Biochemistry Biochemistry最新文献

{"title":"Decoding the Biochemical Complexities Using Raman Spectroscopy","authors":"Jiro Karlo,&nbsp;Dhananjaya Giriraju,&nbsp;Nilkamal Mahanta,&nbsp;Surya Pratap Singh","doi":"10.1021/acs.biochem.6c00389","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00389","url":null,"abstract":"<p>Obtaining biochemical information and monitoring thebiomoleculesin complex biological systems and environments remains a challengeand requires methods that can detect biomolecules. Raman spectroscopyhas emerged as a powerful analytical tool for biochemical analysisowing to its ability to provide label-free, nondestructive, and chemicallyspecific molecular information with minimal sample preparation. Thisreview summarizes the principles and recent advances of Raman spectroscopyfor the characterization of biomolecules and complex biological systems.We discuss its applications in the detection and mapping of majorbiochemical constituents, including proteins, lipids, nucleic acids,carbohydrates, and metabolites. Recent developments in Raman mappinghave significantly enhanced the sensitivity and spatial resolutionof biochemical investigations. The review further highlights the growinguse of Raman-active probes and stable-isotope-based strategies andcurrent challenges for monitoring metabolic processes and intracellularmolecular dynamics. Collectively, this review provides an overviewof the capabilities, applications, and emerging opportunities of Ramanspectroscopy as a versatile platform for biochemical analysis.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 16","pages":"2456–2469"},"PeriodicalIF":2.7,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769687","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":"Bilayer Elastic Properties Modulate Interface Energetics of Transmembrane β-Barrels","authors":"Swadha Gupta,&nbsp;Radhakrishnan Mahalakshmi","doi":"10.1021/acs.biochem.6c00283","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00283","url":null,"abstract":"<p>Assembly energetics of transmembrane proteins occursthrough theorchestrated molecular interplay of the protein’s membranepartitioning free energy and the mechanical and physicochemical propertiesof the bilayer. While amphipathic residues help accommodate diametricallyopposite forces and the steep energy gradient at the membrane–waterinterface, the magnitude to which the intrinsic curvature and elasticenergy of the bilayer regulates protein thermodynamics at the interfaceremains poorly understood. Here, using PagP as a model β-barrel,we compare the per-residue energetics of an interface site acrossmembranes with(out) distinct curvature stress, lipid packing, andheadgroup chemistry. In vesicles, the measured interface energeticscorrelates well with known free energy scales. Interestingly, unlikevesicles, we show that planar bicelles impose fundamentally distinctmembrane–protein coupling regimes: (i) PagP experiences alteredfolding with a stabilized membrane-associated intermediate and (ii)side chain transfer free energies are restructured in a residue-specificmanner. Additionally, the energetics are unaffected by the presenceof nonlamellar lipids. Our results reveal an unexpected dependenceof partitioning energetics on both bilayer elastic energy and lipidpacking defects, demonstrating how subtle changes in membrane physicalproperties could redirect protein folding pathways and thermodynamics.Our findings underscore the need for caution when extrapolating sidechain transfer free energies from simplified model membranes to crowded,compositionally complex in vivo environments.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 16","pages":"2535–2547"},"PeriodicalIF":2.7,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769780","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":"Spontaneous Refolding of Escherichia coli Methionine Adenosyltransferase (MetK) Is Extremely Slow, Indicative of Chaperone Dependence","authors":"Anupam Barai,&nbsp;Pronoy Majumder,&nbsp;Madhurima Sarkar,&nbsp;Tapan K. Chaudhuri","doi":"10.1021/acs.biochem.6c00531","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00531","url":null,"abstract":"<p><em>Escherichia coli</em> methionineadenosyltransferase(MetK) catalyzes the synthesis of S-adenosylmethionine from ATP and <span>l</span>-methionine and is essential for cellular metabolism. To gaininsight into its conformational stability and folding behavior <em>in vitro</em>, we investigated the unfolding and refolding ofMetK using guanidine hydrochloride (GdnHCl), urea, and acid (pH 2.0)as denaturing conditions. Unfolding was monitored using enzymaticactivity, tryptophan fluorescence, far-UV circular dichroism (CD),native-PAGE, and light scattering. GdnHCl, urea, and acid treatmentgenerated structurally distinct denatured ensembles with differentstructural and functional properties. Although unfolding transitionsmonitored individually by tryptophan fluorescence and far-UV CD couldbe described by simple unfolding models, noncoincident transitionstogether with singular value decomposition (SVD) analysis suggestedconformational heterogeneity during unfolding. Refolding of denaturedMetK in native buffer was inefficient and accompanied by aggregation,whereas the inclusion of physiologically relevant osmolytes such asglycerol and trehalose substantially improved refolding yields. Completerecovery of enzymatic activity was achieved only from the 8 M urea-denaturedstate, with a refolding half-time of 4.64 ± 0.23 h. Collectively,these results reveal denaturant-dependent differences in the unfoldingand refolding behavior of MetK and highlight the importance of unfolded-stateproperties and osmolyte-assisted stabilization in determining productivefolding outcomes.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 16","pages":"2522–2534"},"PeriodicalIF":2.7,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769666","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 Kinetic Analysis of Protein–Phosphoinositide Binding by Optimized Liposome-Based Bio-Layer Interferometry","authors":"Pinshuo Yao,&nbsp;Taki Nishimura,&nbsp;Kotaro Tsuboyama","doi":"10.1021/acs.biochem.6c00344","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00344","url":null,"abstract":"<p>Phosphoinositide-binding domains recruit cytosolic proteinstospecific membrane surfaces, and their binding specificity and kineticsare critical determinants of protein localization and signaling. However,most available assays report only equilibrium binding strength anddo not resolve the underlying association and dissociation kinetics.Here, we developed an optimized liposome-based bio-layer interferometry(BLI) assay for specificity profiling and comparative kinetic analysisof protein–phosphoinositide interactions. Buffer optimizationshowed that 0.5% bovine serum albumin and 0.001% Tween-20 almost completelysuppressed nonspecific protein adsorption to the biosensor surfacewhile preserving the expected specific interactions between proteinsand liposomes. Using representative PX and PH domain proteins andbinding-altered mutants, we obtained phosphoinositide-binding profilesconsistent with previous biochemical data. Concentration-dependentBLI analysis further resolved distinct kinetics contributing to affinityenhancement: for a PX-SnxA mutant series, stronger apparent bindingwas associated primarily with increased association rates, whereasfor PH-AKT1 mutants, it was associated primarily with decreased dissociationrates. These results show that the platform can attribute differencesin apparent affinity to specific changes in association and/or dissociationrates, providing a level of mechanistic resolution useful for bothcharacterizing and engineering lipid-binding probes.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 16","pages":"2557–2565"},"PeriodicalIF":2.7,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769836","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":"Energetic Determinants of Expanded PAM Recognition in Engineered FnCas9","authors":"Ajit Kumar,&nbsp;Priyanka Kumari,&nbsp;Shalini Mishra,&nbsp;Shubham Thakur,&nbsp;Sundaram Acharya,&nbsp;Debojyoti Chakraborty,&nbsp;Souvik Maiti,&nbsp;Niyati Jain","doi":"10.1021/acs.biochem.6c00350","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00350","url":null,"abstract":"<p>Expanding protospacer-adjacent motif (PAM) compatibilitywhilepreserving specificity remains a central challenge in CRISPR–Cas9engineering. <em>Francisella novicida</em> Cas9(FnCas9) exhibits high intrinsic specificity but is constrained bystringent PAM requirements. Here, we quantitatively