Phospho- N -acetylmuramoyl-pentapeptide translocase (MraY AA ) from Aquifex aeolicus is the binding target for the nucleotide antibiotic muraymycin D2 (MD2). MraY AA 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 MraY AA 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 MraY AA 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.

Tunicamycin (TUN) is a nucleoside antibiotic with a complex structure comprising uracil, tunicamine sugar, N -acetylglucosamine (GlcNAc), and fatty acyl tail moieties. TUN, known as a canonical inhibitor, blocks vital functions of certain transmembrane protein families, for example, the insect enzyme dolichyl phosphate α- N -acetylglucosaminylphosphotransferase (DPAGT1) of Spodoptera frugiperda and the bacterial enzyme phospho- N -acetylmuramoylpentapeptide translocase (MraY CB ) of Clostridium bolteae . Accurate description of protein–drug interactions has an immense impact on structure-based drug design, while the main challenge is to create proper topology and parameter entries for TUN in modeling protein–TUN interactions given the structural complexity. Starting from DPAGT1–TUN and MraY CB –TUN crystal structures, we first sketched these structural complexes on the basis of the CHARMM36 force field and optimized each of them using quantum mechanics/molecular mechanics (QM/MM) calculations. By continuing calculations on the active site (QM region) of each optimized structure, we specified the characteristics of intermolecular interactions contributing to the binding of TUN to each active site by quantum theory of atoms in molecules (QTAIM) and natural bond orbital (NBO) analyses at the M06-2X/6-31G** level. The results outlined that TUN insertion into each active site requires multiple weak, moderate, and strong hydrogen bonds accompanying charge–dipole, dipole–dipole, and hydrophobic interactions among different TUN moieties and adjacent residues. The water-mediated interactions also play central roles in situating the uracil and tunicamine moieties of TUN within the DPAGT1 active site as well as in preserving the uracil-binding pocket in the MraY CB active site. The TUN binds more strongly to DPAGT1 than to MraY CB . The information garnered here is valuable particularly for better understanding mode of action at the molecular level, as it is conducive to developing next generations of nucleoside antibiotics.

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.

Albofungin, a natural product produced from Streptomycetes, exhibits bioactivities against bacteria, fungi, and tumor cells. The biosynthetic logic, regulations, and resistance of albofungin remain yet to be addressed.

Caprazamycin is a nucleoside antibiotic that inhibits phospho-N-acetylmuramyl-pentapeptide translocase (MraY). The biosynthesis of nucleoside antibiotics has been studied but is still far from completion. The present study characterized enzymes Cpz10, Cpz15, Cpz27, Mur17, Mur23 out of caprazamycin/muraymycin biosynthetic gene cluster, particularly the nonheme αKG-dependent enzyme Cpz10. Cpz15 is a β-hydroxylase converting uridine mono-phosphate to uridine 5′ aldehyde, then incorporating with threonine by Mur17 (Cpz14) to form 5′-C-glycyluridine. Cpz10 hydroxylates synthetic 11 to 12 in vitro. Major product 13 derived from mutant Δcpz10 is phosphorylated by Cpz27. β-Hydroxylation of 11 by Cpz10 permits the maturation of caprazamycin, but decarboxylation of 11 by Mur23 oriented to muraymycin formation. Cpz10 recruits two iron atoms to activate dioxygen with regio-/stereo-specificity and commit electron/charge transfer, respectively. The chemo-physical interrogations should greatly advance our understanding of caprazamycin biosynthesis, which is conducive to pathway/protein engineering for developing more effective nucleoside antibiotics.

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Bakground:Aspergillus fumigatus is an airborne opportunistic fungal pathogen that can cause fatal infections in immunocompromised patients. Although the current anti-fungal therapies are relatively efficient, some issues such as drug toxicity, drug interactions, and the emergence of drug-resistant fungi have promoted the intense research toward finding the novel drug targets.Methods:In search of new antifungal drug targets, we have used a bioinformatics approach to identify novel drug targets. We compared the whole proteome of this organism with yeast Saccharomyces cerevisiae to come up with 153 specific proteins. Further screening of these proteins revealed 50 potential molecular targets in A. fumigatus. Amongst them, RNA-binding protein (RBP) was selected for further examination. The aspergillus fumigatus RBP (AfuRBP), as a peptidylprolyl isomerase, was evaluated by homology modeling and bioinformatics tools. RBP-deficient mutant strains of A. fumigatus were generated and characterized. Furthermore, the susceptibility of these strains to known peptidylprolyl isomerase inhibitors was assessed.Results:AfuRBP-deficient mutants demonstrated a normal growth phenotype. MIC assay results using inhibitors of peptidylprolyl isomerase confirmed a higher sensitivity of these mutants compared to the wild type.Conclusion:Our bioinformatics approach revealed a number of fungal-specific proteins that may be considered as new targets for drug discovery purposes. Peptidylprolyl isomerase, as a possible drug target, was evaluated against two potential inhibitors, and the promising results were investigated mechanistically. Future studies would confirm the impact of such target on the antifungal discovery investigations

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.