: 8VSK), 4-nitrophenol (2.03 Å, PDB: 8VKC), 4-nitrocatechol (1.40 Å, PDB: 8VKD), and 4-bromo-o-cresol (1.64 Å, PDB: 8VZR). For the remaining substrates that bind to DHP B, such as cresols, 5-bromoindole, benzimidazole, 4,4-biphenol, 4.4-ethylidenebisphenol, 2,4-dimethoxyphenol, and guaiacol, the electron density maps in DHP A are not sufficient to determine the presence of the substrates, much less their orientation. In our hands, only phenols, 4-Br-o-cresol, and 4-nitrocatechol can be soaked into crystalline DHP A. None of the larger substrates were observed to bind. A minimum of seven hanging drops were selected for soaking with more than 50 crystals screened for each substrate. The five high-quality examples of direct comparison of modes of binding in DHP A and B for the same substrate provide further support for the hypothesis that the substrate-binding conformation determines the enzyme function of DHP.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489872","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 : 2024-07-03DOI: 10.1021/acs.biochem.4c00176
Kyle M Glockzin, Tamari Narindoshvili, Frank M Raushel
ProTides are nucleotide analogues used for the treatment of specific viral infections. These compounds consist of a masked nucleotide that undergoes in vivo enzymatic and spontaneous chemical transformations to generate a free mononucleotide that is ultimately transformed to the pharmaceutically active triphosphorylated drug. The three FDA approved ProTides are composed of a phosphoramidate (P-N) core coupled with a nucleoside analogue, phenol, and an l-alanyl carboxylate ester. The previously proposed mechanism of activation postulates the existence of an unstable 5-membered mixed anhydride cyclic intermediate formed from the direct attack of the carboxylate group of the l-alanyl moiety with expulsion of phenol. The mixed anhydride cyclic intermediate is further postulated to undergo spontaneous hydrolysis to form a linear l-alanyl phosphoramidate product. In the proposed mechanism of activation, the 5-membered mixed anhydride intermediate has been detected previously using mass spectrometry, but the specific site of nucleophilic attack by water (P-O versus C-O) has not been determined. To further interrogate the mechanism for hydrolysis of the putative 5-membered cyclic intermediate formed during ProTide activation, the reaction was conducted in 18O-labeled water using a ProTide analogue that could be activated by carboxypeptidase Y. Mass spectrometry and 31P NMR spectroscopy were used to demonstrate that the hydrolysis of the mixed anhydride 5-membered intermediate occurs with exclusive attack at the phosphorus center.
{"title":"Regiochemical Analysis of the ProTide Activation Mechanism.","authors":"Kyle M Glockzin, Tamari Narindoshvili, Frank M Raushel","doi":"10.1021/acs.biochem.4c00176","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00176","url":null,"abstract":"<p><p>ProTides are nucleotide analogues used for the treatment of specific viral infections. These compounds consist of a masked nucleotide that undergoes <i>in vivo</i> enzymatic and spontaneous chemical transformations to generate a free mononucleotide that is ultimately transformed to the pharmaceutically active triphosphorylated drug. The three FDA approved ProTides are composed of a phosphoramidate (P-N) core coupled with a nucleoside analogue, phenol, and an l-alanyl carboxylate ester. The previously proposed mechanism of activation postulates the existence of an unstable 5-membered mixed anhydride cyclic intermediate formed from the direct attack of the carboxylate group of the l-alanyl moiety with expulsion of phenol. The mixed anhydride cyclic intermediate is further postulated to undergo spontaneous hydrolysis to form a linear l-alanyl phosphoramidate product. In the proposed mechanism of activation, the 5-membered mixed anhydride intermediate has been detected previously using mass spectrometry, but the specific site of nucleophilic attack by water (P-O versus C-O) has not been determined. To further interrogate the mechanism for hydrolysis of the putative 5-membered cyclic intermediate formed during ProTide activation, the reaction was conducted in <sup>18</sup>O-labeled water using a ProTide analogue that could be activated by carboxypeptidase Y. Mass spectrometry and <sup>31</sup>P NMR spectroscopy were used to demonstrate that the hydrolysis of the mixed anhydride 5-membered intermediate occurs with exclusive attack at the phosphorus center.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489871","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 : 2024-07-02Epub Date: 2024-06-13DOI: 10.1021/acs.biochem.3c00728
