Biochemistry Biochemistry最新文献

The catalytic site of photosynthetic water oxidation, the water-oxidizing complex (WOC), which contains the Mn₄CaO₅ cluster as its inorganic core, is assembled in photosystem II (PSII) through a light-driven process known as photoactivation. Despite extensive study, the detailed molecular mechanism underlying photoactivation remains elusive. Here, we investigated the mechanism of photoactivation by focusing on the “dark rearrangement process” that occurs following the first flash illumination, using time-resolved Fourier transform infrared (FTIR) measurements of apo-WOC PSII both in crystals, where the protein conformation remains nearly unchanged upon Mn depletion, and in solution, where Mn removal induces substantial conformational changes. Time-resolved FTIR spectra of apo-WOC PSII in solution, following single-flash illumination in the presence of Mn²⁺, revealed two distinct decay phases. The fast phase was characterized by increased relative intensities of amide I bands accompanied by shifts in carboxylate stretching bands, while the slow phase exhibited minimal spectral changes. In contrast, FTIR spectra of apo-WOC PSII in crystals showed only a single slow decay phase, with a time constant comparable to that of the slow component in solution, and with negligible change in spectral shape. This striking contrast between PSII in solution and in crystals provides definitive evidence that significant protein conformational changes, accompanied by Mn³⁺ relocation via carboxylate groups, occur during the dark rearrangement process following the initial photooxidation of Mn²⁺ under physiological conditions.

G-rich sequences of DNA and RNA can form G-quadruplex (G4) structures, modulating a myriad of biological processes. Thus, it is imperative to understand the structural topologies, location, and function of G4s under cell-free conditions and in the cellular milieu. In the present study, we report three small-molecule fluorescent probes based on azlactones (AZL1-3) that significantly light up (∼65–135-fold) the parallel topology of the c-MYC, c-KIT1, and mitochondrial HRCC G4 DNAs. The lead probe AZL1 exhibits a 2:1 binding stoichiometry with c-KIT1 G4 DNA by accessing the 5′ and 3′-G-quartets. It shows limited cytotoxicity and exhibits fluorescence light-up in the cytoplasm of the HeLa cells due to weak colocalization with the mitochondrial G4 DNAs along with strong colocalization with lipid droplets. These results demonstrate that azlactone-based probes are useful tools to sense G4 structures in a cell-free environment and could be further engineered for potential bioimaging and diagnostic applications.

Dietary β-carotene is the most common carotenoid in the world. Naturally occurs in vegetables and fruits (e.g., carrots, tomatoes). Recently, β-carotene has been studied for its effects on the human body; however, the effect of this carotenoid on brain tumor metabolism at the cellular level is still unknown. Here, we consider whether β-carotene influences brain tumor cell metabolism and, if so, whether this effect stimulates or inhibits tumor growth. To find out the effect of β-carotene on brain cells (normal human astrocytes, astrocytoma, and glioblastoma), we applied Raman spectroscopy and imaging. We focused our analysis on biological changes in particular cell organelles such as the nucleus, mitochondria, lipid droplets/endoplasmic reticulum, and cytoplasm. Our Raman results demonstrated that cancer cell metabolism is altered following β-carotene supplementation, as reflected in changes to Raman bands associated with cytochrome c (1310 and 1583 cm^–1), lipids (1337 and 1444 cm^–1), and proteins (1337 and 1654 cm^–1). The response to supplementation is different not only for normal cells compared to cancer cells (the effects vary depending on the cell type) but also for supplementation timing and doses.

While humans utilize approximately ten building blocks, hundreds of “rare” sugars exist, which are absent in mammals but present in microbes, plants, and other natural sources. In addition to the common sugars found across organisms, more than 700 different rare monosaccharides exist, many of which are prokaryote-specific and utilized across bacteria to decorate natural products and various other glycoconjugates. As the outer glycocalyx layer of bacterial cells is composed of glycolipids, glycoproteins, and polysaccharides, rare sugars are enriched on the cell surface and are major components of structures known to mediate interactions with other cells and the environment. Despite their importance in biology, there remain many open questions in the field of biochemistry regarding the biosynthesis and functions of rare sugars. This perspective highlights ongoing biochemical work on prokaryotic rare sugars, including approaches to study the incorporation of rare sugars into cellular glycans, to develop chemical and enzymatic routes for generating rare sugar probes and glycans, and to analyze rare sugar–protein interactions. Opportunities to improve the sequencing efforts of microbial glycans through experimental and computational approaches are also discussed, along with potential therapeutic applications of rare sugar-containing molecules. In covering these topics, we emphasize tools that have not yet been utilized to study rare sugars but may be used for future approaches that will expand our knowledge of their distinct roles in microbes and the interplay between pathogens and their hosts.

