Biochemistry Biochemistry最新文献

Disruption of Molecular Interactions between the G3BP1 Stress Granule Host Protein and the Nucleocapsid (NTD-N) Protein Impedes SARS-CoV-2 Virus Replication.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-21 DOI: 10.1021/acs.biochem.4c00536

Preeti Dhaka, Ankur Singh, Sanketkumar Nehul, Shweta Choudhary, Prasan Kumar Panda, Gaurav Kumar Sharma, Pravindra Kumar, Shailly Tomar

{"title":"Disruption of Molecular Interactions between the G3BP1 Stress Granule Host Protein and the Nucleocapsid (NTD-N) Protein Impedes SARS-CoV-2 Virus Replication.","authors":"Preeti Dhaka, Ankur Singh, Sanketkumar Nehul, Shweta Choudhary, Prasan Kumar Panda, Gaurav Kumar Sharma, Pravindra Kumar, Shailly Tomar","doi":"10.1021/acs.biochem.4c00536","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00536","url":null,"abstract":"The Ras GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) serves as a formidable barrier to viral replication by generating stress granules (SGs) in response to viral infections. Interestingly, viruses, including SARS-CoV-2, have evolved defensive mechanisms to hijack SG proteins like G3BP1 for the dissipation of SGs that lead to the evasion of the host's immune responses. Previous research has demonstrated that the interaction between the NTF2-like domain of G3BP1 (G3BP1NTF-2) and the intrinsically disordered N-terminal domain (NTD-N1-25) of the N-protein plays a crucial role in regulating viral replication and pathogenicity. Interestingly, the current study identified an additional upstream stretch of residues (128KDGIIWVATEG138) (N128-138) within the N-terminal domain of the N-protein (NTD-N41-174) that also forms molecular contacts with the G3BP1 protein, as revealed through in silico analysis, site-directed mutagenesis, and biochemical analysis. Remarkably, WIN-62577, and fluspirilene, the small molecules targeting the conserved peptide-binding pocket in G3BP1NTF-2, not only disrupted the protein-protein interactions (PPIs) between NTD-N41-174 and G3BP1NTF-2 but also exhibited significant antiviral efficacy against SARS-CoV-2 replication with EC50 values of ∼1.8 and ∼1.3 μM, respectively. The findings of this study, validated by biophysical thermodynamics and biochemical investigations, advance the potential of developing therapeutics targeting the SG host protein against SARS-CoV-2, which may also serve as a broad-spectrum antiviral target.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2024-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142870575","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

Insights into the Activation and Self-Association of Arrestin-1.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-20 DOI: 10.1021/acs.biochem.4c00632

David Salom, Philip D Kiser, Krzysztof Palczewski

{"title":"Insights into the Activation and Self-Association of Arrestin-1.","authors":"David Salom, Philip D Kiser, Krzysztof Palczewski","doi":"10.1021/acs.biochem.4c00632","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00632","url":null,"abstract":"Arrestins halt signal transduction by binding to the phosphorylated C-termini of activated G protein-coupled receptors. Arrestin-1, the first subtype discovered, binds to rhodopsin in rod cells. Mutations in SAG, the gene encoding Arrestin-1, are linked to Oguchi disease, characterized by delayed dark adaptation. Since the discovery of Arrestin-1, substantial progress has been made in understanding the role of these regulatory proteins in phototransduction, including the characterization of visual phenotypes of animals and humans lacking this protein, discovery of splice variants, and documentation of its binding to inositol-polyphosphates. Arrestin-1 was one of the first structurally characterized proteins in the phototransduction cascade. However, there are knowledge gaps regarding the conformational intermediates leading to its binding to phosphorylated rhodopsin. Among various mammalian Arrestin-1 conformations captured via crystallography, the preactivated state is represented by the mutant R175E-Arrestin-1 and by a C-terminally truncated splice variant (p44). This report describes a novel purification method of Arrestin-1 from bovine retinas followed by limited proteolysis to obtain a protein resembling p44. We solved the crystal structure of this preactivated, shortened 3-367Arrestin-1 at a resolution of 1.40 Å. The structure reveals a more complete picture of the finger loop structure and of the role of the polar core in the activation of Arrestin-1. The structure of 3-367Arrestin-1 captures an intermediate form halfway between the inactive and fully activated conformations of Arrestin-1. Finally, we addressed the question of Arrestin-1 oligomerization by comparing the packing interfaces in different Arrestin-1 crystals and dimer models predicted by AlphaFold 3.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142862518","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

