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Cytochrome P450 2U1 (CYP2U1) identified from the human genome remains poorly known since few data are presently available on its physiological function(s) and substrate(s) specificity. CYP2U1 mutations are associated with complicated forms of hereditary spastic paraplegia, alterations of mitochondrial architecture and bioenergetics. In order to better know the biological roles of CYP2U1, we used a bioinformatics approach. The analysis of the data invited us to focus on leukotriene B[sub.4] (LTB[sub.4]), an important inflammatory mediator. Here, we show that CYP2U1 efficiently catalyzes the hydroxylation of LTB[sub.4] predominantly on its ω-position. We also report docking experiments of LTB[sub.4] in a 3D model of truncated CYP2U1 that are in agreement with this hydroxylation regioselectivity. The involvement of CYP2U1 in the metabolism of LTB[sub.4] could have strong physiological consequences in cerebral pathologies including ischemic stroke because CYP2U1 is predominantly expressed in the brain.

The steroid superfamily includes a wide range of compounds that are essential for living organisms of the animal and plant kingdoms. Structural modifications of steroids highly affect their biological activity. In this review, we focus on hydroxylation of steroids by bacterial hydroxylases, which take part in steroid catabolic pathways and play an important role in steroid degradation. We compare three distinct classes of metalloenzymes responsible for aerobic or anaerobic hydroxylation of steroids, namely: cytochrome P450, Rieske-type monooxygenase 3-ketosteroid 9α-hydroxylase, and molybdenum-containing steroid C25 dehydrogenases. We analyze the available literature data on reactivity, regioselectivity, and potential application of these enzymes in organic synthesis of hydroxysteroids. Moreover, we describe mechanistic hypotheses proposed for all three classes of enzymes along with experimental and theoretical evidences, which have provided grounds for their formulation. In case of the 3-ketosteroid 9α-hydroxylase, such a mechanistic hypothesis is formulated for the first time in the literature based on studies conducted for other Rieske monooxygenases. Finally, we provide comparative analysis of similarities and differences in the reaction mechanisms utilized by bacterial steroid hydroxylases.

The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile 1 , promote hepatocellular carcinoma 2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. 3 ). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids 4 ; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A–B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe–S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes , conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool. The biosynthetic pathway that produces the secondary bile acids DCA and LCA in human gut microbes has been fully characterized, engineered into another bacterial host, and used to confer DCA production in germ-free mice—an important proof-of-principle for the engineering of gut microbial pathways.

Under conditions of hypoxia, cancer cells with hypoxia inducible factor-1[alpha] (HIF-1[alpha]) from heterogeneous tumor cells show greater aggression and progression in an effort to compensate for harsh environmental conditions. Extensive study on the stability of HIF-1[alpha] under conditions of acute hypoxia in cancer progression has been conducted, however, understanding of its involvement during the chronic phase is limited. In this study, we investigated the effect of SIRT1 on HIF1 stability in a typical chronic hypoxic conditon that maintains cells for 24 h under hypoxia using Western blotting, co-IP, measurement of intracellular NAD + and NADH levels, semi-quantitative RT-PCR analysis, invasion assay, gene knockdown. Here we demonstrated that the high concentration of pyruvate in the medium, which can be easily overlooked, has an effect on the stability of HIF-1[alpha]. We also demonstrated that NADH functions as a signal for conveyance of HIF-1[alpha] degradation via the SIRT1 and VHL signaling pathway under conditions of chronic hypoxia, which in turn leads to attenuation of hypoxically strengthened invasion and angiogenic activities. A steep increase in the level of NADH occurs during chronic hypoxia, leading to upregulation of acetylation and degradation of HIF-1[alpha] via inactivation of SIRT1. Of particular interest, p300-mediated acetylation at lysine 709 of HIF-1[alpha] is recogonized by VHL, which leads to degradation of HIF-1[alpha] via ubiquitin/proteasome machinary under conditions of chronic hypoxia. In addition, we demonstrated that NADH-elevation-induced acetylation and subsequent degradation of HIF-1[alpha] was independent of proline hydroxylation. Our findings suggest a critical role of SIRT1 as a metabolic sensor in coordination of hypoxic status via regulation of HIF-1[alpha] stability. These results also demonstrate the involvement of VHL in degradation of HIF-1[alpha] through recognition of PHD-mediated hydroxylation in normoxia and p300-mediated HIF-1[alpha] acetylation in hypoxia.