Flexizymes enable the stoichiometric acylation of tRNAs with a variety of compounds, enabling the in vitro translation of peptides with both non-natural backbones and side chains. However, flexizyme reactions have several drawbacks, including single-turnover kinetics, high Mg(II) carryover, inhibiting in vitro translation, and rapid product hydrolysis. Here we present flexizyme reactions utilizing an ice-eutectic phase, with high yields, 30 times lower Mg(II), and long-term product stability. The eutectic flexizyme reactions increase the ease of use, yield and flexibility of aminoacylation and significantly increase the in vitro protein production.

PrnB, a long-standing member of the recently reclassified heme-dependent aromatic oxygenase (HDAO) superfamily of histidine-ligated heme enzymes, catalyzes the conversion of 7-chloro-l-tryptophan (7-Cl-Trp) to monodechloroaminopyrrolnitrin (MCAP). This unique ring rearrangement is an essential step in the biosynthesis of the broad-spectrum antifungal pyrrolnitrin. The conversion of 7-Cl-Trp to MCAP by PrnB differs from other HDAOs, which typically affect the mono- or dioxygenation of aromatic substrates. However, the molecular basis for this transformation has remained enigmatic due to the inability to reconstitute enzymatic activity in vitro. Transient kinetic approaches reveal that the sequential binding of 7-Cl-Trp and dioxygen results in the formation of a long-lived oxy-ferrous complex. Despite similarity to intermediates found in other HDAOs, the PrnB-oxy ternary species does not react with bound 7-Cl-Trp. However, the provision of a surrogate redox delivery system supports turnover, and single-turnover studies reveal that activation of the oxy-complex is required. Together, these studies reveal the molecular basis for functional expansion of the HDAO structural framework through alteration of the strategy of oxygen activation.

The steroid aldehyde dehydrogenase (Sad) from Proteobacteria is a class 3 aldehyde dehydrogenase (ALDH3) that catalyzes the oxidation of C₃ steroid side chain aldehydes during bile acid catabolism. The 1.8 Å structure of the enzyme revealed an expanded active site that was able to accommodate bulky steroids, including bile acid intermediates and cholesterol derivatives, with minimal selectivity for ring-conformation or hydroxylation. Sad can utilize both NAD⁺ and NADP⁺ as coenzymes, likely due to a truncated N-terminus and a flexible Glu149 residue, which can avoid steric and electrostatic repulsion with the 2′-phosphate of NADP⁺ while retaining the ability to hydrogen bond to the C2′-OH of NAD⁺. Sad was over 1000-fold more specific for steroid aldehyde substrates than for smaller molecules such as benzaldehyde. Structural comparison with the homologousPseudomonas putida benzaldehyde dehydrogenase (PpBADH) suggested residues that might contribute to the ability of Sad to utilize bulky steroid substrates. Replacement of these residues in an F400A/L125T PpBADH double-variant resulted in a ∼39-fold increase in catalytic efficiency toward steroid aldehyde compared with the wild-type enzyme. This study advances our understanding of the molecular determinants of substrate specificity within the ALDH3 family and lays the groundwork for biocatalytic applications of steroid aldehyde dehydrogenases in the production of steroid pharmaceuticals and the bioremediation of steroidal pollutants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to accumulate mutations in the spike receptor-binding domain (RBD) region, leading to the emergence of new variants that potentially change the binding affinity for the human angiotensin converting enzyme 2 (hACE2) receptor. Deep mutational scanning (DMS) is a powerful biochemical experimental technique that can characterize the impact of mutations on protein sequence–function relationships, allowing for rapid assessment of new mutations. Herein, machine learning (ML) models were built using the SARS-CoV-2 DMS data set, with the input features derived from the Rosetta-computed decomposition energy terms. To improve the performance of this physics-based model, we further incorporated local environment information (the number of residue pair-specific contacts within shells at different distances) as the input features. Alternatively, a convolutional neural network (CNN) model based on amino-acid sequence information as well as their physicochemical and biochemical properties was also employed, yielding predictions that achieved good agreement with the experimental data. In addition, compared to three popular protein language models, the dual-encoding CNN model demonstrated consistently superior performance on the SARS-CoV-2 DMS data set and seven additional DMS data sets for different biological properties. Furthermore, a transfer-learning strategy was applied to fine-tune the CNN model using recently reported DMS data sets for the Alpha, Delta, and Omicron BA.1, BA.2, and XBB.1.5 variants, enabling the development of variant-specific prediction models. These ML models trained on DMS data sets can not only identify the effects of single-point mutations in mutagenesis data sets but also be useful in predicting the effects of multiple-point mutations and providing valuable information for ongoing viral surveillance efforts. Moreover, this dual-encoding CNN model, without including 3D geometric information, has the potential to be a robust and alternative ML model for other DMS studies.

