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Flavin transferase: the maturation factor of flavin-containing oxidoreductases.
Bogachev, AV, Baykov, AA, Bertsova, YV
Biochemical Society transactions. 2018;(5):1161-1169
Abstract
Flavins, cofactors of many enzymes, are often covalently linked to these enzymes; for instance, flavin adenine mononucleotide (FMN) can form a covalent bond through either its phosphate or isoalloxazine group. The prevailing view had long been that all types of covalent attachment of flavins occur as autocatalytic reactions; however, in 2013, the first flavin transferase was identified, which catalyzes phosphoester bond formation between FMN and Na+-translocating NADHquinone oxidoreductase in certain bacteria. Later studies have indicated that this post-translational modification is widespread in prokaryotes and is even found in some eukaryotes. Flavin transferase can occur as a separate ∼40 kDa protein or as a domain within the target protein and recognizes a degenerate DgxtsAT/S motif in various target proteins. The purpose of this review was to summarize the progress already achieved by studies of the structure, mechanism, and specificity of flavin transferase and to encourage future research on this topic. Interestingly, the flavin transferase gene (apbE) is found in many bacteria that have no known target protein, suggesting the presence of yet unknown flavinylation targets.
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Phosphorus concentration coordinates a respiratory bypass, synthesis and exudation of citrate, and the expression of high-affinity phosphorus transporters in Solanum lycopersicum.
Del-Saz, NF, Romero-Munar, A, Cawthray, GR, Palma, F, Aroca, R, Baraza, E, Florez-Sarasa, I, Lambers, H, Ribas-Carbó, M
Plant, cell & environment. 2018;(4):865-875
Abstract
Plants exhibit respiratory bypasses (e.g., the alternative oxidase [AOX]) and increase the synthesis of carboxylates in their organs (leaves and roots) in response to phosphorus (P) deficiency, which increases P uptake capacity. They also show differential expression of high-affinity inorganic phosphorus (Pi) transporters, thus avoiding P toxicity at a high P availability. The association between AOX and carboxylate synthesis was tested in Solanum lycopersicum plants grown at different soil P availability, by using plants grown under P-sufficient and P-limiting conditions and by applying a short-term (24 hr) P-sufficient pulse to plants grown under P limitation. Tests were also performed with plants colonized with arbuscular mycorrhizal fungi, which increased plant P concentration under reduced P availability. The in vivo activities of AOX and cytochrome oxidase were measured together with the concentration of carboxylates and the P concentration in plant organs. Gene transcription of Pi transporters (LePT1 and LePT2) was also studied. A coordinated response between plant P concentration with these traits was observed, indicating that a sufficient P availability in soil led to a suppression of both AOX activity and synthesis of citrate and a downregulation of the transcription of genes encoding high-affinity Pi transporters, presumably to avoid P toxicity.
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Mechanisms for enzymatic reduction of nitric oxide to nitrous oxide - A comparison between nitric oxide reductase and cytochrome c oxidase.
Blomberg, MRA, Ädelroth, P
Biochimica et biophysica acta. Bioenergetics. 2018;(11):1223-1234
Abstract
Cytochrome c oxidases (CcO) reduce O2 to H2O in the respiratory chain of mitochondria and many aerobic bacteria. In addition, some species of CcO can also reduce NO to N2O and water while others cannot. Here, the mechanism for NO-reduction in CcO is investigated using quantum mechanical calculations. Comparison is made to the corresponding reaction in a "true" cytochrome c-dependent NO reductase (cNOR). The calculations show that in cNOR, where the reduction potentials are low, the toxic NO molecules are rapidly reduced, while the higher reduction potentials in CcO lead to a slower or even impossible reaction, consistent with experimental observations. In both enzymes the reaction is initiated by addition of two NO molecules to the reduced active site, forming a hyponitrite intermediate. In cNOR, N2O can then be formed using only the active-site electrons. In contrast, in CcO, one proton-coupled reduction step most likely has to occur before N2O can be formed, and furthermore, proton transfer is most likely rate-limiting. This can explain why different CcO species with the same heme a3-Cu active site differ with respect to NO reduction efficiency, since they have a varying number and/or properties of proton channels. Finally, the calculations also indicate that a conserved active site valine plays a role in reducing the rate of NO reduction in CcO.
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Nitrogen-fixation activity and the abundance and taxonomy of nifH genes in agricultural, pristine, and urban prairie stream sediments chronically exposed to different levels of nitrogen loading.
