Biochemistry Biochemistry最新文献

The ubiquitous flavoenzymes typically function as oxidoreductasesthat comprise several distinct main types, including flavoproteinoxidases (FPOs), flavoprotein dehydrogenases (FPDs), and flavoproteinmonooxygenases (FPMOs). FPOs and FPDs catalyze two-electron oxidationreactions of organic substrates, typically dehydrogenations, therebyconverting oxidized flavin (Fl_ox) into its fully reducedstate (Fl_red). Prior to the next catalytic cycle, molecularoxygen (=dioxygen or O₂) or (protein-bound) cofactors facilitatethe required Fl_red reoxidation for FPOs and FPDs. Remarkably,members of these two flavoenzyme types can be homologous with highlysimilar amino acid compositions and overall structures, as minor proteinalterations, particularly in the vicinity of the flavin cofactor,can drastically affect O₂ reactivity. Finally, FPMOs incorporateone O₂-derived oxygen atom into their substrate. To thisend, required electrons for Fl_red formation and O₂ activation either come from NAD(P)H (external FPMOs) or, more rarely,the substrate itself (internal FPMOs). External FPMOs steer O₂ reactivity toward the formation of covalent flavin-oxygenadducts primarily at the C4a atom of the flavin’s isoalloxazinering or, in some cases, at the adjacent N5. In contrast, typical internalFPMOs forego the formation of covalent oxygen adducts entirely, althoughan exception in the form of a flavin-N5-oxide-forming enzyme has beenreported. Consequently, natural selection has led to three distinctO₂ reactivity patterns in flavoenzymes, which either suppress(FPDs), stimulate (FPOs), or steer (FPMOs) this challenging process.In this review, current knowledge on the relationship between flavoenzymesand O₂ is summarized, emphasizing strategies to insertoxygen into organic substrates and counteract uncoupling, while alsohighlighting open questions and future challenges.

Fluorinated succinate analogues were evaluated as mechanisticprobesof Mycobacterium tuberculosis isocitratelyase (MtICL). However, 2,2-difluorosuccinate (1; K_i = 6.1 mM) and 2,2,3-trifluorosuccinate(2; K_i = 23.5 μM) actas reversible noncompetitive inhibitors and meso-2,3-difluorosuccinate(3) displayed slow-onset reversible inhibition (K_i = 30 μM), the 2-fluorosuccinate enantiomers((R)-4 and (S)-4) produced time-dependent irreversible inactivation. Inactivationby 4 was observable under turnover conditions in thepresence of glyoxylate and succinate, consistent with a two-step kineticmechanism. The S enantiomer inactivated more efficientlythan (R)-4, consistent with stereoelectronicalignment required for elimination of HF following abstraction ofthe pro-S proton. ¹H NMR analysis detectedmaleate formation from (S)-4, and massspectrometry revealed a +132 Da adduct consistent with covalent modificationof Cys191. Notably, k_inact/K_I values for 4 exceeded that measured formaleate, indicating that covalent capture occurs from an enzyme-boundintermediate prior to product release. These results support a mechanismin which fluorine substitution redirects the enolate-generating half-reactionof MtICL toward elimination and covalent modification.(S)-2-Fluorosuccinate therefore represents a succinate-analoguemechanism-based inactivator that exploits a catalytic step distinctfrom previously described isocitrate-analogue inhibitors.

Parkinson's disease (PD) is characterized by the pathological aggregation of α-synuclein (α-syn) into β-sheet-rich fibrils, contributing to neuronal toxicity and oxidative stress. In this study, we investigated the inhibitory and disaggregating effects of Triprolidine (TC) on α-syn fibrillation through a combined experimental and computational approach. Biophysical assays, including ThT assay, DLS, and ANS assays, demonstrated that TC inhibits α-syn fibrillation in a concentration-dependent manner (IC50 ≈ 255 μM), disrupts preformed fibrils, and maintains the protein's native form. CD data further revealed that TC prevents the transition of α-syn to its toxic β-sheet-rich form and facilitates partial structural reversal during disaggregation. To elucidate the molecular mechanism of inhibition, we performed all-atom molecular dynamics (MD) simulations followed by Markov State Model (MSM) construction. The simulations revealed that TC binding remodels the conformational landscape of α-syn by stabilizing compact, disordered states and reducing the population of β-sheet-prone intermediates, particularly in the aggregation-prone NAC region. MSM analysis identified metastable states with diminished aggregation potential and reduced inter-residue contact probability, offering mechanistic insights into how TC interferes with early nucleation events. TC attenuates seeded fibrillation in a concentration-dependent manner too. Complementary cellular assays, including MTT and hemolytic assays, confirmed a significant reduction in α-syn-induced cytotoxicity upon TC treatment, with a decrease in ROS levels as confirmed by the DCFH-DA assay. Together, these findings demonstrate that TC modulates both the structural dynamics and functional toxicity of α-synuclein, and highlight its potential as a promising chemical modulator for further investigation in PD-related protein aggregation.