examine the energeticand catalytic consequences of PAM-interacting mutations in three engineeredvariants, en1 (E1369R), en15 (E1603H), and en31 (G1243T/E1369R/E1449H),using a <em>VEGFA3</em> DNA substrate framework. Microscalethermophoresis and isothermal titration calorimetry reveal that theengineered variants enhance binding affinity toward the canonicalNGG PAM relative to wild-type FnCas9, with modest gains in bindingfree energy. Selected noncanonical PAM substrates, particularly TGAand TAG, also show improved binding by en15 and en31, with en31 displayingthe strongest overall binding among the substrates tested. Thermodynamicprofiles indicate that enhanced affinity is associated with more favorableenthalpic contributions, consistent with altered interactions at thePAM interface; however, the specific molecular contributions underlyingthese changes remain to be directly established. Despite improvedbinding, active-site titration reveals reduced fractions of catalyticallycompetent enzyme in engineered variants, particularly en31, necessitatinghigher enzyme concentrations to achieve cleavage efficiencies comparableto wild-type. Cleavage assays demonstrate that en31 most effectivelycouples improved recognition of the tested noncanonical PAM substratesto productive catalysis, enabling robust cleavage of both TGA andTAG substrates while maintaining minimal off-target activity underthe conditions examined. Together, these results suggest that PAM-interactingmutations in FnCas9 can alter the energetic coupling between DNA bindingand catalytic activation within the <em>VEGFA3</em> substrateframework tested, highlighting the importance of balancing substrateaffinity with conformational activation in the design of high-precisiongenome-editing nucleases.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 16","pages":"2495–2509"},"PeriodicalIF":2.7,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769517","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":"Mena EVH1 Interacts with PTP1B Via a Distributed Interface Without Regulating Enzymatic Activity","authors":"Lanette LaComb,&nbsp;Sean M. Cahill,&nbsp;Jeffrey B. Bonanno,&nbsp;David Cowburn,&nbsp;Steven C. Almo,&nbsp;Agnidipta Ghosh","doi":"10.1021/acs.biochem.6c00299","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00299","url":null,"abstract":"<p>The interaction between mammalianenabled protein (Mena),an actinregulatory protein, and protein tyrosine phosphatase 1B (PTP1B) iscritical for maintaining epidermal growth factor receptor (EGFR) signalinghomeostasis and regulating cellular motility. Mena recruits PTP1Bto activated EGFR in vivo, facilitating receptor dephosphorylationand limiting invasive signaling. However, overexpression of wild-typeMena or the invasive isoform, Mena^INV, perturbs this regulatorymechanism by altering PTP1B localization and EGFR signaling dynamics.Here, we report the solution NMR structure of the unliganded MenaEVH1 domain, which contains a conserved aromatic triad (Y16, W23,F77) that forms the canonical polyproline-binding cleft. Buildingon prior evidence that the PTP1B polyproline region serves as an EVH1ligand, we investigated the structural basis of the interaction betweenEVH1 and wild-type PTP1B. NMR chemical shift perturbation analysesusing multiple PTP1B variants, including full-length and truncatedconstructs, revealed that binding affects not only the canonical EVH1hydrophobic cleft but also residues on the opposite surface, suggestingan expanded interaction interface. Diffusion-ordered spectroscopy(DOSY) and size-exclusion chromatography further indicate that PTP1Bvariants containing the disordered C-terminal tail form more compactcomplexes with EVH1, consistent with a disorder-to-order transition.Complementary perturbation studies using [²H,¹⁵N] PTP1B demonstrate that EVH1 binding induces changes not only inthe polyproline region but also across the phosphatase core, α7helix, and C-terminal tail. Despite these structural perturbations,enzymatic assays show that EVH1 binding does not affect the catalyticactivity of PTP1B, supporting a model in which Mena functions as ascaffold to spatially organize PTP1B within EGFR signaling complexes.