Biochemistry Biochemistry最新文献

{"title":"Multiple Copper Ions Bind to and Promote the Oligomerization of Huntingtin Protein with Nonpathological Repeat Expansions.","authors":"Deepa Neupane, Miguel Santos-Fernandez, Francisco Fernandez-Lima, Katlyn K Meier","doi":"10.1021/acs.biochem.5c00012","DOIUrl":"10.1021/acs.biochem.5c00012","url":null,"abstract":"<p><p>Huntington's disease (HD) is a fatal neurodegenerative disease characterized by the expression of huntingtin protein (htt) that has a polyglutamine (CAG; polyQ) repeat domain consisting of 36 or more glutamines (mhtt). Historically, mhtt is more broadly associated with HD severity, as are elevated metal levels observed in HD patients. The depletion of wild-type (WT) htt (fewer than 36Qs) is also recognized as a contributing factor to HD progression; however, many questions remain about the interactions of biorelevant metals with WT htt and the impact of the interactions on protein aggregation. In the present work, we utilize a combination of biochemical assays and spectroscopic techniques to provide insights into the interaction of copper with an <i>in vitro</i> htt model (N171-17Q). Herein, we use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering to show that the addition of equimolar or higher concentrations of Cu(II) to htt induces time- and temperature-dependent protein oligomerization/aggregation. Additionally, chelation assays, trapped ion mobility spectrometry, and mass spectrometry confirm the (i) rapid reduction of Cu(II) in the presence of N171-17Q htt, (ii) direct binding of multiple copper ions per protein, and (iii) complex Cu:htt speciation profile with a preference for three distinct Cu:htt states. These findings contribute to our molecular level understanding of copper's role in the depletion and oligomerization/aggregation of WT htt while underscoring the physiological significance of our work, its potential relevance to metal binding in mhtt, and its significance for identifying new avenues for biomarker exploration and therapeutic design strategies.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1121-1135"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143472008","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}

{"title":"Aβ40 Fibril Assembly on Human Cerebral Smooth Muscle Cells Impairs Cell Viability.","authors":"Brandon Irizarry, Judianne Davis, Jitika Rajpoot, Xiaoyue Zhu, Feng Xu, Steven O Smith, William E Van Nostrand","doi":"10.1021/acs.biochem.4c00478","DOIUrl":"10.1021/acs.biochem.4c00478","url":null,"abstract":"<p><p>Cerebral vascular deposition of the amyloid-β (Aβ) peptide, a condition known as cerebral amyloid angiopathy (CAA), is associated with intracerebral hemorrhaging and contributes to disease progression in Alzheimer's disease (AD) and vascular cognitive impairment and dementia (VCID). Familial mutations at positions 22 and 23 within the Aβ peptide lead to early onset and severe CAA pathology. Here, we evaluate the effects of fibrillar Aβ peptides on the viability of primary-cultured human cerebral smooth muscle (HCSM) cells, which are the major site of amyloid deposition in cerebral blood vessel walls. Comparisons are made of the familial E22Q (Dutch) mutant of Aβ40 with wild-type Aβ40 and Aβ42. In agreement with previous studies, we find that there is a significant reduction in cell viability when Aβ40-Dutch or Aβ42-WT peptides are added to HCSM cell cultures as monomeric Aβ, whereas Aβ40-WT is relatively nontoxic. The binding of Aβ fibrils derived from sporadic CAA or familial Dutch-type CAA brain tissue to the membrane surface of HCSM cells does not result in a significant loss of cell viability. In contrast, when Aβ40-WT monomers and sporadic CAA fibrils are coincubated in HCSM cell cultures, there is a significant reduction in HCSM cell viability that is accompanied by an increase in cell surface fibril formation. Lastly, intrathecal administration of Aβ40-Dutch fibrillar seeds promotes fibrillar amyloid accumulation in the smooth muscle of meningeal vessels in the rTg-D transgenic rat model of CAA. Together, the present findings suggest that fibrillar Aβ seeds propagate the expansion of new amyloid fibrils on cerebral vascular smooth muscle, leading to membrane disruption and cell death.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 5","pages":"1065-1078"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539490","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}

