Biochemistry Biochemistry最新文献

{"title":"Probing the Human 4-Oxo-l-proline Reductase-Catalyzed Reaction by Deuterium Kinetic and Equilibrium Isotope Effects","authors":"Ennio Pečaver,&nbsp;Myriam Kabu,&nbsp;Greice M. Zickuhr,&nbsp;David J. Harrison,&nbsp;Rafael G. da Silva","doi":"10.1021/acs.biochem.6c00491","DOIUrl":"10.1021/acs.biochem.6c00491","url":null,"abstract":"<p>Human 4-oxo-<span>l</span>-proline reductase (<em>Hs</em>BDH2),a short-chain dehydrogenase/reductase (SDR) superfamily member possessinga canonical N–S–Y–K catalytic tetrad, catalyzesthe formation of the endogenous anticancer compound <em>cis</em>-4-hydroxy-<span>l</span>-proline via the NADH-dependent reduction of4-oxo-<span>l</span>-proline. Except for hydride-transfer stereochemistry,information on the <em>Hs</em>BDH2 chemical step remains elusive.Here, deuterium isotope effects are employed to gather informationon the hydride-transfer step of the reaction. Primary and α-secondaryequilibrium isotope effects were inverse and normal, respectively,consistent with the 4<em>S</em>-[²H] accumulatingon <em>cis</em>-4-hydroxy-<span>l</span>-proline and the 4<em>R</em>-[²H] on NADH. Primary kinetic isotope effects,with 4<em>S</em>-[4-²H]NADH, on <em>k</em>_cat (^D<em>k</em>_cat) and <em>k</em>_cat/<em>K</em>_M (^D(<em>k</em>_cat/<em>K</em>_M^4OLP)) were small, indicating that hydride transfer is fastrelative to other catalytic steps. Internal-competition equilibriumbinding isotope effects were inverse with 4<em>S</em>-[4-²H]NADH and normal with 4<em>R</em>-[4-²H]NADH, suggesting that <em>Hs</em>BDH2 tight binding distortsthe pro-<em>S</em> hydrogen to a more constrained bondingenvironment, while the opposite happens to the pro-<em>R</em> hydrogen. Both levulinate and pyruvate were low-affinity, slow-reactionsubstrates; while a ^D(<em>k</em>_cat/<em>K</em>_M) of 2.6 and a ^D<em>k</em>_cat of 2.5 were determined with the former, modest valueswere obtained with the latter. Using NADPH as a coenzyme resultedin a similar <em>k</em>_cat, but drastically increased <em>K</em>_M. Accordingly, 4<em>S</em>-[4-²H]NADPH increased ^D(<em>k</em>_cat/<em>K</em>_M^NADPH) to 1.5, while ^D<em>k</em>_cat remained small. The N105Asubstitution, proposed to affect coenzyme binding in SDRs, increased <em>K</em>_M for NADH and doubled <em>k</em>_cat. The ^D(<em>k</em>_cat/<em>K</em>_M) increased to 2.5 and ^D<em>k</em>_cat to 2.0. The ^D₂O<em>k</em>_cat with 4<em>S</em>-[4-²H]NADH decreased from its value with NADH, suggesting stepwise hydride-transferand proton-transfer steps. These results expand the role of N105 in <em>Hs</em>BDH2 and possibly other SDRs.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 15","pages":"2419–2430"},"PeriodicalIF":2.7,"publicationDate":"2026-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148467746","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":"Fluorescence-Activated Cell Sorting Enables Rapid Screening of RNA Polymerase–Promoter Interaction in E. coli","authors":"Lilian Göldel,&nbsp;Ralph Krafczyk,&nbsp;Romina Rathberger,&nbsp;Thomas Brück,&nbsp;Hagen Richter","doi":"10.1021/acs.biochem.6c00326","DOIUrl":"10.1021/acs.biochem.6c00326","url":null,"abstract":"<p>RNA polymerases are essential in the enzymatic synthesisof RNAvia <em>in vitro</em> transcription. The resulting mRNA transcriptshave been investigated more and more as therapeutics in clinical studies.Therefore, it is important that these enzymes possess a high specificity.Despite their utility, many commercially available RNA polymerasesare limited by their tendency to generate abortive transcripts andundesired byproducts. To ensure high specificity, optimal pairingbetween the RNA polymerase and its cognate promoter is vital. However,current promoter characterization techniques remain laborious andtime-consuming, thereby limiting efficient and rapid RNA-polymeraseapplications. In this work, we describe a high-throughput strategyfor rapid promoter identification using a two-plasmid screening platformin <em>E. coli</em>. Cell populations are analyzed and sortedvia fluorescence-activated cell sorting (FACS), followed by sequenceverification. Functionality of the screening platform was validatedvia cell sorting, based on high fluorescence, of a mixed populationcontaining T7 RNA polymerase paired with three different promoters.Moreover, the screening system was evaluated by using two recentlyidentified RNA polymerases in combination with their respective cognatepromoters. This proof-of-concept facilitates the identification ofboth RNA polymerase and promoter. Further, it substantially acceleratespromoter characterization, thereby supporting improvements regardingmRNA manufacturing workflows.