[FeFe]-hydrogenases are very activebiocatalysts for H₂ conversion. However, their active siteis vulnerable to irreversibledegradation initiated by O₂ binding at the catalytic ironion (Fe_d) of the active center. CbA5H, the [FeFe]-hydrogenasefrom Clostridium beijerinckii, exhibitsstability toward oxygen (O₂) due to its ability to reversiblyenter an inactive state termed H_inact upon contact withO₂. We previously proposed that the close distance of approximately3.1 Å between the thiol of a nearby cysteine (C367) and Fe_d, based on a 2.9 Å crystal structure of CbA5H in theH_inact state, enables their binding to each other. Thisbinding therefore was suggested to shield Fe_d from O₂ damage. However, there is currently a lack of evidence tosupport this hypothesis. Furthermore, density functional theory (DFT)calculations based on a homologous model favored hydroxide as thebinding ligand of Fe_d over the thiol of C367. In this study,we present the crystal structure of CbA5H in the H_inact state at an improved resolution of 2.15 Å. The structure revealsa direct binding between the thiol of C367 and Fe_d witha distance of approximately 2.77 Å, which is well supported byour DFT calculations based on the new crystallographic data. It isnoteworthy that the 2.77 Å bond distance is strikingly long whencompared with other iron–sulfur bonds. This finding may providea crucial foundation for understanding the rapid reversibility ofthe H_inact state.

Tamavidin 2 is a biotin-binding proteinof the avidin-like family,discovered in Tamogitake mushrooms, that has found a number of usesin biotechnology. Unlike avidin or streptavidin, it can be expressedin Escherichia coli as a soluble protein,making it a good partner in chimaeric proteins. Surprisingly, we foundthat the full-length protein purified from E. coli has a much lower affinity for biotin than that of avidin or streptavidin.In fact, a micromolar K_d was determinedfrom fluorescence measurements, in contrast to the femtomolar K_d of avidin. Interestingly, treatments withtrypsin or denaturing agents resulted in a large increase in the affinity.Modeling of the structure suggested that the C-terminal five aminoacids obscured the biotin-binding site in the complete protein. Indeed,alterations of these five amino acids, either by truncation or individualmutation to alanine, resulted in a thousand-fold or greater increasein affinity for biotin. A truncated tamavidin was much more effectivein forming complexes with biotinylated target proteins and as a greenfluorescent protein (GFP) conjugate in binding assays. The inhibitedstate of the full-length protein and its activation by proteolysissuggest that tamavidin functions as a defensive protein to discouragepredation.

Auxiliary activity family 7 (AA7) oxidoreductases arefungal flavoenzymesthat catalyze the C1 oxidation of diverse oligosaccharides coupledto the reduction of molecular oxygen (oxidase activity) or organicmolecules (dehydrogenase activity). These enzymes are predominantlyderived from Ascomycota, with a smaller, less-explored fraction fromBasidiomycota and Oomycota plant pathogens. Moreover, AA7 membersare promising biocatalysts for the selective oxidation of carbohydrates,which is an enduring challenge in catalysis. However, the sequencespace of AA7 enzymes remains largely uncharted, particularly withinbasidiomycota members, and the molecular determinants shaping substrateselectivity remain ill-defined. Focusing on the basidiomycete maizepathogen Ustilago maydis, we exploredthe boundaries of the AA7 sequence space by identifying and characterizingtwo enzymes, UmAA7A and UmAA7B,that are divergent from hitherto described members. We found that UmAA7s oxidize both chitooligosaccharides (CHOS) and theirdeacetylated forms (dCHOS) in an acetylation-site-dependent manner.The X-ray crystal structure of UmAA7A, sequence,and structural comparisons with a model of UmAA7Band other known CHOS-active AA7s, combined with docking analyses onCHOS and dCHOS, revealed specific active-site residues behind thisunprecedented substrate specificity. Moreover, a previously not reportedcombination of a bicovalently tethered FAD and an atypical arrangementof residues at re-side of the FAD cofactor was associatedwith a mainly dehydrogenase activity profile, contrasting the majorityof oxidases in AA7. Altogether, this work reveals the enzymatic oxidationof dCHOS, extending the substrate scope of AA7s and laying the biochemicalfoundation for uncovering their biological functions during plantinfection.