{"title":"Sortase-Mediated Fluorescent Labeling of eIF4E for Investigating Translation Initiation Mechanisms.","authors":"Justin Pi, Simpson Joseph","doi":"10.1021/acs.biochem.4c00851","DOIUrl":"10.1021/acs.biochem.4c00851","url":null,"abstract":"<p><p>Translation initiation represents a critical regulatory step in gene expression, orchestrated by the interaction of eukaryotic initiation factor 4E (eIF4E) with the 7-methylguanosine (m⁷G) cap structure at the 5' end of mRNA. This interaction enables eIF4F, composed of eIF4E, eIF4G, and eIF4A, to recruit the 43S preinitiation complex to the mRNA 5' end. The activity of eIF4E is tightly regulated and often dysregulated in cancer, neurological disorders, and viral infections. To investigate the interactions of human eIF4E with m⁷G-RNA and eIF4G, we engineered single-cysteine mutants of eIF4E to enable fluorescent dye attachment. However, these mutants presented challenges in purification and exhibited diminished activity. To overcome these issues, we developed a method to fluorescently label eIF4E via sortase-mediated transpeptidation. Our results demonstrate that sortase-labeled eIF4E retains activity comparable to wild-type eIF4E. This approach provides a valuable tool for studying the dynamic mechanisms of translation initiation and its regulation.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1099-1108"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11887568/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143447436","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}

{"title":"Unraveling the Binding Mode of TSC2-Rheb through Protein Docking and Simulations.","authors":"Berith F Pape, Shraddha Parate, Leif A Eriksson, Vibhu Jha","doi":"10.1021/acs.biochem.4c00562","DOIUrl":"10.1021/acs.biochem.4c00562","url":null,"abstract":"<p><p>Proteasome inhibitors (PIs) constitute the first line of therapy for multiple myeloma (MM). Despite the impressive clinical efficacy, MM remains fatal due to the development of drug resistance over time. During MM progression, stress responses to hypoxia and PIs suppress mammalian target of rapamycin complex 1 (mTORC1) activity by releasing tuberous sclerosis complex 2 (TSC2), which deactivates Ras homologue enriched in brain (Rheb), a crucial regulator of mTORC1. The efficacy of PIs targeting MM is enhanced when mTORC1 is hyperactivated. We thus propose that the inhibition of TSC2 will improve the efficacy of PIs targeting MM. To the best of our knowledge, no cocrystallized structure of the TSC2-Rheb complex has been reported. We therefore developed a representative model using the individual structures of TSC2 (PDB: 7DL2) and Rheb (PDB: 1XTS). Computational modeling involving an extensive protein-protein docking consensus approach was performed to determine the putative binding mode of TSC2-Rheb. The proposed docking poses were refined, clustered, and evaluated by MD simulations to explore the conformational dynamics and protein mobility, particularly at the drug-binding interface of TSC2-Rheb. Our results agree with the suggested binding mode of TSC2-Rheb previously reported in the literature. The results reported herein establish a basis for the development of new inhibitors blocking the binding of TSC2 and Rheb, aiming to reinstate mTORC1 activation and facilitate improved efficacy of PIs against multiple myeloma.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1006-1019"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11883811/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143412269","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}