Caton, IR, Caton, TM, Schneegurt, MA
Archives of microbiology. 2018;(4):623-633
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Abstract
Small streams exert great influences on the retention and attenuation of nitrogen (N) within stream networks. Human land use can lead to increased transport of dissolved inorganic N compounds and downstream eutrophication. Microbial activity in streams is important for maintaining an actively functioning N cycle. Chronically high N loading in streams affects the rates of the central processes of the N cycle by increasing rates of nitrification and denitrification, with biota exhibiting decreased efficiency of N use. The LINXII project measured N-cycle parameters in small streams using 15NO3- tracer release experiments. We concurrently measured N2 fixation rates in six streams of three types (agricultural, pristine, and urban prairie streams) as part of this broader study of major N-cycle processes. Nitrogen fixation in streams was significantly negatively correlated with nitrate levels, dissolved inorganic N levels, and denitrification rates. Algal mat and leaf litter samples generally exhibited the highest rates of N2 fixation. The abundance of nifH genes, as measured by real-time PCR, was marginally correlated with N2-fixation rates, but not to other N-cycle processes or stream characteristics. The nifH sequences observed were assigned to cyanobacteria, Deltaproteobacteria, Methylococcus, and Rhizobia. Seasonal changes, disturbances, and varying inputs may encourage a diverse, flexible, stable N2-fixing guild. Patchiness in the streams should be considered when assessing the overall impact of N2 fixation, since algal biomass exhibited high rates of N2 fixation.
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Photoirradiation Generates an Ultrastable 8-Formyl FAD Semiquinone Radical with Unusual Properties in Formate Oxidase.
Robbins, JM, Geng, J, Barry, BA, Gadda, G, Bommarius, AS
Biochemistry. 2018;(40):5818-5826
Abstract
Formate oxidase (FOX) was previously shown to contain a noncovalently bound 8-formyl FAD (8-fFAD) cofactor. However, both the absorption spectra and the kinetic parameters previously reported for FOX are inconsistent with more recent reports. The ultraviolet-visible (UV-vis) absorption spectrum reported in early studies closely resembles the spectra observed for protein-bound 8-formyl flavin semiquinone species, thus suggesting FOX may be photosensitive. Therefore, the properties of dark and light-exposed FOX were investigated using steady-state kinetics and site-directed mutagenesis analysis along with inductively coupled plasma optical emission spectroscopy, UV-vis absorption spectroscopy, circular dichroism spectroscopy, liquid chromatography and mass spectrometry, and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, these experimental results demonstrate that FOX is deactivated in the presence of light through generation of an oxygen stable, anionic (red) 8-fFAD semiquinone radical capable of persisting either in an aerobic environment for multiple weeks or in the presence of a strong reducing agent like sodium dithionite. Herein, we study the photoinduced formation of the 8-fFAD semiquinone radical in FOX and report the first EPR spectrum of this radical species. The stability of the 8-fFAD semiquinone radical suggests FOX to be a model enzyme for probing the structural and mechanistic features involved in stabilizing flavin semiquinone radicals. It is likely that the photoinduced formation of a stable 8-fFAD semiquinone radical is a defining characteristic of 8-formyl flavin-dependent enzymes. Additionally, a better understanding of the radical stabilization process may yield a FOX enzyme with more robust activity and broader industrial usefulness.
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6.
Halogenase engineering and its utility in medicinal chemistry.
Fraley, AE, Sherman, DH
Bioorganic & medicinal chemistry letters. 2018;(11):1992-1999
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Abstract
Halogenation is commonly used in medicinal chemistry to improve the potency of pharmaceutical leads. While synthetic methods for halogenation present selectivity and reactivity challenges, halogenases have evolved over time to perform selective reactions under benign conditions. The optimization of halogenation biocatalysts has utilized enzyme evolution and structure-based engineering alongside biotransformation in a variety of systems to generate stable site-selective variants. The recent improvements in halogenase-catalyzed reactions has demonstrated the utility of these biocatalysts for industrial purposes, and their ability to achieve a broad substrate scope implies a synthetic tractability with increasing relevance in medicinal chemistry.
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7.
Pyrroloquinoline quinone-dependent dehydrogenases of acetic acid bacteria.