The direct hydroxylation of benzene is a green and economical-efficient alternative to the existing cumene process for phenol production. However, the undesired phenol selectivity at high benzene conversion hinders its wide application. Here, we develop a one-pot synthesis of protonated g-C[sub.3]N[sub.4] supporting vanadia catalysts (V-pg-C[sub.3]N[sub.4]) for the efficient and selective hydroxylation of benzene. Characterizations suggest that protonating g-C[sub.3]N[sub.4] in diluted HCl can boost the generation of amino groups (NH/NH[sub.2]) without changing the bulk structure. The content of surface amino groups, which determines the dispersion of vanadia, can be easily regulated by the amount of HCl added in the preparation. Increasing the content of surface amino groups benefits the dispersion of vanadia, which eventually leads to improved H[sub.2]O[sub.2] activation and benzene hydroxylation. The optimal catalyst, V-pg-C[sub.3]N[sub.4]-0.46, achieves 60% benzene conversion and 99.7% phenol selectivity at 60 [sup.o]C with H[sub.2]O[sub.2] as the oxidant.

The extracellular matrix is a major component of the local environment—that is, the niche—that determines cell behaviour 1 . During metastatic growth, cancer cells shape the extracellular matrix of the metastatic niche by hydroxylating collagen to promote their own metastatic growth 2 , 3 . However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen, because nutrients dictate which enzymatic reactions are active in cancer cells 4 , 5 . Here we show that breast cancer cells rely on the nutrient pyruvate to drive collagen-based remodelling of the extracellular matrix in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activates collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast-cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodelling and the nutrient environment in the metastatic niche. Exogenous pyruvate is needed for breast cancer cells to form metastases, and the inhibition of pyruvate metabolism impairs collagen hydroxylation and the growth of lung metastases in different mouse models.

Plastic contamination currently threatens a wide variety of ecosystems and presents damaging repercussions and negative consequences for many wildlife species. Sustainable plastic waste management is an important approach to environmental protection and a necessity in the current life cycle of plastics in nature. Plastic biodegradation by microorganisms is a notable possible solution. This opinion article includes a proposal to use hypothetical P450 enzymes with an engineered active site as potent trigger biocatalysts to biodegrade polyethylene (PE) via in-chain hydroxylation into smaller products of linear aliphatic alcohols and alkanoic acids based on cascade enzymatic reactions. Furthermore, we propose the adoption of P450 into plastic-eating synthetic bacteria for PE biodegradation. This strategy can be applicable to other dense plastics, such as polypropylene (PP) and polystyrene (PS). Plastic pollution causes harmful environment effects, but the development of biocatalysts can solve them.Cascading enzymatic systems that are started via in-chain hydroxylation and catalyzed by a hypothetical P450 enzyme with an engineered active site can depolymerize polyethylene into small degradation molecules.Synthetic artificial bacteria are emerging interdisciplinary technologies that can solve plastic issues by applying degrading enzymes that contribute to the breakdown of plastics.This biodegradation enzyme cascade system will require directed evolution and rational engineering to enhance its degradation activity.

