Structural and mechanistic basis of the high catalytic activity of monooxygenase Tet(X4) on tigecycline

Background Tigecycline is a tetracycline derivative that constitutes one of the last-resort antibiotics used clinically to treat infections caused by both multiple drug-resistant (MDR) Gram-negative and Gram-positive bacteria. Resistance to this drug is often caused by chromosome-encoding mechanisms including over-expression of efflux pumps and ribosome protection. However, a number of variants of the flavin adenine dinucleotide (FAD)-dependent monooxygenase TetX, such as Tet(X4), emerged in recent years as conferring resistance to tigecycline in strains of Enterobacteriaceae, Acinetobacter sp., Pseudomonas sp., and Empedobacter sp. To date, mechanistic details underlying the improvement of catalytic activities of new TetX enzymes are not available. Results In this study, we found that Tet(X4) exhibited higher affinity and catalytic efficiency toward tigecycline when compared to Tet(X2), resulting in the expression of phenotypic tigecycline resistance in E. coli strains bearing the tet(X4) gene. Comparison between the structures of Tet(X4) and Tet(X4)-tigecycline complex and those of Tet(X2) showed that they shared an identical FAD-binding site and that the FAD and tigecycline adopted similar conformation in the catalytic pocket. Although the amino acid changes in Tet(X4) are not pivotal residues for FAD binding and substrate recognition, such substitutions caused the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain of Tet(X4), resulting in the formation of a larger number of loops in the structure. These changes in turn render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrate molecules, thereby improving catalytic efficiency. Conclusions Our works provide a better understanding of the molecular recognition of tigecycline by the TetX enzymes; these findings can help guide the rational design of the next-generation tetracycline antibiotics that can resist inactivation of the TetX variants.

Essential elements of radical pair magnetosensitivity in Drosophila

Many animals use the Earth magnetic field (geoMF) for navigation. The favored mechanism for magnetosensitivity involves a blue-light (BL) activated electron transfer reaction between flavin adenine dinucleotide (FAD) and a chain of tryptophan (Trp) residues within the photoreceptor protein, CRYPTOCHROME (CRY). The spin-state of the resultant radical pair (RP) and hence the concentration of CRY in its active state is influenced by the geoMF. The canonical CRY-centric radical pair mechanism (RPM) does not, however, explain many physiological and behavioural observations. Here, using electrophysiology and behavioural analyses, we assay magnetic field (MF) responses at single neuron and organismal level. We show that the 52 C-terminal (CT) amino acids of CRY, which are missing the FAD binding domain and the Trp chain, are sufficient to facilitate magnetoreception. We also show that increasing intracellular FAD potentiates both BL-induced and MF-dependent effects on the activity mediated by the CT. Additionally, high levels of FAD alone are sufficient to cause BL neuronal sensitivity and, remarkably, potentiation of this response in the co-presence of a MF. These unexpected results reveal the essential components of a primary magnetoreceptor in flies, providing strong evidence that non-canonical (i.e., non-CRY-dependent) RPs can elicit MF responses in cells.

Hot Spots of Phytoene Desaturase from Rhodobacter Sphaeroides Influencing the Desaturation of Phytoene

Phytoene desaturase (CrtI, E.C. 1.3.99.31) shows variable desaturation activity, thereby introducing different numbers of conjugated double bonds (CDB) into the substrate phytoene. In particular, Rhodobacter sphaeroides CrtI is known to introduce additional 6 CDBs into the phytoene with 3 CDBs, generating neurosporene with 9 CDBs. Although in-depth studies have been conducted on the function and phylogenetic evolution of CrtI, little information exists on its range of CDB-introducing capabilities. We investigated the relationship between the structure and CDB-introducing capability of CrtI. CrtI of R. sphaeroides KCTC 12085 was randomly mutagenized to produce carotenoids of different CDBs (neurosporene for 9 CDBs, lycopene for 11 CDBs, and 3,4-didehydrolycopene for 13 CDBs). From six CrtI mutants producing different ratios of neurosporene/lycopene/3,4-didehydrolycopene, three amino acids (Leu163, Ala171, and Ile454) were identified that significantly determined carotenoid profiles. While the L163P mutation was responsible for producing neurosporene as a major carotenoid, A171P and I454T produced lycopene as the major product. Finally, according to the in silico model, the mutated amino acids are gathered in the membrane-binding domain of CrtI, which could distantly influence the FAD binding region and consequently the degree of desaturation in phytoene.

Molecular Dynamics Analysis of Glucose Oxidase Stability against Temperature

Glucose oxidase (GOD) from local isolated Aspergillus niger IPBCC.08.610 shows a widespread application, specifically as a bioanode in glucose-based biofuel cells. Enzymes with adequate thermal stability are necessary for enhancing product efficiency. Also, evaluating the structural dynamics to improve temperature helps to determine the residue. The molecular dynamics simulation of GOD_IPBCC_1CF3 at temperatures of 300, 400, and 500 K was carried out to analyze important amino acid residues for thermal stability. The results showed that the amino acid residues responsible for thermal stability were dispersed into several essential regions, including D576 at the C terminal, E266-R250, and E38-R237 in the FAD-binding domain E485-R470 in the substrate-binding antiparallel beta system. However, the FAD molecular flexibility against temperature depends on conserve E48 by stabilizing the ribose sugar moiety.