Elijah D Humphrey, Maxim V Sukhodolets
In growing E. coli cells, the transcription-translation complexes (TTCs) form characteristic foci; however, the exact molecular composition of these superstructures is not known with certainty. Herein, we report that, during our recently developed "fast" procedures for purification of E. coli RNA polymerase (RP), a fraction of the RP's α/RpoA subunits is displaced from the core RP complexes and copurifies with multiprotein superstructures carrying the nucleic acid-binding protein Hfq and the ribosomal protein S6. We show that the main components of these large multiprotein assemblies are fixed protein copy-number (Hfq6)n≥8 complexes; these complexes have a high level of structural uniformity and are distinctly unlike the previously described (Hfq6)n "head-to-tail" polymers. We describe purification of these novel, structurally uniform (Hfq6)n≥8 complexes to near homogeneity and show that they also contain small nonprotein molecules and accessory S6. We demonstrate that Hfq, S6, and RP have similar solubility profiles and present evidence pointing to a role of the Hfq C-termini in superstructure formation. Taken together, our data offer new insights into the composition of the macromolecular assemblies likely acting as scaffolds for transcription complexes and ribosomes during bacterial cells' active growth.
在生长中的大肠杆菌细胞中,转录-翻译复合物(TTC)会形成特征性的病灶;然而,这些超结构的确切分子组成尚不确定。在此,我们报告说,在我们最近开发的大肠杆菌 RNA 聚合酶(RP)"快速 "纯化程序中,一部分 RP 的 α/RpoA 亚基脱离了核心 RP 复合物,并与携带核酸结合蛋白 Hfq 和核糖体蛋白 S6 的多蛋白超结构共聚。我们的研究表明,这些大型多蛋白集合体的主要成分是固定蛋白拷贝数(Hfq6)n≥8 的复合物;这些复合物的结构高度一致,与之前描述的(Hfq6)n "头对尾 "聚合物截然不同。我们描述了如何纯化这些结构均匀的新型 (Hfq6)n≥8 复合物,使其接近均一,并证明它们还含有小的非蛋白分子和附属 S6。我们证明 Hfq、S6 和 RP 具有相似的溶解度曲线,并提出证据表明 Hfq C 端在超结构形成中的作用。总之,我们的数据为了解细菌细胞活跃生长过程中可能作为转录复合物和核糖体支架的大分子组装体的组成提供了新的见解。
{"title":"Isolation and Partial Characterization of Novel, Structurally Uniform (Hfq<sub>6</sub>)<sub>n≥8</sub> Assemblies Carrying Accessory Transcription and Translation Factors.","authors":"Elijah D Humphrey, Maxim V Sukhodolets","doi":"10.1021/acs.biochem.3c00728","DOIUrl":"10.1021/acs.biochem.3c00728","url":null,"abstract":"<p><p>In growing <i>E. coli</i> cells, the transcription-translation complexes (TTCs) form characteristic foci; however, the exact molecular composition of these superstructures is not known with certainty. Herein, we report that, during our recently developed \"fast\" procedures for purification of <i>E. coli</i> RNA polymerase (RP), a fraction of the RP's α/RpoA subunits is displaced from the core RP complexes and copurifies with multiprotein superstructures carrying the nucleic acid-binding protein Hfq and the ribosomal protein S6. We show that the main components of these large multiprotein assemblies are fixed protein copy-number (Hfq<sub>6</sub>)<sub>n≥8</sub> complexes; these complexes have a high level of structural uniformity and are distinctly unlike the previously described (Hfq<sub>6</sub>)<sub>n</sub> \"head-to-tail\" polymers. We describe purification of these novel, structurally uniform (Hfq<sub>6</sub>)<sub>n≥8</sub> complexes to near homogeneity and show that they also contain small nonprotein molecules and accessory S6. We demonstrate that Hfq, S6, and RP have similar solubility profiles and present evidence pointing to a role of the Hfq C-termini in superstructure formation. Taken together, our data offer new insights into the composition of the macromolecular assemblies likely acting as scaffolds for transcription complexes and ribosomes during bacterial cells' active growth.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141309488","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 : 2024-07-02Epub Date: 2024-06-17DOI: 10.1021/acs.biochem.4c00165
Jake W Saunders, Adam M Damry, Vanessa Vongsouthi, Matthew A Spence, Rebecca L Frkic, Chloe Gomez, Patrick A Yates, Dana S Matthews, Nobuhiko Tokuriki, Malcolm D McLeod, Colin J Jackson
The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.