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 15","pages":"2343–2349"},"PeriodicalIF":2.7,"publicationDate":"2026-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148443432","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":"Kinetically Divergent Dimanganese–Tyrosyl Radical Cofactor Assembly in Listeria monocytogenes Class I Ribonucleotide Reductase","authors":"Paoning G. Chong,&nbsp;Saborni Biswas,&nbsp;Chihjung Chen,&nbsp;Joseph A. Cotruvo, Jr.","doi":"10.1021/acs.biochem.6c00369","DOIUrl":"10.1021/acs.biochem.6c00369","url":null,"abstract":"<p>Ribonucleotide reductases (RNRs) catalyze the reductionof nucleotidesto deoxynucleotides in all organisms. Class I RNRs comprise two subunits,α2 (containing the active site) and β2 (containing theradical cofactor essential for initiating catalysis). The associationof α2 and β2 is well known to regulate RNR activity, buta role for α2 in kinetically controlling cofactor assembly inβ2 has not been reported. The class I RNR from the importanthuman intracellular pathogen, <em>Listeria monocytogenes</em> (<em>Lmo</em>) is a member of the largest cluster of βsubunit sequences (the “2RCC cluster”) remaining tobe biochemically characterized. This protein was previously suggestedto be a class Ia enzyme, active with a diferric-tyrosyl radical (Y•)cofactor. Here, we demonstrate that the <em>Lmo</em> classI RNR, in fact, is most active with a dimanganese-Y• cofactor,assembled with the help of the flavoprotein, NrdI. While superficiallysimilar to previously characterized manganese-dependent class Ib RNRs,the <em>Lmo</em> RNR features a number of unusual properties.Most strikingly, the Mn^IIIMn^IV intermediateformed from reaction of the dimanganese(II) cluster, NrdI, and O₂ converts extremely slowly (<em>t</em>_1/2 = 11 min) to an active dimanganese(III)-Y• cofactor. However,in the presence of α2, cofactor assembly is accelerated by &gt;10-fold(<em>t</em>_1/2 = 0.6 min). Therefore, α2-drivenconformational changes in <em>Lmo</em> β2 are likelyrequired physiologically for not only catalysis but also activation.The properties of <em>Lmo</em>’s distinctive classIb RNR expand the known mechanisms of RNR activity regulation andmay have implications for the observed sensitivity of <em>L. monocytogenes</em> to iron excess during infection.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 15","pages":"2387–2399"},"PeriodicalIF":2.7,"publicationDate":"2026-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148434353","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":"Hyperfusogenic Mutations Destabilize the Postfusion Six-Helix Bundle of the Measles Virus Fusion Glycoprotein","authors":"Nisansala Vithanage,&nbsp;Victor K. Outlaw","doi":"10.1021/acs.biochem.6c00182","DOIUrl":"10.1021/acs.biochem.6c00182","url":null,"abstract":"<p>Fusion of the host membrane and viral envelope by classI viralfusion proteins is driven by the assembly of a postfusion six-helixbundle formed through antiparallel interactions between N-terminal(HR1) and C-terminal (HR2) heptad-repeat regions. Although mutationsin these regions of the measles virus (MeV) fusion (F) glycoproteinare known to promote neuropathogenic and hyperfusogenic phenotypes,their effects on postfusion core stability have not been systematicallyexamined. Here, we combine peptide biophysics and X-ray crystallographyto interrogate how mutations within the HR2 domain, present in nativeneuropathogenic MeV isolates (<em>e.g</em>., L454W and N462K)and laboratory-generated hyperfusogenic variants (<em>e.g</em>., L454M and T461A), influence postfusion 6HB assembly. Circulardichroism (CD) spectroscopy reveals that, with few exceptions, thesemutations decrease postfusion core stability, despite their associationwith enhanced fusion activity. We also report the first crystal structureof the wild-type MeV postfusion core as well as structures of sixhyperfusogenic variants, enabling high-resolution comparison of themolecular basis of destabilization. Structural analysis shows thatthese effects arise from localized perturbations to steric packing,hydrogen bonding networks, and helix-stabilizing interactions withinHR2, while the overall 6HB architecture remains conserved. Together,these results indicate that hyperfusogenic mutations are not associatedwith stabilization of the postfusion state and are instead consistentwith models in which hyperfusogenicity arises from a reduction inthe energetic barrier to fusion, potentially through effects on prefusionstability or triggering efficiency. These findings establish key sequence-structure–stabilityrelationships governing coiled-coil assembly and provide a frameworkfor the design of HR1- and HR2-based fusion inhibitors.