Autoregulation of TRF2 through G-Quadruplex-Specific Interaction between the Gene and N-Terminal Domain of the Protein.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-20 DOI: 10.1021/acs.biochem.4c00287

Xiaojuan Xu, Tao Wang

{"title":"Autoregulation of TRF2 through G-Quadruplex-Specific Interaction between the Gene and N-Terminal Domain of the Protein.","authors":"Xiaojuan Xu, Tao Wang","doi":"10.1021/acs.biochem.4c00287","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00287","url":null,"abstract":"Telomere repeat-binding factor 2 (TRF2) is a key component of the shelterin complex which guards the integrity of the telomere. Most of the TRF2 discussed previously was focused on the telomere, and relatively less is discussed on aspects other than that. It is proved that TRF2 also localizes to other potential G-quadruplex-forming sequences among the whole genome besides the telomere. Therefore, it may participate in regulating genes generally except for the well-known function of protecting telomeres. Here, we demonstrate that the N-terminal basic domain of TRF2 (TRF2B) can interact with the G-quadruplex formed by the 5'-UTR sequence of its gene. Subsequently, this interaction was identified as G-quadruplex-specific. Using a reporter gene system, we proved that the translation of the reporter gene was dramatically reduced, triggered by the interaction between TRF2B and the G-quadruplex. Altogether, we propose that TRF2 can be \"auto-regulated\" through the G-quadruplex formed by its own gene sequence. This finding indicates a potential feedback mechanism in the regulation of the TRF2 gene. Additionally, it suggests a common mode in gene regulation involving the cooperation of TRF2 and the G-quadruplex.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142869271","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

Determining the Electrostatic Contributions of GTPase-GEF Complexes on Interfacial Drug Binding Specificity: A Case Study of a Protein-Drug-Protein Complex. 确定 GTPase-GEF 复合物对界面药物结合特异性的静电贡献：蛋白质-药物-蛋白质复合物案例研究。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-17 Epub Date: 2024-11-26 DOI: 10.1021/acs.biochem.4c00313

Frank A Jermusek, Lauren J Webb

{"title":"Determining the Electrostatic Contributions of GTPase-GEF Complexes on Interfacial Drug Binding Specificity: A Case Study of a Protein-Drug-Protein Complex.","authors":"Frank A Jermusek, Lauren J Webb","doi":"10.1021/acs.biochem.4c00313","DOIUrl":"10.1021/acs.biochem.4c00313","url":null,"abstract":"Understanding the factors that contribute to specificity of protein-protein interactions allows for design of orthosteric small molecules. Within this environment, a small molecule requires both structural and electrostatic complementarity. While the structural contribution to protein-drug-protein specificity is well characterized, electrostatic contributions require more study. To this end, we used a series of protein complexes involving Arf1 bound to guanine nucleotide exchange factors (GEFs) that are sensitive or resistant to the small molecule brefeldin A (BFA). By comparing BFA-sensitive Arf1-Gea1p and Arf1-ARNO with different combinations of four BFA sensitizing ARNO mutations (ARNOwt, ARNO1M, ARNO3M, and ARNO4M), we describe how electrostatic environments at each interface guide BFA binding specificity. We labeled Arf1 with cyanocysteine at several interfacial sites and measured by nitrile adsorption frequencies to map changes in electric field at each interface using the linear Stark equation. Temperature dependence of nitrile vibrational spectra was used to investigate differences in hydrogen bonding environments. These comparisons showed that interfacial electric field at the surface of Arf1 varied substantially depending on the GEF. The greatest differences were seen between Arf1-ARNOwt and Arf1-ARNO4M, suggesting a greater change in electric field is required for BFA binding to Arf1-ARNO. Additionally, rigidity of the interface of the Arf1-ARNO complex correlated strongly with BFA sensitivity, indicating that flexible interfaces are sensitive to disruption upon orthosteric small molecule binding. These findings demonstrate a qualitatively consistent electrostatic environment for Arf1 binding and more subtle differences preventing BFA specificity. We discuss how these results will guide improved design of other small molecules that can target protein-protein interfaces.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"3220-3235"},"PeriodicalIF":2.9,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142714763","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

Insights into the Mechanism of Paraoxonase-1: Comparing the Reactivity of the Six-Bladed β-Propeller Hydrolases. 对氧杂环戊烯酶-1 机制的启示：比较六叶片 β-螺旋桨水解酶的反应性。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-17 DOI: 10.1021/acs.biochem.8b01115

Timothy J Grunkemeyer, David G Mata, Kiran Doddapaneni, Srividya Murali, Thomas J Magliery