{"title":"Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.","authors":"Jake W Saunders, Adam M Damry, Vanessa Vongsouthi, Matthew A Spence, Rebecca L Frkic, Chloe Gomez, Patrick A Yates, Dana S Matthews, Nobuhiko Tokuriki, Malcolm D McLeod, Colin J Jackson","doi":"10.1021/acs.biochem.4c00165","DOIUrl":"10.1021/acs.biochem.4c00165","url":null,"abstract":"<p><p>The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from <i>Ideonella sakaiensis</i> carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416540","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 : 2024-07-02Epub Date: 2024-06-18DOI: 10.1021/acs.biochem.4c00066
Jessica L Taylor, Pedro H Ayres-Galhardo, Breann L Brown
The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme in vitro, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.
在某些细菌和真核生物中,氨基乙酰丙酸合成酶(ALAS)通过催化甘氨酸和琥珀酰-CoA缩合生成氨基乙酰丙酸,从而启动血红素的生物合成。在人类,负责在红细胞发育过程中产生血红素的 ALAS 同工酶是红细胞特异性 ALAS2 同工酶。由于 ALAS2 在红细胞生成过程中的重要作用,人类 ALAS2(hALAS2)功能的变化可导致两种不同的血液疾病。X连锁性红细胞性贫血是由ALAS2功能缺失引起的,而X连锁性原卟啉症则是由ALAS2功能获得引起的。有趣的是,ALAS2 C端延伸部分的突变可能与这两种疾病有关。在这里,我们研究了之前报道的两种 C 端功能缺失变体(hALAS2 V562A 和 M567I)介导的酶功能障碍的分子基础。我们发现,这些变异不会导致严重的结构紊乱,但 V562A 的酶稳定性会降低。此外,我们还发现这两个变体在加入 5'-磷酸吡哆醛(PLP)辅助因子后,酶的稳定性会适度增加。与野生型 hALAS2 相比,这些变体与 PLP 和单个底物的结合存在差异。虽然 hALAS2 V562A 在体外是一种更活跃的酶,但它与琥珀酰-CoA 结合的效率较低。相比之下,M567I 突变显著改变了底物结合的合作性。结合之前报道的基于细胞的研究,我们的工作揭示了 hALAS2 C 端突变对正常血红素生物合成所需的 ALA 生成产生负面影响的分子基础。
{"title":"Elucidating the Role of Human ALAS2 C-terminal Mutations Resulting in Loss of Function and Disease.","authors":"Jessica L Taylor, Pedro H Ayres-Galhardo, Breann L Brown","doi":"10.1021/acs.biochem.4c00066","DOIUrl":"10.1021/acs.biochem.4c00066","url":null,"abstract":"<p><p>The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme <i>in vitro</i>, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416539","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 : 2024-07-02Epub Date: 2024-06-17DOI: 10.1021/acs.biochem.4c00177
Joshua R Miller, Elizabeth C Schnorrenberg, Cole Aschenbrener, Brian G Fox, Thomas C Brunold
In mammals, l-cysteine (Cys) homeostasis is maintained by the mononuclear nonheme iron enzyme cysteine dioxygenase (CDO), which oxidizes Cys to cysteine sulfinic acid. CDO contains a rare post-translational modification, involving the formation of a thioether cross-link between a Cys residue at position 93 (Mus musculus CDO numbering) and a nearby tyrosine at position 157 (Cys-Tyr cross-link). As-isolated CDO contains both the cross-linked and non-cross-linked isoforms, and formation of the Cys-Tyr cross-link during repeated enzyme turnover increases CDO's catalytic efficiency by ∼10-fold. Interestingly, while the C93G CDO variant lacks the Cys-Tyr cross-link, it is similarly active as cross-linked wild-type (WT) CDO. Alternatively, the Y157F CDO variant, which also lacks the cross-link but maintains the free thiolate at position 93, exhibits a drastically reduced catalytic efficiency. These observations suggest that the untethered thiolate moiety of C93 is detrimental to CDO activity and/or that Y157 is essential for catalysis. To further assess the roles of residues C93 and Y157, we performed a spectroscopic and kinetic characterization of Y157F CDO and the newly designed C93G/Y157F CDO variant. Our results provide evidence that the non-cross-linked C93 thiolate stabilizes a water at the sixth coordination site of Cys-bound Y157F Fe(II)CDO. A water is also present, though more weakly coordinated, in Cys-bound C93G/Y157F Fe(II)CDO. The presence of a water molecule, which must be displaced by cosubstrate O2, likely makes a significant contribution to the ∼15-fold and ∼7-fold reduced catalytic efficiencies of the Y157F and C93G/Y157F CDO variants, respectively, relative to cross-linked WT CDO.