{"title":"Dynamics in the <i>Phytophthora capsici</i> Effector AVR3a11 Confirm the Core WY Domain Fold.","authors":"James Tolchard, Vicki S Chambers, Laurence S Boutemy, Mark J Banfield, Tharin M A Blumenschein","doi":"10.1021/acs.biochem.4c00660","DOIUrl":"10.1021/acs.biochem.4c00660","url":null,"abstract":"<p><p>Oomycete pathogens cause large economic losses in agriculture through diseases such as late blight (<i>Phytophthora infestans</i>), and stem and root rot of soybean (<i>Phytophthora sojae</i>). The effector protein AVR3a, from <i>P. infestans</i>, and its homologue AVR3a11 from <i>Phytophthora capsici</i>, are host-translocated effectors that interact with plant proteins to evade defense mechanisms and enable infection. Both proteins belong to the family of RXLR effectors and contain an N-terminal secretion signal, an RXLR motif for translocation into the host cell, and a C-terminal effector domain. Within this family, many proteins have been predicted to contain one or more WY domains as their effector domain, which is proposed to encompass a conserved minimal core fold containing three helices, further stabilized by additional helices or dimerization. In AVR3a11, a helical N-terminal extension to the core fold forms a four-helix bundle, as determined by X-ray crystallography. For a complete picture of the dynamics of AVR3a11, we have determined the solution structure of AVR3a11, and studied its dynamics in the fast time scale (ns-ps, from NMR relaxation parameters) and in the slow time scale (seconds to minutes, from hydrogen/deuterium exchange experiments). Hydrogen/deuterium exchange showed that the N-terminal helix is less stable than the other three helices, confirming the core fold originally proposed. Relaxation measurements confirm that AVR3a11 undergoes extensive conformational exchange, despite the uniform presence of fast motions in the spectral density function throughout most of its sequence. As functional residues are in the more mobile regions, flexibility in the slow/intermediate time scale may be functionally important.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1146-1156"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11883810/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143456263","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}

{"title":"Role of the Palmitoyl Group and of the Amphipathic α Helix in the Membrane Binding of the C-Terminus of G-Protein Receptor Kinase 4α/β.","authors":"Marc-Antoine Millette, Ana Coutinho, Manuel Prieto, Christian Salesse","doi":"10.1021/acs.biochem.4c00492","DOIUrl":"10.1021/acs.biochem.4c00492","url":null,"abstract":"<p><p>Membrane binding of monotopic proteins can involve various post-translational modifications or a combination of some membrane-binding elements. For example, amphipathic α helices and palmitoylation could drive the membrane attachment of proteins. G-protein-coupled receptor kinases (GRKs) regulate the activity of G-protein-coupled receptors. Several members of the family of GRKs are acylated. Moreover, the C-terminus of GRK6 contains an amphipathic α helix and a palmitoyl group, which could also be the case for GRK4 isoforms. In our experiments, GRK4α/β-derived peptides of differing C-terminal lengths (Cter-GRK4α/β variants) were thus studied to discriminate the individual role of the palmitoyl group and amphipathic α helix of Cter-GRK4α/β in its membrane binding. The membrane binding of the Cter-GRK4α/β variants was studied by comparing their maximum insertion pressure (MIP) to lipid monolayers as well as their intrinsic fluorescence properties using large unilamellar vesicles. The MIP data show a higher level of binding of the palmitoylated longest GRK4α/β variant. Moreover, MIP measurements in the absence and presence of 15 mol % of the negatively charged phosphoserine demonstrated that the amphipathic α helix of Cter-GRK4α/β plays a major role in its membrane binding. Accordingly, partition studies of the Cter-GRK4α/β variants to membranes by fluorescence spectroscopy demonstrate the involvement of the palmitoyl group and the amphipathic α helix of the C-terminus of GRK4α/β in its membrane binding. Altogether, the data show that both the palmitoyl group and the amphipathic helix highly favor membrane binding of the C-terminus of GRK4α/β, which should facilitate the proper anchoring of GRK4α/β and phosphorylation of GPCRs.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"987-1005"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143466675","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}