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 16","pages":"2470–2484"},"PeriodicalIF":2.7,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769594","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":"The Effects of Phosphorylation on the Structure and Function of Motif A, an Intrinsically Disordered Region within SIRT1","authors":"Sabrina M. Richter,&nbsp;Hoang-Long Bui,&nbsp;Addison Chen,&nbsp;Chloe Tannous,&nbsp;Benjamin R. Butler,&nbsp;Sophia D. Bennett,&nbsp;Samson Quang-Anh Nguyen,&nbsp;Justin Prado,&nbsp;Ryan Nhan,&nbsp;Ayan Mohamed,&nbsp;Isaac A. DuBois,&nbsp;Emile Tadros,&nbsp;Nhu Tam Thai,&nbsp;Selina Lima Guan,&nbsp;Carla Marie Peralta,&nbsp;Andy Kwong,&nbsp;Laura M.L. Hawk,&nbsp;Gianmarc Grazioli,&nbsp;Ningkun Wang","doi":"10.1021/acs.biochem.6c00333","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00333","url":null,"abstract":"<p>The NAD⁺-dependent deacetylase sirtuin-1 (SIRT1)isknown to elicit cellular defenses against aging, cancer, and otheraberrant pathologies. Previous studies have identified an intrinsicallydisordered region of SIRT1 comprised of N-terminal residues 1–52,herein referred to as motif A, which activates SIRT1 activity, likelythrough intramolecular interactions. Additionally, phosphorylationof N-terminal residues Ser27 and Ser47 has been shown to be importantfor regulating SIRT1 activity and stability. The lack of <em>in vitro</em> characterization of these effects hampers our furtherunderstanding of the role of motif A in SIRT1 regulation. In thisstudy, we elucidate the role phosphorylation plays in motif A’sstructure as well as its regulatory effects on SIRT1 activity againstAc-p65. We find that a phosphomimetic mutation at Ser27 significantlyincreases the activation effect of motif A toward SIRT1. This resultis supported by molecular dynamics simulations of the phosphomimetics,which reveal stabilization of different transient structures for motifA depending on whether Ser27 and Ser47 have been modified. A key findingsuggested by this study is that phosphorylation of S27 appears toactivate SIRT1 by causing motif A, which is intrinsically disorderedin the WT, to become more rigid. This conclusion is based on boththe experimental findings and simulation results. While more experimentsare necessary to confirm the MD simulation results, these findingsstill bring us a step closer to understanding SIRT1 regulation, specificallythe role played by phosphorylation within the N-terminal disorderedregion.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 16","pages":"2485–2494"},"PeriodicalIF":2.7,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769845","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":"Suppression of Thermal Protein Aggregation by Acetylation of Lysine Side Chains","authors":"Yuen Ki Ng,&nbsp;Lars Konermann","doi":"10.1021/acs.biochem.6c00424","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00424","url":null,"abstract":"<p>The association of proteins into amorphous aggregatesis linkedto various detrimental phenomena, ranging from disease mechanismsto the degradation of biopharmaceuticals. The development of aggregationsuppression strategies is of considerable interest. Experiments onaggregation (and aggregation suppression) are commonly performed underaccelerated conditions, where proteins are exposed to elevated temperatures.Focusing on the thermal aggregation of myoglobin (Mb, a paradigmaticmodel protein), our group previously reported that intermolecularLys⁺/Glu^– and Lys⁺/Asp^– salt bridges are a key driver of aggregate formation.Additives such as free arginine and guanidinium salts suppress aggregationby “blocking” Glu^– and Asp^– side chains, thereby preventing them from participating in saltbridges (Ng &amp; Konermann (2024) JACS 146, 8394). However, the useof such additives, e.g., as excipients in biopharmaceutical formulations,can be problematic. The current work highlights a complementary approach.Using spectroscopic experiments and molecular dynamics simulations,we show that acetylation of Lys side chains is highly effective forsuppressing the thermal aggregation of Mb. Acetylation (R–NH₃⁺ → R–NH–CO–CH₃) eliminates the positive charge of Lys, thereby renderingthe corresponding side chains incapable of salt bridge formation.It is demonstrated that acetylation only has relatively minor effectson the native protein structure under physiological conditions (roomtemperature, pH 7). In summary, Lys acetylation is a simple yet effectiveaggregation suppression approach that may also be applicable to otherproteins, including biopharmaceuticals such as monoclonal antibodies.