Biochemistry Biochemistry最新文献

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.

Bacteroides are often the most abundant, commensal species in the gut microbiome of industrialized human populations. One of the most commonly detected species is Bacteroides ovatus. It has been linked to benefits like the suppression of intestinal inflammation but is also correlated with some autoimmune disorders, for example irritable bowel disorder (IBD). Bacterial cell surface carbohydrates, like capsular polysaccharides (CPS), may play a role in modulating these varied host interactions. Recent studies have begun to explore the diversity of CPS loci in Bacteroides; however, there is still much unknown. Here, we present structural and functional characterization of a putative polysaccharide deacetylase from Bacteroides ovatus (BoPDA) encoded in a CPS biosynthetic locus. We solved four high resolution crystal structures (1.36-1.56 Å) of the enzyme bound to divalent cations Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺ and performed carbohydrate binding and deacetylase activity assays. Structural analysis of BoPDA revealed an atypical domain architecture that is unique to this enzyme, with a carbohydrate esterase 4 (CE4) superfamily catalytic domain inserted into a carbohydrate binding module (CBM). Additionally, BoPDA lacks the canonical CE4 His-His-Asp metal binding motif and our structures show it utilizes a noncanonical His-Asp dyad to bind metal ions. BoPDA is the first protein involved in CPS biosynthesis from B. ovatus to be characterized, furthering our understanding of significant biosynthetic processes in this medically relevant gut microbe.

Plants make pyrimidine base substitutions in organellar mRNAs through the action of sequence-specific nuclear-encoded enzymes. Pentatricopeptide repeat (PPR) proteins are essential for ensuring specificity, while the enzymatic DYW domain is often present at the C-terminus of a PPR protein and dependent on the variant possessing C-to-U and/or U-to-C RNA editing activities. Expression of exogenous DYW-KP variant enzymes in bacteria leads to the modification of RNAs suggestive of U-to-C base changes. The modified RNAs could only be purified from the interphase of an acidic guanidinium thiocyanate-phenol-chloroform experiment. It was projected that in bacteria stable RNA-enzyme cross-links form from a lysyl attack. In this study, RNA editing was examined for dual U-to-C/C-to-U editing enzyme KP6 with conserved lysine residues substituted by alanine. A single lysine was found to be essential for U-to-C editing and, based on the crystal structures of DYW domains, would likely be present in the active site. Crystal structures also suggest that the lysine can potentially form an ion pair with the catalytic glutamate critical for C-to-U RNA editing. Mutation of lysine to alanine greatly stimulated the C-to-U RNA editing by KP6. A ∼319 Da adduct observed on DYW-KP proteins could not be detected on the U-to-C-deficient lysine to alanine point mutant enzymes. This work establishes the critical role for a single lysine in the DYW-KP domain specifically for U-to-C editing activity but also highlights a secondary role for the lysine in modulating C-to-U editing through the formation of an inhibitory ion pair with the catalytic glutamate.

Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. Chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to eight used in this study. Enzyme kinetics performed in vitro combined with metabolic labeling in cellulo, resulted in the identification of 8 proteins for C10AlkOPP, 70 proteins for C15AlkOPP, 41 proteins for C15hAlkOPP, and 7 proteins for C20AlkOPP. While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes was relatively small. In contrast, C15AlkOPP labeled the most proteins including representatives from all classes of prenylated proteins. Functional analysis of these analogues demonstrated that C15AlkOPP was particularly well suited for biological studies since it was efficiently incorporated in cellulo, was able to confer correct plasma membrane localization of H-Ras protein and complement the effects of GGPP depletion in macrophages to yield correct cell polarization and filopodia. Collectively, these results indicate that C15AlkOPP is a biologically functional, universal probe for metabolic labeling experiments that has minimal effects on cellular physiology.

Sensing of peptidoglycan fragments is essential for inducing downstream signaling in both mammalian and fungal systems. The hexokinases NagK and Hxk1 are crucial enzymes for the phosphorylation of peptidoglycan molecules in order to activate specific cellular responses; however, biochemical characterization of their enzymatic specificity and efficiency has yet to be investigated in depth. Here a mass spectrometry enzymatic screen was implemented to assess substrate specificity, and an ATP coupled assay provided the quantitative kinetic profiles of these two homologous, eukaryotic enzymes. The data show, that while homologous, NagK and Hxk1 have vastly different substrate profiles. NagK accepts a variety of different peptidoglycan-based substrates albeit with reduced efficiency but are still valuable as a tool in large scale chemoenzymatic settings. Conversely, Hxk1 has a smaller substrate scope but can turnover these alternative substrates at similar levels to its natural substrate. These results allow for deeper understanding into the biosynthetic machinery responsible for essential cellular processes including UDP-GlcNAc regulation and immune recognition events in the cell.

This study explores the ion selectivity and conduction mechanisms of the hNa_V1.5 sodium channel using classical molecular dynamics simulations under an externally applied electric field. Our findings reveal distinct conduction mechanisms for Na⁺ and K⁺, primarily driven by differences in their hydration states when they diffuse close to the channel's selective filter (DEKA) and extracellular ring (EEDD). The Na⁺ ions undergo partial dehydration in the EEDD region, followed by a rehydration step in the DEKA ring, resulting in longer retention times and a deeper free energy minimum compared to K⁺. Conversely, the K⁺ ions exhibit a continuous dehydration process, facilitating a smoother passage through these key regions. These results indicate that ion selectivity and conductance are primarily governed by solvation dynamics, which, in turn, depend on the interactions with key charged residues within the channel. Additionally, we show that the delicate energetic balance between the interactions of the ions with the protein residues and with their solvation shells during the dehydration and rehydration processes is not properly captured by the force field. As a consequence, the selectivity of the channel is not well described, indicating that more accurate computational models must be applied to simulate ion conduction through eukaryotic Na_V channels.

