Biochemistry Biochemistry最新文献

Some species of the Nocardia genus harbor a highly conserved biosynthetic gene cluster designated as the NOCardiosis-Associated Polyketide (NOCAP) synthase that produces a unique glycolipid natural product. The NOCAP glycolipid is composed of a fully substituted benzaldehyde headgroup linked to a polyfunctional alkyl tail and an O-linked disaccharide composed of 3-α-epimycarose and 2-O-methyl-α-rhamnose. Incorporation of the disaccharide unit is preceded by a critical step involving hydroxylation by NocapM, a flavin monooxygenase. In this study, we employed biochemical, spectroscopic, and kinetic analyses to explore the substrate scope of NocapM. Our findings indicate that NocapM catalyzes hydroxylation of diverse aromatic substrates, although the observed coupling between NADPH oxidation and substrate hydroxylation varies widely from substrate to substrate. Our in-depth biochemical characterization of NocapM provides a solid foundation for future mechanistic studies of this enzyme as well as its utilization as a practical biocatalyst.

The in vitro transcription (IVT) of messenger ribonucleic acid (mRNA) from the linearized deoxyribonucleic acid (DNA) template of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) was optimized for total mRNA yield and purity (by percent intact mRNA) utilizing machine learning in conjunction with automated, high-throughput liquid handling technology. An iterative Bayesian optimization approach successfully optimized 11 critical process parameters in 42 reactions across 5 experimental rounds. Once the optimized conditions were achieved, an automated, high-throughput screen was conducted to evaluate commercially available T7 RNA polymerases for rate and quality of mRNA production. Final conditions showed a 12% yield improvement and a 50% reduction in reaction time, while simultaneously significantly decreasing (up to 44% reduction) the use of expensive reagents. This novel platform offers a powerful new approach for optimizing IVT reactions for mRNA production.

To combat the permanent exposure to potential pathogens every organism relies on an immune system. Important factors in innate immunity are antimicrobial peptides (AMPs) that are structurally highly diverse. Some AMPs are known to belong to the saposin-like proteins (SAPLIPs), a group of polypeptides with a broad functional spectrum. The model organism Dictyostelium discoideum possesses a remarkably large arsenal of potential SAPLIPs, which are termed amoebapore-like peptides (Apls), but the knowledge about these proteins is very limited. Here, we report about the biochemical characterization of AplE1, AplE2, AplK1, and AplK2, which are derived from the two precursor proteins AplE and AplK, thereby resembling prosaposins of vertebrates. We produced these Apls as recombinant polypeptides in Escherichia coli using a self-splicing intein to remove an affinity tag used for purification. All recombinant Apls exhibited pore-forming activity in a pH-dependent manner, as evidenced by liposome depolarization, showing higher activities the more acidic the setting was. Lipid preference was detected for negatively charged phospholipids and in particular for cardiolipin. Antimicrobial activity against various bacteria was found to be inferior in classical microdilution assays. However, all of the Apls studied permeabilized the cytoplasmic membrane of live Bacillus subtilis. Collectively, we assume that the selected Apls interact by their cationic charge with negatively charged bacterial membranes in acidic environments such as phagolysosomes and eventually lyse the target cells by pore formation.