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 15","pages":"2350–2358"},"PeriodicalIF":2.7,"publicationDate":"2026-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148560214","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":"Combinatorial Residue Recognition in SsTx-4 Inhibition of the Kir4.1/5.1 Channel","authors":"Dongfang Tang,&nbsp;Peng Feng,&nbsp;Yanming Liu,&nbsp;Zixu Lu,&nbsp;Xin Liu,&nbsp;Xiaoxia Liu,&nbsp;Linyan Cao,&nbsp;Caiqin Yang,&nbsp;Yuhong Guo,&nbsp;Wenzi Tang,&nbsp;Yujun Wang,&nbsp;Changjian Li,&nbsp;Zuodong Qin","doi":"10.1021/acs.biochem.6c00346","DOIUrl":"10.1021/acs.biochem.6c00346","url":null,"abstract":"<p>TheKir4.1/5.1 channel is predominantly expressed in renal tubularepithelial cells and glial cells of the nervous system, where it playsessential physiological roles. In our previous work, the centipedevenom peptide SsTx-4 was shown to strongly inhibit the homomeric Kir4.1channel; however, its activity toward the heteromeric Kir4.1/5.1 channelremained unclear. Here, we show that SsTx-4 inhibits the Kir4.1/5.1channel with a potency comparable to that of Kir4.1. Electrophysiologicalrecordings combined with site-directed mutagenesis, structural modeling,and molecular dynamics simulations demonstrate that SsTx-4 blocksthe Kir4.1/5.1 channel via a conserved pore-occlusion mechanism, whileutilizing a distinct, subtype-dependent combination of peptide residuesfor channel binding. Structural analyses indicate that K13 insertsinto the pore and interacts with the selectivity filter, whereas K10forms a hydrogen bond with Y135 of the Kir4.1 subunit and F44 formsa hydrophobic contact with Y136 of the Kir5.1 subunit. Additionalresidues, including K4, R12, F14, Y16, K40, and K45, further stabilizethe toxin–channel complex through hydrogen bonds and hydrophobicinteractions. Notably, the basic residues K4, K40, and K45 may cooperativelycontribute to binding to the Kir4.1/5.1 channel. Together, these findingssupport a combinatorial residue recognition mechanism underlying SsTx-4inhibition of Kir channels and provides mechanistic insights intounderstanding peptide–Kir channel interactions.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 15","pages":"2376–2386"},"PeriodicalIF":2.7,"publicationDate":"2026-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148519534","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":"Biochemical Characterization of ABHD14A, an Outlying Member of the Metabolic Serine Hydrolase Family","authors":"Sonali Gupta,&nbsp;Mahamaya Dhaware,&nbsp;Siddhesh S. Kamat","doi":"10.1021/acs.biochem.6c00515","DOIUrl":"10.1021/acs.biochem.6c00515","url":null,"abstract":"<p>Certain uncharacterized members of the metabolic serinehydrolaseenzyme family remain difficult to annotate due to poor tractability,context-dependent expression, and the absence of defined biochemicalactivities. Here, we provide the first functional characterizationof the human enzyme ABHD14A. By engineering a soluble N-terminallytruncated variant, we demonstrate by gel-based activity-based proteinprofiling and <em>p</em>-nitrophenyl-ester hydrolysis assaysthat ABHD14A is an active enzyme that can turn over short-chain esters.Notably, ABHD14A exhibits a coenzyme A-dependent enhancement of <em>p</em>-nitrophenyl acetate hydrolysis, indicative of a ping-pongtype acetyltransferase mechanism similar to that previously describedfor another homologous ABHD14 enzyme, ABHD14B. To investigate thesubcellular localization of ABHD14A, heterologous overexpression ofan ABHD14A-GFP fusion variant in HEK293T cells was performed. Here,we found that the ABHD14A-GFP fusion protein is catalytically activeand localizes specifically to the Golgi apparatus, suggesting a specializedrole in the secretory pathway biology. Together, these findings establishthe enzymatic activity, mechanistic features, and subcellular localizationof ABHD14A while providing essential biochemical tools that can nowenable the systematic discovery of its physiological substrates andregulatory contexts.