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Halogenases with Potential Applications for the Synthesis of Halogenated Pharmaceuticals
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Georgette Rebollar-Pérez, Cynthia Romero-Guido, Antonino Baez, Eduardo Torres
Flavin dependent halogenases (Fl-Hal) are part of the monooxygenases group that have been identified in bacterial and fungal biosynthetic pathways (Latham et al., 2018). They are another type of enzymes that catalyze halogenation through hypohalous acid, although an additional step in the catalytic cycle takes place because these enzymes are highly specific and regioselective catalysts. Most of these enzymes oxidize free reduced flavin (FADH2) using O2 to generate a FAD-OOH activated intermediate (Fig. 16.1c). In addition to the free diffusable flavin reductase described, flavin halogenases from NRPS and PKS pathways require other enzymes for substrate activation or tethering (Weichold et al., 2016). Then, chloride ions react with this species to produce hypohalous acid (Fig. 16.1c). An interesting mechanistic aspect of flavin halogenases is that there is a 10 Å gap between the site where HOCL is produced and the site where the subsequent halogenating step takes place; therefore, HOCL has to migrate through the protein (Sirimulla et al., 2013). At this point, the alternative step among haloperoxidases takes place, a lysine residue in the active site participates orienting hypohalous acid, through H-bonding of the amino group, towards the reduced substrate, improving the regiospecificity of the reaction (Dong et al., 2005). Another possibility is that HOCl may react with the primary amine group in lysine to produce an electrophilic chloramine with a specific orientation which is responsible for the substrate halogenation (Yeh et al., 2007).
Metronidazole
Published in M. Lindsay Grayson, Sara E. Cosgrove, Suzanne M. Crowe, M. Lindsay Grayson, William Hope, James S. McCarthy, John Mills, Johan W. Mouton, David L. Paterson, Kucers’ The Use of Antibiotics, 2017
The mechanism of resistance in T. vaginalis has been best studied among the protozoa. Athough phenotypic resistance is described for aerobic and anaerobic conditions, the mechanisms that lead to resistance under both conditions are likely to have some overlap (Leitsch et al., 2014a). In general, in anaerobic resistance, in which the presence of oxygen is not a prerequisite for this type of resistance, there is a usually a defect in the activation of metronidazole. This may be mediated by a reduction in activity of the PFOR pathway, occurring in the hydrogenosome, a mitochondrion-derived organelle (Kulda et al., 1993; Kulda, 1999; Rasoloson et al., 2002). Reduced metronidazole activation may also occur as a result of reduced activity of thioredoxin reductase (Leitsch et al., 2009). Recently, reduced flavin reductase 1 activity resulting in elevated intracellular oxygen levels and futile cycling of metronidazole have been demonstrated in metronidazole-resistant T. vaginalis (Leitsch et al., 2012a; Leitsch et al., 2014a). Another mechanism of resistance may be related to stop codons in nitroreductase genes (Paulish-Miller et al., 2014).
Host cell protein profiling of commercial therapeutic protein drugs as a benchmark for monoclonal antibody-based therapeutic protein development
Published in mAbs, 2021
Rosalynn Molden, Mengqi Hu, Sook Yen E, Diana Saggese, James Reilly, John Mattila, Haibo Qiu, Gang Chen, Hanne Bak, Ning Li
Some of the HCP were identified less frequently, either only present in specific drugs or were identified at much higher abundance only in specific ones compared to others. Examples of this include 40 S ribosomal protein S20, flavin reductase and Protein S100-A11, which were identified at higher abundance in specific drugs compared to the rest (Figure 2). In addition, some drugs had specific HCP species that contributed more to the total HCP contents than any other identified protein. Nine of the 29 drugs had one dominant HCP contributing to over 50% of the total HCP amount whereas the remaining products had the levels of individual HCP distributed more evenly (Figure 3), indicating that these dominant HCP are likely to bind with a higher affinity to specific antibody sequences. Other studies have shown that HCP that persist in the final drug product are often “hitch-hiker” proteins.34 These HCP likely associate to the antibody molecule through nonspecific ionic and/or hydrophobic interactions, and as a result are carried through the purification process.31,35 A known example of this is Phospholipase B-like protein (PLBL2), which has been reported in publications to bind to IgG4 molecules and can be enriched in the first affinity purification step.36 In fact, PLBL2 was identified as the dominant HCP species in two drugs, drug 9 and drug 15 that we profiled (Figure 3).
In vivo evaluation of electron mediators for the reduction of methemoglobin encapsulated in liposomes using electron energies produced by red blood cell glycolysis
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Semhar Ghirmai, Leif Bülow, Hiromi Sakai
In red blood cells (RBCs), ferric metHb is mainly reduced by NADH-cytochrome b5 reductase via cytochrome b5, NADPH metHb reductase and NADPH-flavin reductase. According to a simulation study of metHb reduction, the NADPH-flavin pathway is used under normal physiological conditions where the oxidative stress is low and metHb levels are low. Conversely, the NADH-cytochrome b5 pathway plays a major role when oxidative stress is high [11]. These electron-energy-rich molecules are re-energized repeatedly during the glycolysis of RBCs. Glucose, the main energy source for the cells, is metabolized through glycolysis and the hexose monophosphate shunt (HMP), also known as the pentose phosphate pathway [12,13]. In the presence of metHb, the electron-energy-rich molecule NADH produced in the Embden–Meyerhof pathway can be a resource to reduce metHb by NADP-cytochrome b5 to its functional form. The HMP shunt, the only source for NADPH, is generated by reduction of NADP+ [11,14].