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 16","pages":"2510–2521"},"PeriodicalIF":2.7,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769676","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":"Fluorogenic RNA Aptamer-Based Activity Quantification of T7 RNA Polymerase","authors":"Khalil Mimouni,&nbsp;Weiqi Qiu,&nbsp;Huiqing Zhou","doi":"10.1021/acs.biochem.6c00336","DOIUrl":"https://doi.org/10.1021/acs.biochem.6c00336","url":null,"abstract":"<p>T7 RNA polymerase(T7 RNAP) is a central molecular biologytoolthat is broadly used for biomedical research and the RNA therapeuticindustry. T7 RNAP and its engineered variants provide valuable toolsin synthetic biology and gene expression regulation. T7 RNAP activityunit is traditionally quantified by a radioactivity-based polymerizationassay, which is often not accessible to many laboratories and raisessafety concerns because of exposure to radioactivity. Here, we developeda fluorescence-based <em>in vitro</em> transcription (IVT)assay that enables facile and quantitative determination of T7 RNAPactivity units, using the Broccoli RNA aptamer as a transcriptionalreporter and commercially available T7 RNAPs as standard references,which are readily accessible materials for most biochemical laboratories.This method will improve the consistency and reproducibility assessmentof T7 RNAP prepared from different batches or different laboratoriesand benefit research communities that rely on the robust performanceof RNA polymerases.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 16","pages":"2579–2586"},"PeriodicalIF":2.7,"publicationDate":"2026-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769853","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":"Substrate Specificity and Kinetic Mechanism of the NAD+-Dependent Deacylase from Mycobacterium tuberculosis, Rv1151c (Mt-Sirt)","authors":"Drake M. Mellott,&nbsp;Jiyun Zhu,&nbsp;Hudson Fulcher,&nbsp;Thomas D. Meek","doi":"10.1021/acs.biochem.6c00374","DOIUrl":"10.1021/acs.biochem.6c00374","url":null,"abstract":"<p>The pathogen <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) relies on extensive metabolic remodelingand post-translational regulation of its proteins to retain persistencein its host, yet these biochemical functions remain largely uncharacterized,including the actions of lysine deacylases. Here, we characterizedRv1151c, the sole annotated NAD⁺-dependent deacylase in <em>Mtb</em>, which we designate <em>Mt</em>-Sirt. Usingsynthetic substrates, peptide libraries, and mutational analysis,we showed that <em>Mt</em>-Sirt exhibits strong selectivityfor peptide substrates containing N^ε-succinyl-lysineand long-chain fatty-acyl-lysine groups. Substitution of conservedactive-site residues (Y53F and R56M) confirmed their roles in substraterecognition and acyl-group discrimination. Inhibition studies revealedthat nicotinamide, NADH, ADP-ribose, and mechanism-based thioamide/thioureaanalogs inhibited <em>Mt</em>-Sirt activity with low-micromolarto nanomolar potency. Initial-velocity, product-inhibition, and dead-endinhibition analyses confirmed that <em>Mt</em>-Sirt operatesby an equilibrium-ordered kinetic mechanism in which NAD⁺ binds first, followed by the N^ε-acylated peptidesubstrate. We further demonstrated that chemically succinylated <em>Mtb</em> isocitrate lyase 1 (ICL1), a key enzyme in <em>Mtb</em> metabolism, was a substrate of <em>Mt</em>-Sirt <em>in vitro</em>. Lastly, metabolic labeling with tetradec-13-ynoicacid (Alk-12) revealed widespread lysine and N-terminal fatty acylationin <em>Mtb</em> cells, suggesting that <em>Mt</em>-Sirt may regulate previously unrecognized fatty-acyl PTMs <em>in vivo</em>. Together, these findings provide a kinetic andsubstrate-specificity analysis of <em>Mt</em>-Sirt, and highlightits potential role in controlling reversible lysine acylation in <em>Mtb</em>.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 15","pages":"2400–2418"},"PeriodicalIF":2.7,"publicationDate":"2026-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148434318","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}