{"title":"Regulation of GLP-1 and Glucagon Receptor Function by β-Arrestins in Metabolically Important Cell Types.","authors":"Liu Liu, Misbah Rashid, Jürgen Wess","doi":"10.1021/acs.biochem.4c00867","DOIUrl":"10.1021/acs.biochem.4c00867","url":null,"abstract":"<p><p>Glucagon-like peptide-1 (GLP-1) and glucagon (GCG) are polypeptides derived from a common precursor (preproglucagon) that modulates the activity of numerous cell types involved in regulating glucose and energy homeostasis. GLP-1 and GCG exert their biological functions via binding to specific G protein-coupled receptors (GLP-1Rs and GCGRs). Ligand-activated GLP-1Rs and GCGRs preferentially activate the heterotrimeric G protein G_s, resulting in increased cytosolic cAMP levels. However, activation of the two receptors also leads to the recruitment of β-arrestin-1 and -2 (βarr1 and βarr2, respectively) to the intracellular surface of the receptor proteins. The binding of β-arrestins to the activated receptors contributes to the termination of receptor-stimulated G protein coupling. In addition, receptor-β-arrestin complexes can act as signaling nodes in their own right by modulating the activity of many intracellular signaling pathways. In this Review, we will discuss the roles of βarr1 and βarr2 in regulating key metabolic functions mediated by activated GLP-1Rs and GCGRs. During the past decade, GLP-1R agonists have emerged as highly efficacious antidiabetic and antiobesity drugs. Moreover, dual agonists that stimulate both GLP-1Rs and GCGRs are predicted to offer additional therapeutic benefits as compared to GLP-1R agonist monotherapy. We will summarize and try to synthesize a series of studies suggesting that the development of G protein-biased GLP-1R and/or GCGR agonists, which do not lead to the recruitment of β-arrestins, may lead to even more efficacious therapeutic agents.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"978-986"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143472012","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}

{"title":"From Negative to No Cooperativity: Effects of Mutations on Intersubunit Communication within F420H2:NADP+ Oxidoreductase Using Steady-State and Pre-Steady-State Kinetic Methods","authors":"Jamariya A. Howard,&nbsp;Alaa Aziz,&nbsp;Lindsay A. Davis,&nbsp;Denzel Pugh,&nbsp;Md Sabid Ahamed,&nbsp;Ravi Ramkissoon,&nbsp;Juan Corrales,&nbsp;Nathan T. Nguyen,&nbsp;Charlene Mandimutsira,&nbsp;Tekleab Beyene,&nbsp;Co Ha,&nbsp;Calvin Dao,&nbsp;Parth Nikumbh,&nbsp;Adway O. Zacharias,&nbsp;Saiful M. Chowdhury and Kayunta Johnson-Winters*,&nbsp;","doi":"10.1021/acs.biochem.4c0041610.1021/acs.biochem.4c00416","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00416https://doi.org/10.1021/acs.biochem.4c00416","url":null,"abstract":"<p >F₄₂₀H₂:NADP⁺ oxidoreductase (Fno) catalyzes the reversible production of NADPH by transferring a hydride from the reduced F₄₂₀ cofactor to NADP⁺. Previous kinetic studies suggest that wild-type Fno (<i>wt</i>Fno) displays half-site reactivity and negative cooperativity, making Fno regulatory within methanogenic and sulfate-reducing archaea. These studies identified four amino acids; R186, T192, S190, and H133, as potential candidates involved in intersubunit communication due to their location either at or within close proximity to the interface of the dimer. Therefore, a library of Fno variants─R186K, R186Q, R186I, T192V, T192A, S190A, H133A, and H133N─was generated and characterized using binding, steady-state, and pre-steady-state kinetic experiments to understand their involvement in communication. The Hill coefficient for <i>wt</i>Fno was previously reported as 0.61 ± 0.03, while the R186K, R186Q, R186I, and T192V Fno variant values were close or equal to 1, indicating a switch to no cooperativity behavior. The S190A variant displayed increased Hill coefficients of 0.8 ± 0.1 when compared to <i>wt</i>Fno, showing that cooperativity was affected. The steady-state double reciprocal plots of the R186 variants, S190A, and T192V Fno variants were linear, which is indicative of no cooperativity, departing from the negative cooperativity shape displayed by <i>wt</i>Fno. Unlike <i>wt</i>Fno, the pre-steady-state kinetic experiments did not display half-site reactivity for the variants. Additionally, the hydride transfer step became rate-limiting in catalysis for the R186K Fno variant only. Our data suggest that negative cooperativity can be disrupted and that the amino acids R186, T192, and S190 are involved in intersubunit communication.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 6","pages":"1338–1347 1338–1347"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143636020","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}