{"title":"Kinetic and Spectroscopic Investigation of the Y157F and C93G/Y157F Variants of Cysteine Dioxygenase: Dissecting the Roles of the Second-Sphere Residues C93 and Y157.","authors":"Joshua R Miller, Elizabeth C Schnorrenberg, Cole Aschenbrener, Brian G Fox, Thomas C Brunold","doi":"10.1021/acs.biochem.4c00177","DOIUrl":"10.1021/acs.biochem.4c00177","url":null,"abstract":"<p><p>In mammals, l-cysteine (Cys) homeostasis is maintained by the mononuclear nonheme iron enzyme cysteine dioxygenase (CDO), which oxidizes Cys to cysteine sulfinic acid. CDO contains a rare post-translational modification, involving the formation of a thioether cross-link between a Cys residue at position 93 (<i>Mus musculus</i> CDO numbering) and a nearby tyrosine at position 157 (Cys-Tyr cross-link). As-isolated CDO contains both the cross-linked and non-cross-linked isoforms, and formation of the Cys-Tyr cross-link during repeated enzyme turnover increases CDO's catalytic efficiency by ∼10-fold. Interestingly, while the C93G CDO variant lacks the Cys-Tyr cross-link, it is similarly active as cross-linked wild-type (WT) CDO. Alternatively, the Y157F CDO variant, which also lacks the cross-link but maintains the free thiolate at position 93, exhibits a drastically reduced catalytic efficiency. These observations suggest that the untethered thiolate moiety of C93 is detrimental to CDO activity and/or that Y157 is essential for catalysis. To further assess the roles of residues C93 and Y157, we performed a spectroscopic and kinetic characterization of Y157F CDO and the newly designed C93G/Y157F CDO variant. Our results provide evidence that the non-cross-linked C93 thiolate stabilizes a water at the sixth coordination site of Cys-bound Y157F Fe(II)CDO. A water is also present, though more weakly coordinated, in Cys-bound C93G/Y157F Fe(II)CDO. The presence of a water molecule, which must be displaced by cosubstrate O<sub>2</sub>, likely makes a significant contribution to the ∼15-fold and ∼7-fold reduced catalytic efficiencies of the Y157F and C93G/Y157F CDO variants, respectively, relative to cross-linked WT CDO.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416541","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 : 2024-07-02Epub Date: 2024-06-22DOI: 10.1021/acs.biochem.4c00149
Mackenzie C R Denton, Natasha P Murphy, Brenna Norton-Baker, Mauro Lua, Harrison Steel, Gregg T Beckham
Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.