Pseudouridimycin (PUM) is a C-nucleoside antibiotic that selectively inhibits bacterial RNA polymerase (RNAP) with remarkable potency. It binds to the nucleoside triphosphate (NTP) entry region in the RNAP active site by mimicking uridine-5′-triphosphate (UTP), thus blocking RNA synthesis in bacteria. Since PUM does not inhibit human RNAP, it presents a highly selective scaffold for clinical applications. Besides its unique mode of action, PUM’s peptidyl C-nucleoside structure comprises a rare pseudouridine (PU) moiety linked to an N-hydroxylated dipeptide, which is crucial for binding interactions with RNAP. Recently, the biosynthetic gene cluster (BGC) and a putative pathway have been reported for PUM biosynthesis. However, the investigation of the biosynthetic enzymes is still in its infancy. Here, we report detailed biochemical characterization of a flavin-dependent glucose–methanol–choline (GMC) family oxidoreductase, PumI, from Streptomyces rimosus (SrPumI) through substrate scope, computational modeling, mutational, kinetic, and mechanistic studies. Our studies have indicated that PumI preferentially accepts the native C-nucleoside substrate (PU) over N-nucleosides and acts as a gatekeeper in PUM biosynthesis. Our mutational analysis identified two active site histidines (His454 and His455) and two asparagines (Asn90 and Asn499) in SrPumI as potential flavin-binding residues. We propose His455 as the critical base for initiating catalysis based on our biochemical experiments and bioinformatics analysis. Additionally, Gln297 and Met58 were found to be important for substrate (PU) coordination. Based on these experiments, a mechanism has been proposed for PumI. We believe this work will provide new insights into PUM biosynthesis, enabling pathway engineering to prepare novel PUM derivatives for prospective therapeutic applications.

The emergence of drug-resistant viruses is a significant concern for the treatment of human immunodeficiency virus type-1 (HIV-1) infection, despite the availability of various drugs that block viral replication and propagation. Drugs that act upon unexploited targets of the viral replicative cycle may be able to circumvent resistance. The RNase H activity of HIV-1 reverse transcriptase is a viral enzymatic function for which no approved inhibitors are available. The active site of RNase H contains two metal cations that are required for catalysis. In this study, we describe the X-ray crystal structure of p15Ec (an HIV-1 RNase H domain recombinant protein) bound to an active-site inhibitor containing a pyrogallol moiety with chelating properties. The analysis revealed three hydroxyl oxygen atoms on the pyrogallol that firmly chelate two metal ions at the catalytic site. Molecular mechanics (MM) calculations were performed to determine the contributions of the respective compound atoms to the binding score. The analysis suggested that a piperazine moiety connected to the pyrogallol was not required to interact with the RNase H domain. A total of 6,757 derivatives were generated by replacing piperazine with other chemical groups. This was reduced to 5,567 following optimization of their binding poses by MM calculations, which indicated that the pyrogallol moiety maintained coordination with metal ions at the active site. Twelve candidate compounds with the best binding scores were selected as novel galloyl derivatives with improved RNase H inhibitory activity.