Antibiotics are essential medicines threatened by the emergence of resistance in all relevant bacterial pathogens. The engagement of the molecular targets of antibiotics offers multiple opportunities for resistance to emerge. Successful target engagement often requires passage of the antibiotic from outside into the cell interior through one or two distinct membrane barriers. Resistance can occur by preventing the accumulation of antibiotics in sufficient quantities outside the cell, decreasing the rates of entry into the cell, and modifying the antibiotic or the target once inside the cell. These competing equilibria and rates are the lens through which the balance of antibiotic efficacy or failure can be viewed. The two faces of antibiotics, cell growth inhibition or resistance, are reminiscent of Janus, the Roman god of doorways and beginnings and endings, and offer a framework through which antibiotic discovery, use, and the emergence of resistance can be rationally viewed.

Photosystem I (PSI) from Acaryochloris marina utilizes chlorophyll d (Chld) with a formyl group as its primary pigment, which is more red-shifted than chlorophyll a (Chla) in PSI from Thermosynechococcus elongatus. Using the cryo-electron microscopy structure and solving the linear Poisson-Boltzmann equation, here we report the redox potential (E_m) values in A. marina PSI. The E_m(Chld) values at the paired chlorophyll site, [P_AP_B], are nearly identical to the corresponding E_m(Chla) values in T. elongatus PSI, despite Chld having a 200 mV lower reduction power. The accessory chlorophyll site, A_-1, in the B branch exhibits an extensive H-bond network with its ligand water molecule, contributing to E_m(A_-1B) being lower than E_m(A_-1A). The substitution of pheophytin a (Pheoa) with Chla at the electron acceptor site, A₀, decreases E_m(A₀), resulting in an uphill electron transfer from A_-1. The impact of the A_-1 formyl group on E_m(A₀) is offset by the reorientation of the A₀ ester group. It seems likely that Pheoa is necessary for A. marina PSI to maintain the overall electron-transfer cascade characteristic of PSI in its unique light environment.

1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) is a unique thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the formation of DXP, a branchpoint metabolite required for the biosynthesis of vitamins and isoprenoids in bacterial pathogens. DXPS has relaxed substrate specificity and utilizes a gated mechanism, equipping DXPS to sense and respond to diverse substrates. We speculate that pathogens utilize this distinct gated mechanism in different ways to support metabolic adaptation during infection. DXPS is susceptible to time-dependent inhibition by bisubstrate analogs. We suggest that potential differences in the ligand-gated mechanism that may accompany alternative activities of DXPS homologues may enable the development of species-specific bisubstrate analog inhibitors. Here, we evaluate known bisubstrate analog inhibitors of Escherichia coli DXPS (EcDXPS) against DXPS from Pseudomonas aeruginosa (PaDXPS), a Gram-negative pathogen with a remarkable capacity to adapt to diverse environments. Our results indicate that these inhibitors are significantly less potent against PaDXPS compared to EcDXPS. Acceptor site residues that stabilize the phosphonolactyl-ThDP adduct (PLThDP) of bisubstrate analog d-PheTrAP on EcDXPS are not as critical for stabilization of this PLThDP adduct on PaDXPS. Substitution of EcR99 or the analogous PaR106 reduces the potency of both d-PheTrAP and the simpler BAP scaffold, suggesting a common role of these arginine residues in stabilizing PLThDP adducts. However, although EcR99 is required for potent, time-dependent inhibition of EcDXPS by d-PheTrAP, PaR106 does not appear to govern slow-onset inhibition. This work demonstrates that species-specific targeting of DXPS by bisubstrate analogs is possible and highlights mechanistic differences that should be considered in the design of homologue-specific inhibitors, toward narrow-spectrum approaches targeting DXPS.

Human UDP-glucose dehydrogenase (hUGDH) catalyzes the oxidation of UDP-glucose into UDP-glucuronic acid, an essential substrate in the Phase II metabolism of drugs. hUGDH is a hexamer that exists in an equilibrium between an active (E) state and an inactive (E^Ω) state, with the latter being stabilized by the binding of the allosteric inhibitor UDP-xylose (UDP-Xyl). The allosteric transition between E^Ω and E is slow and can be observed as a lag in progress curves. Previous analysis of the lag suggested that unliganded hUGDH exists mainly as E^Ω, but two unique crystal forms suggest that the enzyme favors the E state. Resolving this discrepancy is necessary to fully understand the allosteric mechanism of hUGDH. Here, we used cryo-EM to show that recombinant hUGDH expressed in Escherichia coli copurifies with UDP-4-keto-xylose (UX4O), which mimics the UDP-Xyl inhibitor and favors the E^Ω state. Cryo-EM studies show that removing UX4O from hUGDH shifts the ensemble to favor the E state. This shift is consistent with progress curve analysis, which shows the absence of a lag for unliganded hUGDH. Inhibition studies show that hUGDH has similar affinities for UDP-Xyl and UX4O. The discovery that UX4O inhibits allosteric hUGDH suggests that UX4O may be the physiologically relevant inhibitor of allosteric UGDHs in bacteria that do not make UDP-Xyl.