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 15","pages":"2431–2440"},"PeriodicalIF":2.7,"publicationDate":"2026-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148467809","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":"Structural and Biochemical Insights into the Arabinose and Xylose Derivatives of Cyclic-GAMP-Mediated Inhibition of Mycobacterium tuberculosis Cyclic-di-AMP Phosphodiesterase","authors":"Dagur Singh Hanuman,&nbsp;Singh Neeharika,&nbsp;Kulhar Nitin,&nbsp;Suman Abhishek,&nbsp;Krishna Murari Sinha,&nbsp;Eerappa Rajakumara","doi":"10.1021/acs.biochem.6c00106","DOIUrl":"10.1021/acs.biochem.6c00106","url":null,"abstract":"<p>Bacterialinfection, including that of<em>Mycobacterium tuberculosis</em>, leads to the accumulation of the bacterialcyclic dinucleotides (CDN), c-di-AMP and c-di-GMP, and the host cGAMPsynthase-catalyzed CDN, 2′3′-cGAMP in the cytosol, whichactivates STING-dependent type I interferon (IFN) and NF-κBimmune responses. The mycobacterial cyclic dinucleotide phosphodiesterase(CdnP) secreted into the host macrophages blunts host immunity bydirectly cleaving bacterial- and host-derived CDNs. The arabinose-and xylose-modified 2′3′-cGAMP (2′3′-(A/X)cGAMP)analogues act as potent STING agonists and resist hydrolysis by thehost-PDE ENPP1. Here, we report that 2′3′-(A/X)-cGAMPanalogues bind to CdnP and compete with its substrate binding. Furtherstudies revealed that these analogues inhibit the catalytic activityof CdnP. The cocrystal structure demonstrates that the arabinose-derived2′3′-cGAMP (AR-cGAMP) analogue is trapped in an unusualU-shaped conformation in the substrate-binding pocket, away from thecatalytic residue and Mn²⁺, which suggests that CdnP isincompetent to hydrolyze the analogue and cannot accept the otherCDN substrate for hydrolysis. Given that several bacterial and viralpathogens deploy CDN phosphodiesterase enzymes to hydrolyze both hostand pathogen-derived STING agonists, sugar-modified CDNs can be usedto weaken bacterial and viral defenses and stimulate the STING-mediatedhost immunity against these pathogens.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 15","pages":"2441–2451"},"PeriodicalIF":2.7,"publicationDate":"2026-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148040089","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":"Conformational and Functional Divergence of RnpA Protein Subfamilies in Bacterial Ribonucleoprotein RNase P","authors":"Loc Huynh,&nbsp;Reza Esmaeeli,&nbsp;Tingyi Zhu,&nbsp;Swapnil Joshi,&nbsp;Krushna Mishra,&nbsp;Robin Fournier,&nbsp;Alberto Perez,&nbsp;Michael E. Harris","doi":"10.1021/acs.biochem.6c00277","DOIUrl":"10.1021/acs.biochem.6c00277","url":null,"abstract":"<p>Bacterial ribonuclease P (RNase P) is an essential ribonucleoproteinenzyme that catalyzes 5′ leader removal from precursor tRNAs(ptRNAs) using a catalytic RNA subunit (P RNA) and an essential proteincofactor (RnpA). RnpA binds near the P RNA active site, facilitatingcatalysis and enhancing ptRNA binding by contacting 5′ leadersequences. However, key information regarding how sequence variation,particularly among bacterial pathogens, influences folding, dynamics,P RNA activation, and catalytic function is lacking. Using sequencesimilarity network (SSN) analyses of &gt;1800 RnpA sequences, we identifytwo major subfamilies, a Bacilli-specific class (RnpA-1) that associateswith divergent Type B P RNAs, and a broader class (RnpA-2) that associateswith ancestral Type A P RNAs, with species-specific variation concentratedat the N- and C-termini. Computational and biophysical studies showthat RnpA-1 proteins, including those from <em>Staphylococcus aureus</em> and <em>Enterococcus faecium</em>, exhibit greater conformational dynamics and reduced thermal stabilityrelative to RnpA-2 proteins from representative Gram-negative pathogens.Despite these differences, both families comparably enhance bindingof ptRNA to their cognate Type A or B P RNA. In contrast, kineticstudies reveal higher <em>k</em>_cat values forType B RNase Ps and rate limiting product release, while Type A enzymesare limited by precatalytic steps. Reconstitution with noncognatesubunits produces selective defects in either <em>k</em>_cat or <em>K</em>_M demonstrating that RnpAidentity makes distinct contributions to substrate binding and catalyticactivation. These results further define the conserved and divergentstructural, dynamic and functional features of RnpA proteins and establisha foundation for understanding biological function and inhibitor targeting.