{"title":"Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis.","authors":"Mackenzie C R Denton, Natasha P Murphy, Brenna Norton-Baker, Mauro Lua, Harrison Steel, Gregg T Beckham","doi":"10.1021/acs.biochem.4c00149","DOIUrl":"10.1021/acs.biochem.4c00149","url":null,"abstract":"<p><p>Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141440035","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 : 2024-07-02DOI: 10.1021/acs.biochem.4c00148
Tejaswi Tammareddy, Walid Keyrouz, Ram D Sriram, Harish C Pant, Antonio Cardone, Jeffery B Klauda
Munc18-1 is an SM (sec1/munc-like) family protein involved in vesicle fusion and neuronal exocytosis. Munc18-1 is known to regulate the exocytosis process by binding with closed- and open-state conformations of Syntaxin1, a protein belonging to the SNARE family established to be central to the exocytosis process. Our previous work studied peptide p5 as a promising drug candidate for CDK5-p25 complex, an Alzheimer's disease (AD) pathological target. Experimental in vivo and in vitro studies suggest that Munc18-1 promotes p5 to selectively inhibit the CDK5-p25 complex without affecting the endogenous CDK5 activity, a characteristic of remarkable therapeutic implications. In this paper, we identify several binding modes of p5 with Munc18-1 that could potentially affect the Munc18-1 binding with SNARE proteins and lead to off-target effects on neuronal communication using molecular dynamics simulations. Recent studies indicate that disruption of Munc18-1 function not only disrupts neurotransmitter release but also results in neurodegeneration, exhibiting clinical resemblance to other neurodegenerative conditions such as AD, causing diagnostic and treatment challenges. We characterize such interactions between p5 and Munc18-1, define the corresponding pharmacophores, and provide guidance for the in vitro validation of our findings to improve therapeutic efficacy and safety of p5.
{"title":"Investigation of the Effect of Peptide p5 Targeting CDK5-p25 Hyperactivity on Munc18-1 (P67) Regulating Neuronal Exocytosis Using Molecular Simulations.","authors":"Tejaswi Tammareddy, Walid Keyrouz, Ram D Sriram, Harish C Pant, Antonio Cardone, Jeffery B Klauda","doi":"10.1021/acs.biochem.4c00148","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00148","url":null,"abstract":"<p><p>Munc18-1 is an SM (sec1/munc-like) family protein involved in vesicle fusion and neuronal exocytosis. Munc18-1 is known to regulate the exocytosis process by binding with closed- and open-state conformations of Syntaxin1, a protein belonging to the SNARE family established to be central to the exocytosis process. Our previous work studied peptide p5 as a promising drug candidate for CDK5-p25 complex, an Alzheimer's disease (AD) pathological target. Experimental <i>in vivo</i> and <i>in vitro</i> studies suggest that Munc18-1 promotes p5 to selectively inhibit the CDK5-p25 complex without affecting the endogenous CDK5 activity, a characteristic of remarkable therapeutic implications. In this paper, we identify several binding modes of p5 with Munc18-1 that could potentially affect the Munc18-1 binding with SNARE proteins and lead to off-target effects on neuronal communication using molecular dynamics simulations. Recent studies indicate that disruption of Munc18-1 function not only disrupts neurotransmitter release but also results in neurodegeneration, exhibiting clinical resemblance to other neurodegenerative conditions such as AD, causing diagnostic and treatment challenges. We characterize such interactions between p5 and Munc18-1, define the corresponding pharmacophores, and provide guidance for the <i>in vitro</i> validation of our findings to improve therapeutic efficacy and safety of p5.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489870","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 : 2024-07-02Epub Date: 2024-06-12DOI: 10.1021/acs.biochem.3c00665
Elsa D M Hien, Adrien Chauvier, Patrick St-Pierre, Daniel A Lafontaine
Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing thiC riboswitch in Escherichia coli by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the thiC riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.
{"title":"Structural Characterization of the Cotranscriptional Folding of the Thiamin Pyrophosphate Sensing <i>thiC</i> Riboswitch in <i>Escherichia coli</i>.","authors":"Elsa D M Hien, Adrien Chauvier, Patrick St-Pierre, Daniel A Lafontaine","doi":"10.1021/acs.biochem.3c00665","DOIUrl":"10.1021/acs.biochem.3c00665","url":null,"abstract":"<p><p>Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing <i>thiC</i> riboswitch in <i>Escherichia coli</i> by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the <i>thiC</i> riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141304775","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}
Human serum albumin (HSA) is a protein carrier that transports a wide range of drugs and nutrients. The amount of glycated HSA (GHSA) is used as a diabetes biomarker. To quantify the GHSA amount, the fluorescent graphene-based aptasensor has been a successful method. In aptasensors, the key mechanism is the adsorption/desorption of albumin from the aptamer-graphene complex. Recently, the graphene quantum dot (GQD) has been reported to be an aptamer sorbent. Due to its comparable size to aptamers, it is attractive enough to explore the possibility of GQD as a part of an albumin aptasensor. Therefore, molecular dynamics (MD) simulations were performed here to reveal the binding mechanism of albumin to an aptamer-GQD complex in molecular detail. GQD saturated by albumin-selective aptamers (GQDA) is studied, and GHSA and HSA are studied in comparison to understand the effect of glycation. Fast and spontaneous albumin-GQDA binding was observed. While no specific GQDA-binding site on both albumins was found, the residues used for binding were confined to domains I and III for HSA and domains II and III for GHSA. Albumins were found to bind preferably to aptamers rather than to GQD. Lysines and arginines were the main contributors to binding. We also found the dissociation of GLC from all GHSA trajectories, which highlights the role of GQDA in interfering with the ligand binding affinity in Sudlow site I. The binding of GQDA appears to impair albumin structure and function. The insights obtained here will be useful for the future design of diabetes aptasensors.