{"title":"Insights into the Mechanism of Paraoxonase-1: Comparing the Reactivity of the Six-Bladed β-Propeller Hydrolases.","authors":"Timothy J Grunkemeyer, David G Mata, Kiran Doddapaneni, Srividya Murali, Thomas J Magliery","doi":"10.1021/acs.biochem.8b01115","DOIUrl":"10.1021/acs.biochem.8b01115","url":null,"abstract":"The mammalian protein paraoxonase-1 (PON1) has been explored as a promising bioscavenger treatment for organophosphorus (OP) agent poisoning, but it is not active enough to protect against many agents. Engineering is limited because PON1's catalytic mechanism is poorly understood; moreover, its native activity and substrate are unknown. PON1 is a calcium-bound six-bladed β-propeller hydrolase that shares high structural homology, a conserved metal-coordinating active site, and substrate specificity overlap with other members of a superfamily that includes squid diisopropylfluorophosphatase, bacterial drug responsive protein 35, and mammalian senescence marker protein 30. We hypothesized that, by examining the reactivity of all four hydrolases using a common set of conservative mutations, we could gain further insight into the catalytic mechanism of PON1. We chose a set of mutations to examine conserved Asp and Glu residues in the hydrolase active sites and the ligation sphere around the catalytic calcium and a His-His dyad seen in PON1. The wild-type (WT) and mutant hydrolases were assayed against a set of lactones, aryl esters, and OPs that PON1 is known to hydrolyze. Surprisingly, some mutations of Ca2+-coordinating residues, previously thought to be essential for turnover, resulted in significant activity toward all substrate classes examined. Additionally, merely maintaining WT-like charge in the active site of PON1 was insufficient for high activity. Finally, the H115-H134 dyad does not appear to be essential for catalysis against any substrate. Therefore, previously proposed mechanisms must be re-evaluated.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"3287-3299"},"PeriodicalIF":2.9,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36769318","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

More Pieces of the Puzzle: Transient State Analysis of Dihydroorotate Dehydrogenase B from Lactoccocus lactis.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-17 Epub Date: 2024-12-02 DOI: 10.1021/acs.biochem.4c00475

Corine O Smith, Graham R Moran

{"title":"More Pieces of the Puzzle: Transient State Analysis of Dihydroorotate Dehydrogenase B from Lactoccocus lactis.","authors":"Corine O Smith, Graham R Moran","doi":"10.1021/acs.biochem.4c00475","DOIUrl":"10.1021/acs.biochem.4c00475","url":null,"abstract":"Dihydroorotate dehydrogenases (DHODs) catalyze the transfer of electrons between dihydroorotate and specific oxidant substrates. Class 1B DHODs (DHODBs) use NAD+ as the oxidant substrate and have a heterodimeric structure that incorporates two active sites, each with a flavin cofactor. One Fe2S2 center lies roughly equidistant between the flavin isoalloxazine rings. This arrangement allows for simultaneous association of reductant and oxidant substrates. Here we describe a series of experiments designed to reveal sequences and contingencies in DHODB chemistry. From these data it was concluded that the resting state of the enzyme is FAD•Fe2S2•FMN. Reduction by either NADH or DHO results in two electrons residing on the FMN cofactor that has a 47 mV higher reduction potential than the FAD. The FAD•Fe2S2•FMNH2 state accumulates with a bisemiquinone state that is an equilibrium accumulation formed from a partial transfer of one electron to the FAD. Pyrimidine reduction is reliant on the availability of the Cys135 proton, as the C135S variant slows orotate reduction by ∼40-fold. The rate of pyrimidine reduction is modulated by occupancy of the FAD site; NADH•FAD•Fe2S2•FMNH2•orotate complex can reduce the pyrimidine at 16 s-1, while NAD+•FAD•Fe2S2•FMNH2•orotate complex reduces the pyrimidine at 5.4 s-1 and the FAD•Fe2S2•FMNH2•orotate complex at 0.6 s-1. This set of effector states account for the apparent discrepancy in the slowest rate observed in transient state single turnover reactions with limiting NADH and the limiting rate observed in steady state.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"3324-3335"},"PeriodicalIF":2.9,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142764524","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

PHP-Family Diesterase from Novosphingobium with Broad Specificity and High Catalytic Efficiency against Organophosphate Flame-Retardant Derived Diesters.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-17 Epub Date: 2024-12-02 DOI: 10.1021/acs.biochem.4c00350