{"title":"Quantitative and Systematic NMR Measurements of Sequence-Dependent A-T Hoogsteen Dynamics in the DNA Double Helix.","authors":"Akanksha Manghrani, Atul Kaushik Rangadurai, Or Szekely, Bei Liu, Serafima Guseva, Hashim M Al-Hashimi","doi":"10.1021/acs.biochem.4c00820","DOIUrl":"10.1021/acs.biochem.4c00820","url":null,"abstract":"<p><p>The dynamic properties of DNA depend on the sequence, providing an important source of sequence-specificity in biochemical reactions. However, comprehensively measuring how these dynamics vary with sequence is challenging, especially when they involve lowly populated and short-lived conformational states. Using ¹H CEST supplemented by targeted ¹³C <i>R</i>_1ρ NMR experiments, we quantitatively measured Watson-Crick to Hoogsteen dynamics for an A-T base pair in 13 trinucleotide sequence contexts. The Hoogsteen population and exchange rate varied 4-fold and 16-fold, respectively, and were dependent on both the 3'- and 5'-neighbors but only weakly dependent on monovalent ion concentration (25 versus 100 mM NaCl) and pH (6.8 versus 8.0). Flexible TA and CA dinucleotide steps exhibited the highest Hoogsteen populations, and their kinetics rates strongly depended on the 3'-neighbor. In contrast, the stiffer AA and GA steps had the lowest Hoogsteen population, and their kinetics were weakly dependent on the 3'-neighbor. The Hoogsteen lifetime was especially short when G-C neighbors flanked the A-T base pair. Our results uncover a unique conformational basis for sequence-specificity in the DNA double helix and establish the utility of NMR to quantitatively and comprehensively measure sequence-dependent DNA dynamics.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1042-1054"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143472010","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}

{"title":"Distinct Acyl Carrier Protein Docking Sites Help Mediate the Opposite Stereoselectivities of A- and B-type Modular Polyketide Synthase Ketoreductases.","authors":"Katherine A Ray, Sally N Lin, Adrian T Keatinge-Clay","doi":"10.1021/acs.biochem.4c00565","DOIUrl":"10.1021/acs.biochem.4c00565","url":null,"abstract":"<p><p>The domains of modular polyketide synthases (PKSs) collaborate to extend and process polyketide intermediates; however, most of their interactions with one another remain mysterious. We used AlphaFold 2 to investigate how acyl carrier proteins (ACPs) present intermediates to ketoreductases (KRs), processing domains capable of not only setting the stereochemical orientations of β-hydroxyl substituents but also of α-substituents. In modules that do not contain a dehydratase (DH), the A- and B-type KRs that, respectively, generate l- and d-oriented β-hydroxy groups are predicted to possess distinct ACP docking sites. In modules containing DHs, where A-type KRs are much less common, both KR types are predicted to possess an ACP-docking site equivalent to that of B-type KRs from modules without DHs. To investigate this most common ACP docking site, mutagenesis was performed on 20 residues of the KR from the second pikromycin module within the model triketide synthase <b>P1</b>-<b>P2</b>-<b>P7</b>. The least active variants are those with mutations to a conserved hydrophobe, 2 residues downstream of the LDD motif of B-type KRs, predicted to insert into a hole adjacent to the phosphopantetheinylated serine of ACP.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1136-1145"},"PeriodicalIF":2.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11920649/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143397501","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}