Bacterioferritin nanocage structures uncover the biomineralization process in ferritins.

Iron is an essential element involved in various metabolic processes. The ferritin family of proteins forms nanocage assembly and is involved in iron oxidation, storage, and mineralization. Although several structures of human ferritins and bacterioferritins have been solved, there is still no complete structure that shows both the trapped Fe-biomineral cluster and the nanocage. Furthermore, whereas the mechanism of iron trafficking has been explained using various approaches, structural details on the biomineralization process (i.e. the formation of the mineral itself) are generally lacking. Here, we report the cryo-electron microscopy (cryo-EM) structures of apoform and biomineral bound form (holoforms) of the Streptomyces coelicolor bacterioferritin (ScBfr) nanocage and the subunit crystal structure. The holoforms show different stages of Fe-biomineral accumulation inside the nanocage, in which the connections exist in two of the fourfold channels of the nanocage between the C-terminal of the ScBfr monomers and the Fe-biomineral cluster. The mutation and truncation of the bacterioferritin residues involved in these connections significantly reduced the iron and phosphate binding in comparison with those of the wild type and together explain the underlying mechanism. Collectively, our results represent a prototype for the bacterioferritin nanocage, which reveals insight into its biomineralization and the potential channel for bacterioferritin-associated iron trafficking.

KasQ an Epimerase Primes the Biosynthesis of Aminoglycoside Antibiotic Kasugamycin and KasF/H Acetyltransferases Inactivate Its Activity.

Kasugamycin (KSM), an aminoglycoside antibiotic, is composed of three chemical moieties: D-chiro-inositol, kasugamine and glycine imine. Despite being discovered more than 50 years ago, the biosynthetic pathway of KSM remains an unresolved puzzle. Here we report a structural and functional analysis for an epimerase, KasQ, that primes KSM biosynthesis rather than the previously proposed KasF/H, which instead acts as an acetyltransferase, inactivating KSM. Our biochemical and biophysical analysis determined that KasQ converts UDP-GlcNAc to UDP-ManNAc as the initial step in the biosynthetic pathway. The isotope-feeding study further confirmed that 13C, 15N-glucosamine/UDP-GlcNH2 rather than glucose/UDP-Glc serves as the direct precursor for the formation of KSM. Both KasF and KasH were proposed, respectively, converting UDP-GlcNH2 and KSM to UDP-GlcNAc and 2-N'-acetyl KSM. Experimentally, KasF is unable to do so; both KasF and KasH are instead KSM-modifying enzymes, while the latter is more specific and reactive than the former in terms of the extent of resistance. The information gained here lays the foundation for mapping out the complete KSM biosynthetic pathway.

Theoretical Study of Intermolecular Interactions between Critical Residues of Membrane Protein MraYAA and Promising Antibiotic Muraymycin D2.

Phospho-N-acetylmuramoyl-pentapeptide translocase (MraYAA) from Aquifex aeolicus is the binding target for the nucleotide antibiotic muraymycin D2 (MD2). MraYAA in the presence of the MD2 ligand has been crystallized and released, while the interactions between the ligand and active-site residues remain less quantitatively and qualitatively defined. We characterized theoretically the key residues involved in noncovalent interactions with MD2 in the MraYAA active site. We applied the quantum theory of atoms in molecules and natural bond orbital analyses based on the density functional theory method on the solved crystal structure of MraY with the MD2 to quantitatively estimate the intermolecular interactions. The obtained results revealed the presence of multiple hydrogen bonds in the investigated active site with strength ranging from van der Waals to covalent limits. Lys70, Asp193, Gly194, Asp196, Gly264, Ala321, Gln305, and His325 are key active-site residues interacting with MD2. Conventional and unconventional hydrogen bonds in addition with charge-dipole and dipole-dipole interactions contribute significantly to stabilize the MD2 binding to the MraYAA active site. It was also found that water molecules inside the active site have substantial effects on its structure stability through hydrogen-bonding interactions with MD2 and the interacting residues.

