Biochemistry Biochemistry最新文献

Enzymes of the enolase superfamily (ENS) are mechanistically diverse, yet share a common partial reaction, i.e., the metal-assisted, Bro̷nsted base-catalyzed abstraction of the α-proton from a carboxylate substrate to form an enol(ate) intermediate. Although the catalytic machinery responsible for the initial deprotonation reaction has been conserved, divergent evolution has led to numerous ENS members that catalyze different overall reactions. Using differential scanning calorimetry, we examined the contribution of the Bro̷nsted acid–base catalysts to the thermostability (T_m) of four members of the mandelate racemase (MR)-subgroup of the ENS: MR, d-tartrate dehydratase, l-talarate/galactarate dehydratase, and l-fuconate dehydratase. Each enzyme contains an active-site Lys (part of a KxK motif) and His, which act as Bro̷nsted acid–base catalysts. The KxK → KxM substitutions increased the thermostability in all four enzymes with the effect being most prominent for MR (ΔT_m = +8.6 °C). The KxK → MxK substitutions decreased the thermostability in all four enzymes, and the His → Asn substitution had a significant stabilizing effect only on MR. Thus, the active sites of MR-subgroup enzymes are destabilized by the Lys Bro̷nsted acid–base catalyst, suggesting that the destabilization energy may be used to drive a conformational change of the enzyme to yield a catalytically competent protonation state upon substrate binding.

The mitochondrial outer membrane (OMM) β-barrel proteins link the mitochondrion with the cytosol, endoplasmic reticulum, and other cellular membranes, establishing cellular homeostasis. Their active insertion and assembly in the outer mitochondrial membrane is achieved in an energy-independent yet highly effective manner by the Sorting and Assembly Machinery (SAM) of the OMM. The core SAM constituent is the 16-stranded transmembrane β-barrel Sam50. For over two decades, the primary role of Sam50 has been linked to its function as a chaperone in the OMM, wherein it assembles all β-barrels through a lateral gating and β-barrel switching mechanism. Interestingly, recent studies have demonstrated that despite its low copy number, Sam50 performs various diverse functions beyond assembling β-barrels. This includes maintaining cristae morphology, bidirectional lipid shuttling between the ER and mitochondrial inner membrane, import of select proteins, regulation of PINK1-Parkin function, and timed trigger of cell death. Given these multifaceted critical regulatory functions of SAM across all eukaryotes, we now reason that SAM merely moonlights as the hub for β-barrel biogenesis and has indeed evolved a diverse array of primary roles in maintaining mitochondrial function and cellular homeostasis.

DtpC was isolated from the ditryptophenaline biosynthetic pathway found in filamentous fungi as a cytochrome P450 (P450) that catalyzes the dimerization of diketopiperazines. More recently, several similar P450s were discovered. While a vast majority of such P450s generate asymmetric diketopiperazine dimers, DtpC and other fungal P450s predominantly catalyze the formation of symmetric dimer products. Dimeric compounds can have interesting biological activities, and the mode of dimerization can substantially affect their bioactivities substantially. Here, we set out to examine the mechanism and scope of diketopiperazine dimerization catalyzed by DtpC using both chemically modified substrate molecules and DtpC mutants that were selected by the screening of randomly mutated recombinant variants. Use of N1- and N10-methylated diketopiperazine substrates supports the proposal that the initial radical formation occurs by extraction of the N1 indole nitrogen for this fungal P450 dimerase. Further in vitro studies revealed that DtpC was capable of accepting a range of structurally variable substrates, including N-demethylated diketopiperazines, and forming symmetric homo- and heterodimeric products. Moreover, the introduction of single mutations identified through the screening of random mutants at and around the substrate-binding pocket led to the conversion of DtpC into a catalyst that predominantly generated asymmetric dimers of various diketopiperazines. The versatility of DtpC can serve as a good starting point for directed evolution of P450s that can serve as versatile catalysts for generation of various dimers of not only diketopiperazines derived from standard and nonstandard amino acids but also possibly structurally more divergent analogs of diketopiperazines.

The development of RNA aptamers with high specificity and affinity for target molecules is a critical advancement in the field of therapeutic and diagnostic applications. This study presents the selection of a 2'-fluoro-modified mirror-image RNA aptamer through the in vitro SELEX process. Using a random RNA library, we performed iterative rounds of selection and amplification to enrich aptamers that bind specifically to the viral attenuator hairpin RNA containing the opposite chirality, which is an important part of the frameshift element. The unnatural chirality of the aptamer improved its enzymatic stability, and the incorporation of 2'-fluoro modifications was crucial in enhancing the binding affinity of the aptamers. After nine rounds of SELEX, the enriched RNA pool was sequenced and analyzed, revealing the dominant aptamer sequences. The selected 2'-fluoro-modified mirror-image RNA aptamer demonstrated a dissociation constant of approximately 1.6 μM, indicating moderate binding affinity with the target and exceptional stability against nuclease degradation. Our findings highlight the potential of 2'-fluoro-modified mirror-image RNA aptamers in enhancing the stability and utility of RNA-based therapeutics and diagnostics, paving the way for future applications in diverse biomedical fields.

Mononuclear Fe enzymes such as heme-containing cytochrome P450 enzymes catalyze a variety of C–H activation reactions under ambient conditions, and they represent an attractive platform for engineering reactivity through changes to the native enzyme. Using density functional theory, we study both native Fe and non-native group 8 (Ru, Os) and group 9 (Ir) metal centers in an active site model of P450. We quantify how changing the metal changes spin state preferences throughout the catalytic cycle. Our calculations reveal an intermediate-spin ground state for all Fe intermediates while the heavier metals prefer low-spin ground states across most intermediates in the reaction cycle. We also study the rate-determining hydrogen atom transfer (HAT) step and the subsequent rebound step. We observe comparable HAT barriers for Fe and Ru, a much higher barrier for Os, and the lowest HAT barrier for Ir. Rebound steps are barrierless for all metals, and the rebound intermediate for Fe is most significantly stabilized. Examination of ground spin states of all intermediates in the reaction cycle reveals spin-allowed pathways for the group 8 metals and spin-forbidden energetics for the group 9 Ir with potential two-state reactivity. Our work highlights the differences between the group 8 metals and the group 9 Ir, and it suggests that engineered P450 enzymes with Ru in particular result in improved enzyme reactivity toward C–H hydroxylation.