A subunit of factor XIII (FXIII-A) contains a unique activation peptide (AP) that protects the catalytic triad and prevents degradation. In plasma, FXIII is activated proteolytically (FXIII-A*) by thrombin and Ca²⁺ cleaving AP, while in cytoplasm, it is activated nonproteolytically (FXIII-A°) with increased Ca²⁺ concentrations. This study aimed to elucidate the role of individual parts of the FXIII-A AP in protein stability, thrombin activation, and transglutaminase activity. Recombinant FXIII-A AP variants were expressed, and SDS-PAGE was used to monitor thrombin hydrolysis at the AP cleavage sites R37-G38. Transglutaminase activities were assessed by cross-linking lysine mimics to Fbg αC (233-425, glutamine-substrate) and monitoring reactions by mass spectrometry and in-gel fluorescence assays. FXIII-A AP variants, S19P, E23K, and D24V, degraded during purification, indicating their vital role in FXIII-A₂ stability. Mutation of P36 to L36/F36 abolished the proteolytic cleavage of AP and thus prevented activation. FXIII-A N20S and P27L exhibited slower thrombin activation, likely due to the loss of key interdomain H-bonding interactions. Except N20S and P15L/P16L, all activatable FXIII-A* variants (P15L, P16L, S19A, and P27L) showed similar cross-linking activity to WT. By contrast, FXIII-A° P15L, P16L, and P15L/P16L had significantly lower cross-linking activity than FXIII-A° WT, suggesting that loss of these prolines had a greater structural impact. In conclusion, FXIII-A AP residues that play crucial roles in FXIII-A stability, activation, and activity were identified. The interactions between these AP amino acid residues and other domains control the stability and activity of FXIII.

The secondary plastoquinone (PQ) electron acceptor Q_B in photosystem II (PSII) undergoes a two-step photoreaction through electron transfer from the primary PQ electron acceptor Q_A, converting into plastoquinol (PQH₂). However, the detailed mechanism of the Q_B reactions remains elusive. Here, we investigated the reaction mechanism of Q_B in cyanobacterial PSII core complexes using two time-revolved infrared (TRIR) methods: dispersive-type TRIR spectroscopy and rapid-scan Fourier transform infrared spectroscopy. Upon the first flash, the ∼140 μs phase is attributed to electron transfer from Q_A^•- to Q_B, while the ∼2.2 and ∼440 ms phases are assigned to the binding of an internal PQ in a nearby cavity to the vacant Q_B site and an external PQ traveling to the Q_B site through channels, respectively, followed by immediate electron transfer. The resultant Q_B^•- is suggested to be in equilibrium with Q_BH^•, which is protonated at the distal oxygen. Upon the second flash, the ∼130 μs and ∼3.3 ms phases are attributed to electron transfer to Q_BH^• and the protonation of Q_B^•- followed by electron transfer, respectively, forming Q_BH^-, which then immediately accepts a proton from D1-H215 at the proximal oxygen to become Q_BH₂. The resultant D1-H215 anion is reprotonated in ∼22 ms via a pathway involving the bicarbonate ligand. The final ∼490 ms phase may reflect the release of PQH₂ and its replacement with PQ. The present results highlight the importance of time-resolved infrared spectroscopy in elucidating the mechanism of Q_B reactions in PSII.

CCCH-type tandem zinc finger (TZF) motifs are found in many RNA-binding proteins involved in regulating mRNA stability, translation, and splicing. In Caenorhabditis elegans, several RNA-binding proteins that regulate embryonic development and cell fate determination contain CCCH TZF domains, including POS-1. Previous biochemical studies have shown that despite high levels of sequence conservation, POS-1 recognizes a broader set of RNA sequences compared to the human homologue tristetraprolin. However, the molecular basis of these differences remains unknown. In this study, we refined the consensus RNA sequence and determined the differing binding specificities of the two zinc fingers of POS-1. We also determined the solution structure and characterized the internal dynamics of the TZF domain of POS-1. From the structure, we identified unique features that define the RNA binding specificity of POS-1. We also observed that the TZF domain of POS-1 is in equilibrium between interconverting conformations. Transitions between these conformations require internal motions involving many residues with correlated dynamics in each ZF. We propose that the correlated dynamics are necessary to allow allosteric communication between the nucleotide-binding pockets observed in the N-terminal ZF. Our study shows that both the structure and conformational plasticity of POS-1 are important in ensuring recognition of its RNA binding targets.

The protein periostin is a matricellular protein that is expressed in connective tissue. It is composed of five globular domains arranged in an elongated structure with an extensive disordered C-terminal tail. Periostin contains 11 cysteine residues, of which one is unpaired and the rest form five intramolecular disulfide bonds. Periostin plays an important role during wound healing and is also involved in driving the inflammatory state in atopic diseases. This study provides a comprehensive biochemical characterization of periostin in human skin and in dermal and pulmonary fibroblasts in vitro. Through the application of Western blotting, co-immunoprecipitation, and LC-MS/MS, we show for the first time that periostin is a disulfide-bonded homodimer and engages in a novel disulfide-bonded complex with fibronectin both in vivo and in vitro. This inherent characteristic of periostin holds the potential to redefine our approach to exploring and understanding its functional role in future research endeavors.