人血清白蛋白(HSA)是一种蛋白质载体,可运输多种药物和营养物质。糖化 HSA(GHSA)的含量被用作糖尿病的生物标志物。要量化 GHSA 的含量,基于荧光石墨烯的灵敏传感器是一种成功的方法。在灵敏传感器中,关键机制是白蛋白从灵敏配体-石墨烯复合物中的吸附/解吸。最近,有报道称石墨烯量子点(GQD)可作为一种适配体吸附剂。由于石墨烯量子点的尺寸与吸附剂相当,因此有足够的吸引力来探索将石墨烯量子点作为白蛋白吸附传感器一部分的可能性。因此,我们在此进行了分子动力学(MD)模拟,以揭示白蛋白与一种吸附剂-GQD 复合物的分子结合机制。研究了白蛋白选择性适配体(GQDA)饱和的 GQD,并对 GHSA 和 HSA 进行了比较研究,以了解糖化的影响。观察到白蛋白与 GQDA 快速、自发地结合。虽然在两种白蛋白上都没有发现特定的 GQDA 结合位点,但用于结合的残基仅限于 HSA 的结构域 I 和 III 以及 GHSA 的结构域 II 和 III。研究发现,白蛋白更倾向于与适配体而不是 GQD 结合。赖氨酸和精氨酸是造成结合的主要原因。我们还发现 GLC 从所有 GHSA 轨迹中解离,这突显了 GQDA 在干扰 Sudlow 位点 I 的配体结合亲和力方面的作用。本研究获得的启示将有助于未来糖尿病相应传感器的设计。
{"title":"Aggregation of Apo/Glycated Human Serum Albumins and Aptamer-Saturated Graphene Quantum Dot: A Simulation Study.","authors":"Sirin Sittiwanichai, Chanya Archapraditkul, Deanpen Japrung, Yasuteru Shigeta, Toshifumi Mori, Prapasiri Pongprayoon","doi":"10.1021/acs.biochem.4c00155","DOIUrl":"10.1021/acs.biochem.4c00155","url":null,"abstract":"<p><p>Human serum albumin (HSA) is a protein carrier that transports a wide range of drugs and nutrients. The amount of glycated HSA (GHSA) is used as a diabetes biomarker. To quantify the GHSA amount, the fluorescent graphene-based aptasensor has been a successful method. In aptasensors, the key mechanism is the adsorption/desorption of albumin from the aptamer-graphene complex. Recently, the graphene quantum dot (GQD) has been reported to be an aptamer sorbent. Due to its comparable size to aptamers, it is attractive enough to explore the possibility of GQD as a part of an albumin aptasensor. Therefore, molecular dynamics (MD) simulations were performed here to reveal the binding mechanism of albumin to an aptamer-GQD complex in molecular detail. GQD saturated by albumin-selective aptamers (GQDA) is studied, and GHSA and HSA are studied in comparison to understand the effect of glycation. Fast and spontaneous albumin-GQDA binding was observed. While no specific GQDA-binding site on both albumins was found, the residues used for binding were confined to domains I and III for HSA and domains II and III for GHSA. Albumins were found to bind preferably to aptamers rather than to GQD. Lysines and arginines were the main contributors to binding. We also found the dissociation of GLC from all GHSA trajectories, which highlights the role of GQDA in interfering with the ligand binding affinity in Sudlow site I. The binding of GQDA appears to impair albumin structure and function. The insights obtained here will be useful for the future design of diabetes aptasensors.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416538","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}