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 15","pages":"2359–2375"},"PeriodicalIF":2.7,"publicationDate":"2026-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148560132","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":"Lactate Biology: Subcellular Routing and Chemical Form Define Function","authors":"Nicholas A. Offei,&nbsp; and ,&nbsp;Ahmad A. Cluntun*,&nbsp;","doi":"10.1021/acs.biochem.6c00251","DOIUrl":"10.1021/acs.biochem.6c00251","url":null,"abstract":"<p >Lactate has undergone a major conceptual shift, from a glycolytic waste product to a circulating metabolic currency and, more recently, a multifunctional regulator of physiology. It serves as a mitochondrial fuel, an epigenetic modifier via protein lactylation, a ligand for the G-protein-coupled receptor HCAR1, and a precursor for endocrine-like N-lactoyl amino acids. This convergence raises a central question: how can a single metabolite support such distinct roles without functional conflict? We propose that the resolution lies not in lactate concentration alone but in two complementary organizing principles: subcellular routing and chemical form. Transporter localization, enzyme compartmentalization, donor formation, and metabolic competition bias lactate toward distinct biochemical fates, while conversion into chemically distinct intermediates─including lactyl-CoA, lactoyl-glutathione, <span>d</span>-lactate, and N-lactoyl amino acids─further constrains the biological outcomes that lactate can support. Lactate’s fate is influenced by the cellular compartments it accesses, a process constrained by specific monocarboxylate transporters at the plasma and mitochondrial membranes, isoform-specific localization of lactate dehydrogenases, and compartmentalized enzymatic machinery that converts lactate into distinct biochemical donors. Mitochondrial oxidation, protein lactylation, extracellular signaling, and N-lactoyl amino acid synthesis should therefore not be viewed as parallel consequences of elevated lactate concentration. Instead, they represent interconnected metabolic fates that draw from shared lactate pools and are influenced by compartmental access and local enzymatic context. Here, we integrate evidence from metabolism, epigenetics, and signaling into a spatial framework in which lactate function depends on where it is routed. In this view, lactate is not a promiscuous metabolite but a compartmentalized intermediate whose biological effects are shaped by spatial context. We further distinguish between established, emerging, and speculative aspects of this compartmentalized view to highlight key gaps and prioritize future experimental testing.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2191–2201"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.biochem.6c00251","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148343511","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":"Nature’s Anaerobic Toolkit: Glycyl Radical Enzymes and Their Expanding Functional and Mechanistic Diversity","authors":"Christa N. Imrich,&nbsp;Ankush Chakraborty,&nbsp;Lindsey R. F. Backman,&nbsp;Mary C. Andorfer* and Catherine L. Drennan*,&nbsp;","doi":"10.1021/acs.biochem.6c00318","DOIUrl":"10.1021/acs.biochem.6c00318","url":null,"abstract":"<p >Nature has devised an array of enzymatic cofactors that enable challenging chemical transformations to occur on a time scale compatible with biological life. The glycyl radical enzyme (GRE) superfamily, for example, highlights the catalytic power of protein-based amino acid radicals. GREs utilize a persistent radical housed on the α-carbon of a C-terminally conserved glycine residue to accomplish diverse chemical reactions in anaerobic environments. This radical cofactor is post-translationally installed on the mature, folded protein by a radical <i>S</i>-adenosyl-L-methionine (RS) activating enzyme (GRE-AE; activase) specific to each GRE. The catalytic repertoire of GREs is diverse, spanning ribonucleotide reduction for DNA synthesis, acetyl-CoA formation in anaerobic primary metabolism, dehydration reactions in secondary metabolism, decarboxylation reactions in aromatic amino acid fermentation, and hydrocarbon activation in anaerobic bacteria, enabling their survival on crude oil as a carbon source. Pyruvate formate lyase (PFL), the founding member of the GRE superfamily, was first identified several decades ago. In recent years, sequencing technology has enabled the annotation of thousands of unique GRE-encoding sequences. Several dozen of the identified GREs have been characterized further, providing insight into the structure, mechanism, and regulation of this multitudinous and diverse enzyme family. Here, we introduce the superfamily, summarize what is known about their activation and catalysis, note conserved structural features, expand on the previously identified GRE classes, highlight GRE-associated bacterial microcompartments, and discuss the future and outlook of the GRE superfamily.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2227–2249"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.biochem.6c00318","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148336962","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}