Oligoadenylate synthetase 1 (OAS1) catalyzes the dsRNA-dependent polymerization of ATP to form oligoadenylate, a second messenger of the innate immunity system. This paper reports kinetic and mechanistic studies of OAS1-catalyzed dimerization of ATP to form 2'-5'-diadenylate and pyrophosphate (PP_i), the first step in ATP polymerization. Major findings include the following: (1) Reaction progress curves for the production of PP_i are biphasic, characterized by a presteady-state lag followed by the linear, steady-state production of PP_i. (2) The dependence of steady-state velocity on ATP concentration is sigmoidal and can be described by a rate law derived for a mechanism involving enzyme-catalyzed substrate dimerization. (3) Steady-state velocities were determined as a function of ATP concentration at fixed concentrations of poly(I:C), a synthetic dsRNA activator of OAS1. The data suggest a random mechanism in which either ATP or poly(I:C) can add first to the enzyme. (4) The dependence of k_lag on poly(I:C) and ATP concentration requires expansion of this mechanism to include slow conformational isomerization of various poly(I:C)- and ATP-bound complexes of inactive OAS1 to form complexes comprising an active enzyme, to ultimately form the reactive Michaelis complex of active OAS1, poly(I:C), and two molecules of ATP. Finally, within this complex, the two molecules of ATP dimerize to form 2'-5'-diadenylate and pyrophosphate. (5) The pH dependence and solvent deuterium isotope effect for k_cat suggests that proton transfer occurs in the rate-limiting transition state, which likely involves proton abstraction from the 2'-hydroxyl of the adenylate acceptor ATP as the oxygen of this hydroxyl attacks the a-phosphate of the adenylate donor ATP in an S_N2 fashion.

Biocatalysis is becoming a data science. High-throughput experimentation generates a rapidly increasing stream of biocatalytic data, which is the raw material for mechanistic and novel data-driven modeling approaches for the predictive design of improved biocatalysts and novel bioprocesses. The holistic and molecular understanding of enzymatic reaction systems will enable us to identify and overcome kinetic bottlenecks and shift the thermodynamics of a reaction. The full characterization and modeling of reaction systems is a community effort; therefore, published methods and results should be findable, accessible, interoperable, and reusable (FAIR), which is achieved by developing standardized data exchange formats, by a complete and reproducible documentation of experimentation, by collaborative platforms for developing sustainable software and for analyzing data, and by repositories for publishing results together with raw data. The FAIRification of biocatalysis is a prerequisite to developing highly automated laboratory infrastructures that improve the reproducibility of scientific results and reduce the time and costs required to develop novel synthesis routes.

Single-molecule correlated chemical probing (smCCP) is an experimentally concise strategy for characterizing higher-order structural interactions in RNA. smCCP data yield rich, but complex, information about base pairing, conformational ensembles, and tertiary interactions. To date, through-space communication specifically measuring RNA tertiary structure has been difficult to isolate from structural communication reflective of other interactions. Here, we introduce mutual information as a filtering metric to isolate tertiary structure communication contained within smCCP data and use this strategy to characterize the structural ensemble of the SAM-III riboswitch. We identified an smCCP fingerprint that is selective for states containing a tertiary structure that forms concurrently with cognate ligand binding. We then successfully applied mutual information filters to independent RNAs and isolated through-space tertiary interactions in riboswitches and large RNAs with complex structures. smCCP, coupled with mutual information criteria, can now be used as a tertiary structure discovery tool, including to identify specific states in an ensemble that have a higher-order structure. These studies pave the way for the use of the straightforward smCCP experiment for discovery and characterization of tertiary structure motifs in complex RNAs.