Matsutani, M, Yakushi, T
Applied microbiology and biotechnology. 2018;(22):9531-9540
Abstract
Pyrroloquinoline quinone (PQQ)-dependent dehydrogenases (quinoproteins) of acetic acid bacteria (AAB), such as the membrane-bound alcohol dehydrogenase (ADH) and the membrane-bound glucose dehydrogenase, contain PQQ as the prosthetic group. Most of them are located on the periplasmic surface of the cytoplasmic membrane, and function as primary dehydrogenases in cognate substance-oxidizing respiratory chains. Here, we have provided an overview on the function and molecular architecture of AAB quinoproteins, which can be categorized into six groups according to the primary amino acid sequences. Based on the genomic data, we discuss the types of quinoproteins found in AAB genome and how they are distributed. Our analyses indicate that a significant number of uncharacterized orphan quinoproteins are present in AAB. By reviewing recent experimental developments, we discuss how to characterize the as-yet-unknown enzymes. Moreover, our bioinformatics studies also provide insights on how quinoproteins have developed into intricate enzymes. ADH comprises at least two subunits: the quinoprotein dehydrogenase subunit encoded by adhA and the cytochrome subunit encoded by adhB, and the genes are located in a polycistronic transcriptional unit. Findings on stand-alone derivatives of adhA encourage us to speculate on a possible route for ADH development in the evolutional history of AAB. A combination of bioinformatics studies on big genome sequencing data and wet studies assisted with genetic engineering would unravel biochemical functions and physiological role of uncharacterized quinoproteins in AAB, or even in unculturable metagenome.
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Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in β-lactam biosynthesis.
Rabe, P, Kamps, JJAG, Schofield, CJ, Lohans, CT
Natural product reports. 2018;(8):735-756
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Abstract
Covering: up to 2017 2-Oxoglutarate (2OG) dependent oxygenases and the homologous oxidase isopenicillin N synthase (IPNS) play crucial roles in the biosynthesis of β-lactam ring containing natural products. IPNS catalyses formation of the bicyclic penicillin nucleus from a tripeptide. 2OG oxygenases catalyse reactions that diversify the chemistry of β-lactams formed by both IPNS and non-oxidative enzymes. Reactions catalysed by the 2OG oxygenases of β-lactam biosynthesis not only involve their typical hydroxylation reactions, but also desaturation, epimerisation, rearrangement, and ring-forming reactions. Some of the enzymes involved in β-lactam biosynthesis exhibit remarkable substrate and product selectivities. We review the roles of 2OG oxygenases and IPNS in β-lactam biosynthesis, highlighting opportunities for application of knowledge of their roles, structures, and mechanisms.
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Oxidoreductases and Reactive Oxygen Species in Conversion of Lignocellulosic Biomass.
Bissaro, B, Várnai, A, Røhr, ÅK, Eijsink, VGH
Microbiology and molecular biology reviews : MMBR. 2018;(4)
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Abstract
Biomass constitutes an appealing alternative to fossil resources for the production of materials and energy. The abundance and attractiveness of vegetal biomass come along with challenges pertaining to the intricacy of its structure, evolved during billions of years to face and resist abiotic and biotic attacks. To achieve the daunting goal of plant cell wall decomposition, microorganisms have developed many (enzymatic) strategies, from which we seek inspiration to develop biotechnological processes. A major breakthrough in the field has been the discovery of enzymes today known as lytic polysaccharide monooxygenases (LPMOs), which, by catalyzing the oxidative cleavage of recalcitrant polysaccharides, allow canonical hydrolytic enzymes to depolymerize the biomass more efficiently. Very recently, it has been shown that LPMOs are not classical monooxygenases in that they can also use hydrogen peroxide (H2O2) as an oxidant. This discovery calls for a revision of our understanding of how lignocellulolytic enzymes are connected since H2O2 is produced and used by several of them. The first part of this review is dedicated to the LPMO paradigm, describing knowns, unknowns, and uncertainties. We then present different lignocellulolytic redox systems, enzymatic or not, that depend on fluxes of reactive oxygen species (ROS). Based on an assessment of these putatively interconnected systems, we suggest that fine-tuning of H2O2 levels and proximity between sites of H2O2 production and consumption are important for fungal biomass conversion. In the last part of this review, we discuss how our evolving understanding of redox processes involved in biomass depolymerization may translate into industrial applications.
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Biocatalytic reduction of activated CC-bonds and beyond: emerging trends.
Winkler, CK, Faber, K, Hall, M
Current opinion in chemical biology. 2018;:97-105
Abstract
The biocatalytic reduction of activated CC-bonds is dominated by ene-reductases from the Old Yellow Enzyme family, which gained broad practical use owing to exquisite stereoselectivity combined with wide substrate scope. Protein diversity is fostered by mining distinct protein classes and by implementing protein engineering techniques. Recent efforts are focusing on expanding the chemical complexity of the product portfolio, either through substrate functionalization or design of multi-step reactions. This review also highlights unusual chemistries catalyzed by ene-reductases and presents emerging methodologies developed to bypass the need of natural nicotinamide cofactors.