Chemoenzymatic Synthesis and Biological Evaluation for Bioactive Molecules Derived from Bacterial Benzoyl Coenzyme A Ligase and Plant Type III Polyketide Synthase.

Plant type III polyketide synthases produce diverse bioactive molecules with a great medicinal significance to human diseases. Here, we demonstrated versatility of a stilbene synthase (STS) from Pinus Sylvestris, which can accept various non-physiological substrates to form unnatural polyketide products. Three enzymes (4-coumarate CoA ligase, malonyl-CoA synthetase and engineered benzoate CoA ligase) along with synthetic chemistry was practiced to synthesize starter and extender substrates for STS. Of these, the crystal structures of benzoate CoA ligase (BadA) from Rhodopseudomonas palustris in an apo form or in complex with a 2-chloro-1,3-thiazole-5-carboxyl-AMP or 2-methylthiazole-5-carboxyl-AMP intermediate were determined at resolutions of 1.57 Å, 1.7 Å, and 2.13 Å, respectively, which reinforces its capacity in production of unusual CoA starters. STS exhibits broad substrate promiscuity effectively affording structurally diverse polyketide products. Seven novel products showed desired cytotoxicity against a panel of cancer cell lines (A549, HCT116, Cal27). With the treatment of two selected compounds, the cancer cells underwent cell apoptosis in a dose-dependent manner. The precursor-directed biosynthesis alongside structure-guided enzyme engineering greatly expands the pharmaceutical repertoire of lead compounds with promising/enhanced biological activities.

In silico structure-based design of enhanced peptide inhibitors targeting RNA polymerase PAN-PB1C interaction.

Developing antivirals for influenza A virus (FluA) has become more challenging due to high range of antigenic mutation and increasing numbers of drug-resistant viruses. Finding a selective inhibitor to target highly conserved region of protein-protein interactions interface, thereby increasing its efficiency against drug resistant virus could be highly beneficial. In this study, we used in silico approach to derive FluAPep1 from highly conserved region, PAN-PB1C interface and generated 121 FluAPep1 analogues. Interestingly, we found that the FluAPep1 interaction region in the PAN domain are highly conserved in many FluA subtypes. Especially, FluAPep1 targets two pandemic FluA strains, H1N1/avian/2009 and H3N2/Victoria/1975. All of these FluA subtypes PAN domain (H1N1/H3N2CAN/H3N2VIC/H7N1/H7N2) were superimposed with PAN domain from H17N10 and the calculated root mean standards deviations were less than 3 Å. FlexPepDock analysis revealed that FluAPep1 exhibited higher binding affinity (score -246.155) with the PAN domain. In addition, around 86% of non-hot spot mutated peptides (FluAPep28-122) showed enhanced binding affinity with PAN domain. ToxinPred analysis confirmed that designed peptides were non-toxic. Thus, FluAPep1 and its analogues has potential to be further developed into an antiviral treatment against FluA infection.

Capillary hydrodynamic chromatography reveals temporal profiles of cell aggregates.

Microbial cells are known to form aggregates. Such aggregates can be found in various matrices; for example, functional drinks. Capillary hydrodynamic chromatography (HDC) enables separation of particles by size using nanoliter-scale volumes of samples. Here we propose an approach based on HDC for characterisation of real samples containing aggregated and non-aggregated bacterial and fungal cells. Separation of cells and cell aggregates in HDC arises from the parabolic flow profile under laminar flow conditions. In the presented protocol, hydrodynamic separation is coupled with different on-line and off-line detectors (light absorption/scattering and microscopy). The method has successfully been applied in the monitoring of dynamic changes in the microbiome of probiotic drinks. Chromatographic profiles of yogurt and kefir samples obtained at different times during fermentation are in a good agreement with microscopic images. Moreover, thanks to the implementation of an area imaging detector, capillary HDC could be multiplexed and used to profile spatial gradients in cell suspensions, which arise in the course of sedimentation of cells and cell aggregates. This result shows compatibility of sedimentation analysis and capillary HDC. We believe that the approach may find applications in the profiling of functional foods and other matrices containing aggregated bioparticles.