Preston Garner, Andrew C Davis, Andrew N Bigley

{"title":"PHP-Family Diesterase from Novosphingobium with Broad Specificity and High Catalytic Efficiency against Organophosphate Flame-Retardant Derived Diesters.","authors":"Preston Garner, Andrew C Davis, Andrew N Bigley","doi":"10.1021/acs.biochem.4c00350","DOIUrl":"10.1021/acs.biochem.4c00350","url":null,"abstract":"Organophosphate flame retardants have been widely used in plastic products since the early 2000s. Unfortunately, these compounds leach out of the plastics over time and are carcinogenic, developmental toxins, and endocrine disruptors. Due to the high usage levels and stable nature of the compounds, widespread contamination of the environment has now been observed. Despite their recent introduction into the environment, bacteria from the Sphingomonadaceae family have evolved a three-step hydrolytic pathway to utilize these compounds. The second step in this pathway in Sphingobium sp. TCM1 is catalyzed by Sb-PDE, which is a member of the polymerase and histidinol phosphatase (PHP) family of phosphatases. This enzyme is only the second case of a PHP-family enzyme capable of hydrolyzing phosphodiesters. Bioinformatics analysis has now been used to identify a second PHP diesterase from Novosphingobium sp. EMRT-2 (No-PDE). Kinetic characterization of Sb-PDE and No-PDE with authentic organophosphate flame-retardant diesters demonstrates that these enzymes are true diesterases with more than 1000-fold selectivity for the diesterase activity seen in some cases. Synthesis of a wide array of authentic flame-retardant diesters has allowed the substrate specificity of these enzymes to be determined, and mutagenic analysis of the active site residues has identified key residues that give rise to the high levels of diesterase activity. Despite high sequence identity, No-PDE is found to have a broader substrate specificity against flame-retardant derived diesters, and kcat/Km values greater than 104 M-1 s-1 are seen with the best substrates.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"3189-3193"},"PeriodicalIF":2.9,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142764528","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

HutZ from Aliivibrio fischeri Inhibits HutW-Mediated Anaerobilin Formation by Sequestering Heme.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-17 Epub Date: 2024-12-06 DOI: 10.1021/acs.biochem.4c00624

Alexandra K McGregor, Kirsten R Wolthers

{"title":"HutZ from Aliivibrio fischeri Inhibits HutW-Mediated Anaerobilin Formation by Sequestering Heme.","authors":"Alexandra K McGregor, Kirsten R Wolthers","doi":"10.1021/acs.biochem.4c00624","DOIUrl":"10.1021/acs.biochem.4c00624","url":null,"abstract":"Anaerobilin synthase catalyzes the decyclization of the heme protoporphyrin ring, an O2-independent reaction that liberates iron and produces the linear tetrapyrrole, anaerobilin. The marine bacterium Aliivibrio fischeri, the enteric pathogen Escherichia coli O157:H7, and the opportunistic oral pathogen Fusobacterium nucleatum encode anaerobilin synthase as part of their heme uptake/utilization operons, designated chu (E. coli O157:H7), hmu (F. nucleatum), and hut (A. fischeri). F. nucleatum and E. coli O157:H7 contain accessory proteins (ChuS, ChuY, and HmuF) encoded in their respective operons that mitigate against the cytotoxicity of labile heme and anaerobilin by functioning in heme trafficking and anaerobilin reduction. However, the hut operon of A. fischeri and other members of the Vibrionaceae family including the enteric pathogen Vibrio cholerae do not contain homologues to these accessory proteins, raising questions as to how members of this family mitigate against anaerobilin and heme toxicity. Herein, we show that HutW (anaerobilin synthase) from A. fischeri produces anaerobilin, but that HutX and HutZ, encoded downstream of HutW, do not catalyze anaerobilin reduction in the presence of excess NAD(P)H, FAD, and FMN. However, we show that HutZ prevents labile heme and anaerobilin cytotoxicity by binding tightly to heme, sequestering it from HutW, and preventing anaerobilin formation. Thus, A. fischeri is seemingly unable to extract iron from heme using the hutWXZ gene products. Our results further suggest that the structurally distinct chu, hmu, and hut operons have functionally converged to protect the cell from anaerobilin accumulation and heme cytotoxicity.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"3357-3368"},"PeriodicalIF":2.9,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142789414","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

Evidence of a Catalytic Dyad in F₄₂₀-Dependent Glucose-6-phosphate Dehydrogenase from Mycobacterium tuberculosis.