Multiple Acyl-CoA Dehydrogenase Deficiency with Variable Presentation Due to a Homozygous Mutation in a Bedouin Tribe

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a fatty acid and amino acid oxidation defect caused by a deficiency of the electron-transfer flavoprotein (ETF) or the electron-transfer flavoprotein dehydrogenase (ETFDH). There are three phenotypes of the disease, two neonatal forms and one late-onset. Previous studies have suggested that there is a phenotype–genotype correlation. We report on six patients from a single Bedouin tribe, five of whom were sequenced and found to be homozygous to the same variant in the ETFDH gene, with variable severity and age of presentation. The variant, NM_004453.3 (ETFDH): c.524G>A, p.(R175H), was previously recognized as pathogenic, although it has not been reported in the literature in a homozygous state before. R175H is located near the FAD binding site, likely affecting the affinity of FAD for EFT:QO. The single homozygous ETFDH pathogenic variant was found to be causing MADD in this cohort with an unexpectedly variable severity of presentation. The difference in severity could partly be explained by early diagnosis via newborn screening and early treatment with the FAD precursor riboflavin, highlighting the importance of early detection by newborn screening.

Tools, strains, and strategies to effectively conduct anaerobic and aerobic transcriptional reporter screens and assays in Staphylococcus aureus

Transcriptional reporters are reliable and time-tested tools to study gene regulation. In Staphylococcus aureus, β-galactosidase (lacZ)-based genetic screens are not widely used because of the necessity of selectable markers for strain construction and the production of staphyloxanthin pigment which obfuscates results. We describe a series of vectors that allow for markerless insertion of codon-optimized lacZ-based transcriptional reporters. The vectors encode for different ribosomal binding sites allowing for tailored lacZ expression. A ΔcrtM::kanR deletion insertion mutant was constructed that prevents the synthesis of staphyloxanthin, thereby permitting blue-white screening without the interference of carotenoid production. We demonstrate the utility of these vectors to monitor aerobic and anaerobic transcriptional activity. For the latter, we describe the use of a ferrocyanide-ferricyanide redox system (Fe(CN)63–/4–) permitting blue-white screening in the absence of oxygen. We also describe additional reporter systems and methods for monitoring transcriptional activity during anaerobic culture including a FAD-binding fluorescent protein (EcFbFP), alpha-hemolysin (hla), or lipase (geh). The systems and methods described are compatible with vectors utilized to create and screen high-density transposon mutant libraries.

A qnr-plasmid allows aminoglycosides to induce SOS in Escherichia coli

The plasmid-mediated quinolone resistance (PMQR) genes have been shown to promote high level bacterial resistance to fluoroquinolone antibiotics, potentially leading to clinical treatment failures. In Escherichia coli, sub-inhibitory concentrations (sub-MIC) of the widely used fluoroquinolones are known to induce the SOS response. Interestingly, the expression of several PMQR qnr genes is controlled by the SOS master regulator. During the characterization of a small qnrD-plasmid carried in E. coli, we observed that the aminoglycosides become able to induce the SOS response in this species, thus leading to the transcription of qnrD. We found that induction of the SOS response is due to nitric oxide (NO) accumulation in presence of sub-MIC of aminoglycosides. We demonstrated that the NO accumulation is driven by two plasmid genes, ORF3 and ORF4, whose products act at two levels. ORF3 encode a FAD-binding oxidoreductase which helps NO synthesis, while ORF4 code for an FNR-type transcription factor, related to an O2-responsive regulator of hmp expression, able to repress the Hmp-mediated NO detoxification pathway of E. coli. Thus, this discovery, that other major classes of antibiotics may induce the SOS response could have worthwhile implications for antibiotic stewardship efforts in preventing the emergence of resistance.

Raloxifene, identified as a novel LSD1 inhibitor, suppresses the migration of renal cell carcinoma

Aim: As an important epigenetic modulator, histone lysine-specific demethylase 1 (LSD1) has been proved to be associated with the progression of renal cell carcinoma (RCC). Discovering novel LSD1 inhibitors offers therapeutic potential for RCC treatment. Methods & Results: We identified raloxifene as a novel LSD1 inhibitor (IC50 = 2.08 μM) through small compound library screening. Molecular docking indicated raloxifene might bind LSD1 in the flavin adenine dinucleotide (FAD) binding cavity in a reversible manner. Cell viability and migration assays showed raloxifene could suppress the proliferation and migration of RCC cells bearing overexpressed LSD1. Conclusion: Our findings indicated that LSD1 might be a promising therapeutic target for RCC and that raloxifene could serve as a lead compound for further anti-RCC metastasis drug discovery.