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Chiral methyl mandelates are useful synthons in organic transformation and pharmaceutical synthesis. Green synthesis of these valuable compounds by direct C–H activating oxidative hydroxylation has attracted keen interest. Described herein is achieving the stereoselective and efficient bio-hydroxylation of methyl 2-phenylacetates to the chiral methyl mandelates by directed evolution of the cytochrome P450DA hydroxylase. In the present study, a new colorimetric high-throughput screening assay was successfully developed based on a dualenzyme cascade for the engineering of the P450DA's hydroxylation activity. Several beneficial variants with enhanced bio-hydroxylation activity were created by combining random mutagenesis and site-saturated/directed mutagenesis strategies. Whole-cell bio-hydroxylation of various methyl 2-phenylacetates using the best septupletmutant P450DA-11 yielded the corresponding chiral methyl mandelates in up to 92% isolated yields and >99% ee. [ABSTRACT FROM AUTHOR]

As compounds of natural origin enter human body, it is necessary to investigate their possible interactions with the metabolism of drugs and xenobiotics in general, namely with the cytochrome P450 (CYP) system. Phytic acid (myo-inositol hexaphosphoric acid, IP6) is mainly present in plants but is also an endogenous compound present in mammalian cells and tissues. It has been shown to exhibit protective effect in many pathological conditions. For this paper, its interaction with CYPs was studied using human liver microsomes, primary human hepatocytes, the HepG2 cell line, and molecular docking. Docking experiments and absorption spectra demonstrated the weak ability of IP6 to interact in the heme active site of CYP1A. Molecular docking suggested that IP6 preferentially binds to the protein surface, whereas binding to the active site of CYP1A2 was found to be less probable. Subsequently, we investigated the ability of IP6 to modulate the metabolism of xenobiotics for both the mRNA expression and enzymatic activity of CYP1A enzymes. Our findings revealed that IP6 can slightly modulate the mRNA levels and enzyme activity of CYP1A. However, thanks to the relatively weak interactions of IP6 with CYPs, the chances of the mechanisms of clinically important drug–drug interactions involving IP6 are low. [ABSTRACT FROM AUTHOR]

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14.1; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation processes and how this may help to regulate their functions in health and disease. [ABSTRACT FROM AUTHOR]

The fossil record demonstrates the preservation of porphyrins (e.g., heme) in organic sediments and the fossilized remains of animals. These molecules are essential components in modern metabolic processes, such as electron transport (cytochromes) and oxygen transport (hemoglobin), and likely originated before the emergence of life. The integration and adaptation of porphyrins and structurally similar molecules (e.g., chlorophylls) are key aspects in the evolution of energy production (i.e., aerobic respiration and photosynthesis) and complex life (i.e., eukaryotes and multicellularity). Here, we discuss the evolution and functional diversity of heme-bound hemoglobin proteins in vertebrates, along with the preservation of these molecules in the fossil record. By elucidating the pivotal role of these molecules in the evolution of life, this review lays the groundwork necessary to explore hemoglobin as a means to investigate the paleobiology of extinct taxa, including non-avian dinosaurs. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup. [ABSTRACT FROM AUTHOR]

Silver (Ag) is a pivotal transition metal with applications in multiple industries, necessitating efficient recovery techniques. Despite various proposed methods for silver recovery from wastewaters, challenges persist especially for low concentrations. In this context, bioreduction by bacteria like Geobacter sulfurreducens , offers a promising approach by converting Ag(I) to Ag nanoparticles. To reveal the mechanisms driving microbial Ag(I) reduction, we conducted transcriptional profiling of G. sulfurreducens under Ag(I)-reducing condition. Integrated transcriptomic and protein-protein interaction network analyses identified significant transcriptional shifts, predominantly linked to c -type cytochromes, NADH, and pili. When compared to a pilus-deficient strain, the wild-type strain exhibited distinct cytochrome gene expressions, implying specialized functional roles. Additionally, despite a down-regulation in NADH dehydrogenase genes, we observed up-regulation of specific downstream cytochrome genes, highlighting NADH's potential role as an electron donor in the Ag(I) reduction process. Intriguingly, our findings also highlight the significant influence of pili on the morphology of the resulting Ag nanoparticles. The presence of pili led to the formation of smaller and more crystallized Ag nanoparticles. Overall, our findings underscore the intricate interplay of cytochromes, NADH, and pili in Ag(I) reduction. Such insights suggest potential strategies for further enhancing microbial Ag(I) reduction. [Display omitted] • Transcriptional profiling of G. sulfurreducens under Ag(I)-reducing condition was conducted. • Cytochromes, NADH, and pili were involved in Ag(I) reduction by G. sulfurreducens. • PCA and PCA-Δ pilA showed distinct cytochrome expression, implying specialized roles. • Pili contributed to the formation of small and well-crystallized Ag-NPs. [ABSTRACT FROM AUTHOR]

The fastest-growing microbe Vibrio natriegens is an excellent platform for bioproduction processes. Until now, this marine bacterium has not been examined for bioremediation applications, where the production of substantial amounts of biomass would be beneficial. V. natriegens can perform extracellular electron transfer (EET) to Fe(III) via a single porin-cytochrome circuit conserved in Vibrionaceae. Electroactive microbes capable of EET to Fe(III) usually also reduce toxic metals such as carcinogenic Cr(VI), which is converted to Cr(III), thus decreasing its toxicity and mobility. Here, the performance of V. natriegens was explored for the bioremediation of Cr(VI). At a density of 100 mg/mL, V. natriegens removed 5–20 mg/L Cr(VI) within 30 s and 100 mg/L Cr(VI) within 10 min. In comparison, the model bacterium Escherichia coli grown to a comparable cell density removed Cr(VI) 36 times slower. To eliminate Cr(VI), V. natriegens had to be metabolically active, and functional outer-membrane c -type cytochromes were required. At the end of the Cr(VI) removal process, V. natriegens had reduced all of it into Cr(III) while adsorbing more than half of the metallic ions. These results demonstrate that V. natriegens , with its fast metabolism, is a viable option for the rapid treatment of aqueous pollution with Cr. [Display omitted] • The fast-growing microbe Vibrio natriegens can reduce and adsorb toxic chromium. • High-density V. natriegens removed 5–20 mg/L Cr(VI) within 30 s. • V. natriegens at the same density also removed 100 mg/L Cr(VI) within minutes. • Cr(VI) was reduced extracellularly and Cr(III) was adsorbed by the microbes. • A porin-cytochrome complex is likely involved in Cr(VI) conversion. [ABSTRACT FROM AUTHOR]