Arabinogalactan-proteins (AGPs) are a large, complex, and highly diverse class of heavily glycosylated proteins that belong to the family of cell wall hydroxyproline-rich glycoproteins. Approximately 90% of the molecules consist of arabinogalactan polysaccharides, which are composed of arabinose and galactose as major sugars and minor sugars such as glucuronic acid, fucose, and rhamnose. About half of the AGP family members contain a glycosylphosphatidylinositol (GPI) lipid anchor, which allows for an association with the outer leaflet of the plasma membrane. The mysterious AGP family has captivated the attention of plant biologists for several decades. This diverse family of glycoproteins is widely distributed in the plant kingdom, including many algae, where they play fundamental roles in growth and development processes. The journey of AGP biosynthesis begins with the assembly of amino acids into peptide chains of proteins. An N-terminal signal peptide directs AGPs toward the endoplasmic reticulum, where proline hydroxylation occurs and a GPI anchor may be added. GPI-anchored AGPs, as well as unanchored AGPs, are then transferred to the Golgi apparatus, where extensive glycosylation occurs by the action of a variety glycosyltransferase enzymes. Following glycosylation, AGPs are transported by secretory vesicles to the cell wall or to the extracellular face of the plasma membrane (in the case of GPI-anchored AGPs). GPI-anchored proteins can be released from the plasma membrane into the cell wall by phospholipases. In this review, we present an overview of the accumulated knowledge on AGP biosynthesis over the past three decades. Particular emphasis is placed on the glycosylation of AGPs as the sugar moiety is essential to their function. Recent genetics and genomics approaches have significantly contributed to a broader knowledge of AGP biosynthesis. However, many questions remain to be elucidated in the decades ahead.

Pipecolic acid (Pip) and its derivative hydroxypipecolic acids, such as (2S,3R)-3-hydroxypipecolic acid (cis-3-L-HyPip), are components of many natural and synthetic bioactive molecules. Fe(II)/α-ketoglutaric acid (Fe(II)/2-OG)-dependent dioxygenases can catalyze the hydroxylation of pipecolic acid. However, the available enzymes with desired activity and selectivity are limited. Herein, we compare the possible candidates in the Fe(II)/2-OG-dependent dioxygenase family, and cis-P3H is selected for potentially catalyzing selective hydroxylation of L-Pip. cis-P3H was further engineered to increase its catalytic efficiency toward L-Pip. By analyzing the structural confirmation and residue composition in substrate-binding pocket, a “handlebar” mode of molecular interactions is proposed. Using molecular docking, virtual mutation analysis, and dynamic simulations, R97, E112, L57, and G282 were identified as the key residues for subsequent site-directed saturation mutagenesis of cis-P3H. Consequently, the variant R97M showed an increased catalytic efficiency toward L-Pip. In this study, the k[sub.cat]/K[sub.m] value of the positive mutant R97M was about 1.83-fold that of the wild type. The mutation R97M would break the salt bridge between R97 and L-Pip and weaken the positive-positive interaction between R97 and R95. Therefore, the force on the amino and carboxyl groups of L-Pip was lightly balanced, allowing the molecule to be stabilized in the active pocket. These results provide a potential way of improving cis-P3H catalytic activity through rational protein engineering.

trans-4-Hydroxy-l-proline is highly abundant in collagen (accounting for about one-third of body proteins in humans and other animals). This imino acid (loosely called amino acid) and its minor analogue trans-3-hydroxy-l-proline in their ratio of approximately 100:1 are formed from the post-translational hydroxylation of proteins (primarily collagen and, to a much lesser extent, non-collagen proteins). Besides their structural and physiological significance in the connective tissue, both trans-4-hydroxy-l-proline and trans-3-hydroxy-l-proline can scavenge reactive oxygen species and have both structural and physiological significance in animals. The formation of trans-4-hydroxy-l-proline residues in protein kinases B and DYRK1A, eukaryotic elongation factor 2 activity, and hypoxia-inducible transcription factor plays an important role in regulating their phosphorylation and catalytic activation as well as cell signaling in animal cells. These biochemical events contribute to the modulation of cell metabolism, growth, development, responses to nutritional and physiological changes (e.g., dietary protein intake and hypoxia), and survival. Milk, meat, skin hydrolysates, and blood, as well as whole-body collagen degradation provide a large amount of trans-4-hydroxy-l-proline. In animals, most (nearly 90%) of the collagen-derived trans-4-hydroxy-l-proline is catabolized to glycine via the trans-4-hydroxy-l-proline oxidase pathway, and trans-3-hydroxy-l-proline is degraded via the trans-3-hydroxy-l-proline dehydratase pathway to ornithine and glutamate, thereby conserving dietary and endogenously synthesized proline and arginine. Supplementing trans-4-hydroxy-l-proline or its small peptides to plant-based diets can alleviate oxidative stress, while increasing collagen synthesis and accretion in the body. New knowledge of hydroxyproline biochemistry and nutrition aids in improving the growth, health and well-being of humans and other animals.