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-17 DOI: 10.1021/acs.biochem.4c00557

Alaa Aziz, Lindsay A Davis, Ravi Ramkissoon, Neema Zeighami, Mansi Lohtia, Jamariya A Howard, Edward N Baker, Ghader Bashiri, Kayunta L Johnson-Winters

{"title":"Evidence of a Catalytic Dyad in F420-Dependent Glucose-6-phosphate Dehydrogenase from Mycobacterium tuberculosis.","authors":"Alaa Aziz, Lindsay A Davis, Ravi Ramkissoon, Neema Zeighami, Mansi Lohtia, Jamariya A Howard, Edward N Baker, Ghader Bashiri, Kayunta L Johnson-Winters","doi":"10.1021/acs.biochem.4c00557","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00557","url":null,"abstract":"F420-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using cofactor F420 as the hydride transfer acceptor. Our previous pH dependence studies suggested that E109 serves as an active site acid, donating a proton to the N-1 position of F420, while leaving the role of H40 unanswered, which was previously suggested to serve as the active site base. This work utilizes thermodynamic and kinetic studies to elucidate additional mechanistic details concerning the roles of H40 and E13. The E13 residue had not previously been considered as a key player during catalysis. Therefore, the H40A, H40Q, E13A, and E13Q FGD variants were generated and fully characterized to determine their roles in catalysis. Here, we conducted temperature-dependent pH profiles and inactivation experiments using diethylpyrocarbonate (DEPC) to determine the role of H40 during catalysis. The temperature-dependent experiments suggest that an acidic histidine can donate a proton to E13. The inactivation experiments revealed monophasic kinetics, suggesting that the one active site H40 is covalently modified by DEPC. Therefore, the active site base is a deprotonated H40 that abstracts a proton from G6P, and then a hydride is transferred to the C-5 position of cofactor F420. These data suggest that E13 and H40 act as a catalytic dyad. Global analysis of the pre-steady-state experiments revealed the accumulation of an intermediate, the spectrum of which resembles an enzyme-product complex. The global analysis also reveals fast chemistry and slow product release with cofactor association being rate-limiting in catalysis.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142845300","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

New Insights into the Mechanism of Action of L-681,217, a Medicinally Promising Polyketide Inhibitor of Bacterial Protein Translation. 对L-681,217作用机制的新认识，L-681,217是一种有药用前景的细菌蛋白质翻译多酮抑制剂。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-12-17 Epub Date: 2024-11-22 DOI: 10.1021/acs.biochem.4c00541

Alexander M Soohoo, Rolin A Aguilar, Heewon Cho, Thomas M Privalsky, Lin Liu, Khanh P Nguyen, Christopher T Walsh, Chaitan Khosla

{"title":"New Insights into the Mechanism of Action of L-681,217, a Medicinally Promising Polyketide Inhibitor of Bacterial Protein Translation.","authors":"Alexander M Soohoo, Rolin A Aguilar, Heewon Cho, Thomas M Privalsky, Lin Liu, Khanh P Nguyen, Christopher T Walsh, Chaitan Khosla","doi":"10.1021/acs.biochem.4c00541","DOIUrl":"10.1021/acs.biochem.4c00541","url":null,"abstract":"An attractive strategy for combating antibacterial resistance involves the development of new antibiotics whose mechanisms differ from those of existing ones in the clinic. Elfamycin antibiotics, whose prototypes include kirromycin and aurodox, are illustrative examples based on their ability to target EF-Tu, an essential component for protein translation in bacteria. Our efforts to revisit this antibiotic class were enabled by two developments. First, we produced L-681,217, an understudied member of this polyketide family harboring a terminal carboxylic acid in place of a hydroxypyridone ring, and synthesized a biotinylated derivative with comparable activity to the natural product. Second, we established a sensitive cell-free protein synthesis (CFPS) assay in which superfolder green fluorescent protein (sfGFP) production was inhibited by L-681,217. Biotinyl-L-681,217 was used to drain the CFPS system of endogenous EF-Tu, allowing replenishment with orthologs to interrogate pathogen selectivity and propensity toward resistance. Comparative in vitro analysis of kirromycin and L-681,217 showed that, while both antibiotics are equipotent in CFPS assays, they interact distinctly with purified EF-Tu, a feature that presumably correlates with prior observations that kirromycin enhances GTP hydrolysis by EF-Tu whereas L-681,217 does not. Analysis of L-681,217 and kirromycin accumulation in selected mutant E. coli strains also revealed that antibiotic import and efflux contributed to resistance. The promise of L-681,217 as a medicinal lead was underscored by the observation that, unlike aurodox, this polyketide does not inhibit adenylosuccinate synthase.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"3336-3347"},"PeriodicalIF":2.9,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142692244","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