SARS-CoV-2 infections led to a worldwide pandemic in 2020. As of 2024, therapeutics against SARS-CoV-2 have continued to be desirable. NSP14 is a dual-function methyltransferase (MTase) and exonuclease (ExoN) with key roles in SARS-CoV-2 genome propagation and host immune system evasion. In this work, we developed high-throughput screening (HTS) assays for NSP14 MTase and ExoN activities. We screened both activities against a collection of 40,664 compounds. A total of 1677 initial hit compounds were identified, cherrypicked, counterscreened for assay interference, and screened for off-target selectivity. We identified 396 and 174 high-quality hits against the MTase and ExoN activities, respectively. Along with inhibitors for individual activities, we identified dual-activity inhibitors, including a novel inhibitor that is not competitive with any substrate and interacts with a putative allosteric binding site. This study represents the largest published screen of SARS-CoV-2 NSP14 MTase and ExoN activities to date and culminates in a pipeline for the NSP14 drug discovery.

Dimethyladenosine transferase 1 (DIMT1) is an RNA N^6,6-dimethyladenosine (m₂^6,6A) methyltransferase. DIMT1's role in pre-rRNA processing and ribosome biogenesis is critical for cell proliferation. Here, we investigated the minimal number of residues in a positively charged cleft on DIMT1 required for cell proliferation. We demonstrate that a minimum of four residues in the positively charged cleft must be mutated to alter DIMT1's RNA-binding ability. The variant (4mutA-DIMT1), which presents reduced RNA binding affinity, is diffuse in the nucleoplasm and nucleolus, in contrast with the primarily nucleolar localization of wild-type DIMT1. The aberrant cellular localization significantly impaired 4mutA-DIMT1's role in supporting cell proliferation, as shown in competition-based cell proliferation assays. These results identify the minimum region in DIMT1 to target for cell proliferation regulation.

Post-transcriptional modifications at the anticodon stem-loop of tRNAs are key to the translation function. Metabolic pathways to these modifications often incorporate complex enzymology. A notable example is the hypermodified nucleoside, queuosine, found at the wobble position of Asn, Asp, His, and Tyr encoding tRNAs. The epoxyqueuosine reductase, QueH, catalyzes the final step in the biosynthetic pathway to queuosine. The metalloenzyme catalyzes a two-electron reduction of epoxyqueuosine to provide the modified tRNA. The structure of QueH from T. maritima has previously been determined and unexpectedly contains two metal binding motifs in the active site. This includes a predicted 4Fe-4S cluster, along with a single-metal binding site coordinated by two cysteines along an aspartate carboxylate. In this report, we describe the structural and biochemical analysis of the QueH metal binding sites along with the chemistry of epoxide deoxygenation. To probe the active-site architecture, enzyme mutants of metal binding residues were structurally and biochemically characterized. In addition, structural and binding experiments were used to probe interactions of QueH with tRNA and the in vivo role of QueH and variants in Q-tRNA synthesis was evaluated. Overall, this work provides insight into the chemical mechanism of the final step of the queuosine biosynthetic pathway.

Mycobacterium tuberculosis (Mtb) is a leading cause of death, with an escalating global occurrence of drug-resistant infections that are partially attributed to cell wall mycolic acids derived from type II fatty acid biosynthesis (FAS-II). Here, the central acyl carrier protein, AcpM, contributes to the regulation of complex and specific protein-protein interactions (PPIs), though the orchestration of these events remain largely unresolved due to unique features of AcpM. Limitations include complexities in generating modified AcpM in a single state. Herein, we report a streamlined method to generate homogeneous samples of modified AcpM for applications in structure and functional studies. We apply these to generate solvatochromic labeled crypto-AcpM, where fluorescence response reports cargo sequestration and chain flipping upon interaction with four FAS-II enzymes. We find an increased fluorescence in a truncated form, AcpM80, indicating that the 35-residue C-terminus is involved in modulating the chemical environment surrounding the substrate and contributing to the regulation of PPIs. This study establishes an efficient chemo-enzymatic strategy to generate AcpM analogs for biophysical studies to aid in understanding the processes driving Mtb pathogenicity and drug resistance.

The apoptosome, a critical protein complex in apoptosis regulation, relies on intricate interactions between its components, particularly the proteins containing the Caspase Activation and Recruitment Domain (CARD). This work presents a thorough computational analysis of the stability and specificity of CARD-CARD interactions within the apoptosome. Departing from available crystal structures, we identify important residues for the interaction between the CARD domains of Apaf-1 and Caspase-9. Our results underscore the essential role of these residues in apoptosome activity, offering prospects for targeted intervention strategies. Available experimental complex structures were able to validate the protein-protein docking consensus approach used herein. We furthermore extended our analysis to explore the specificity of CARD-CARD interactions by cross-docking experiments between apoptosome and PIDDosome components, between which there should not be any interaction despite belonging to the same death fold subfamily. Our findings indicate that native interactions within individual complexes exhibit greater stability than the cross-docked complexes, emphasizing the specificity required for effective protein complex formation. This study enhances our understanding of apoptotic regulation and demonstrates the utility of computational approaches in elucidating intricate protein-protein interactions.