Neutral sphingomyelinase2 (nSMase2) is a membrane-bound enzymethat hydrolyzes sphingomyelin (SM) into ceramide and phosphocholine.By generating the bioactive lipid ceramide, nSMase2 plays a criticalrole in cell stress responses and in regulating exosomes that packageand transfer pathogenic factors, including tau protein and amyloidβ. Thus, nSMase2 has been implicated in Alzheimer’s diseaseand other neurological disorders. However, current tools to measurenSMase2 activity are limited, in particular those that could be usedin therapeutic development. Here we developed a high-throughput assayto measure human nSMase2 activity. The assay uses a sphingomyelinsubstrate analogue with a FRET donor and acceptor pair attached tothe headgroup and acyl-chain, respectively. Using recombinant humannSMase2, we sensitively detected both wild-type and mutant activityand demonstrated inhibition by the known nSMase2 inhibitor GW4869.The assay captures the major hallmarks of nSMase2 regulation by anioniclipids and displays sensitivity capable of detecting nSMase2 activityfrom cell lysates. Together, this assay enables rapid screening ofnSMase2 inhibitors to support future therapeutic development.

Bilins, linear tetrapyrroles derived from heme catabolism,areubiquitous across biological systems. The constraints imposed by bindingto a protein confer valuable optical properties on bilins, which areused for sensing and harvesting light, and offer potential for fluorescenceimaging, optogenetics, and biosensing. Recently, bilins have beenused to evaluate methods for designing ligand binding sites, and novelbiliproteins (BPs) that bind phycoerythrobilin (PEB) emerged fromcomputational design using RoseTTAFold Diffusion All-Atom (RFDiffusionAA)and LigandMPNN. Here, we find much lower stability, extinction coefficientsand fluorescence quantum yields for de novo designs compared to thenative CpcA-PEB biliprotein, so we used a LigandMPNN-AlphaFold 3 (AF3)-RosettaRelax pipeline to redesign one BP, C11, which can be implemented quicklyand without in-house GPUs. The top three ligand confidence scoresemerging from this procedure yielded C11-578, C11-620, and C11-756redesigns that retain 50%, 52%, and 57% identities, respectively,with C11. AF3-Rosetta modeling of the BP redesigns predicted similarconformations of the PEB, decreased solvent-accessible surface areaof the bilin binding site, an additional predicted hydrogen bond inC11-620, and a more rigid protein backbone. The new constraints onbilin binding improved the stability, absorption properties and fluorescenceyields for C11-578, C11-620, and C11-756, and the brightness of theredesigns improved up to 13-fold, approaching the values for the nativeCpcA-PEB BP. Further design iterations can be used to create a rangeof absorbing and emitting BPs, including oligomeric assemblies housinginternal energy cascades.

Many bacterial chlorogenic acid esterases (ChlEs) exhibitatypicaltemperature behavior, featuring activities that barely change withtemperature and activity maxima that fall below the thermal denaturationpoint. This work focuses on a ChlE fromLactobacillus helveticus (Lh–ChlE), which has a flat temperaturedependence. First, it was determined that conformational changes duringLh–ChlE turnover are not rate-limiting and that the overallrate depends on the chemical step at all temperatures. Next, Lh–ChlE’stemperature dependence was investigated using a conformational equilibriummodel that assumes the existence of a temperature-dependent equilibriumbetween an active and an inactive conformation and an activation heatcapacity model that postulates a difference in heat capacity betweenthe ground and transition states. Although the equilibrium model recapitulatesthe data well, it yields an unrealistically low inactivation temperaturearound 280 K. Circular dichroism spectroscopy suggests that Lh–ChlEdoes not undergo structural changes at that temperature but may undergosmall structural transitions at moderately elevated temperature. Theactivation heat capacity model describes Lh–ChlE behavior well,yielding an activation heat capacity (ΔC_p^‡) of approximately −1 kJ mol^–1 K^–1. Overall, the results suggestthat the atypical temperature behavior of Lh–ChlE likely arisesfrom a negative activation heat capacity. This work illustrates thatcontrasting thermodynamic models for atypical temperature dependencein enzymes can give rise to similar looking fits, even though theyhave different underlying physical meaning. Our results furthermoreencourage additional analysis of the Lh–ChlE transition statestructure to better understand the structural features that causethe enzyme’s nonzero activation heat capacity.