Riboflavin (vitamin B2) serves as the precursor for FMN and FAD in almost all organisms that utilize the redox‐active isoalloxazine ring system as a coenzyme in enzymatic reactions. The role of ...flavin, however, is not limited to redox processes, as ∼ 10% of flavin‐dependent enzymes catalyze nonredox reactions. Moreover, the flavin cofactor is also widely used as a signaling and sensing molecule in biological processes such as phototropism and nitrogen fixation. Here, we present a study of 374 flavin‐dependent proteins analyzed with regard to their function, structure and distribution among 22 archaeal, eubacterial, protozoan and eukaryotic genomes. More than 90% of flavin‐dependent enzymes are oxidoreductases, and the remaining enzymes are classified as transferases (4.3%), lyases (2.9%), isomerases (1.4%) and ligases (0.4%). The majority of enzymes utilize FAD (75%) rather than FMN (25%), and bind the cofactor noncovalently (90%). High‐resolution structures are available for about half of the flavoproteins. FAD‐containing proteins predominantly bind the cofactor in a Rossmann fold (∼ 50%), whereas FMN‐containing proteins preferably adopt a (βα)8‐(TIM)‐barrel‐like or flavodoxin‐like fold. The number of genes encoding flavin‐dependent proteins varies greatly in the genomes analyzed, and covers a range from ∼ 0.1% to 3.5% of the predicted genes. It appears that some species depend heavily on flavin‐dependent oxidoreductases for degradation or biosynthesis, whereas others have minimized their flavoprotein arsenal. An understanding of ‘flavin‐intensive’ lifestyles, such as in the human pathogen Mycobacterium tuberculosis, may result in valuable new intervention strategies that target either riboflavin biosynthesis or uptake.
Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) serves as a powerful redox‐active cofactor for enzymatic reactions in all kingdoms of life. As expected, the lion’s share of flavin‐dependent enzymes catalyze oxidoreductions. However, ∼ 10% are involved as transferases, lyases, isomerases or ligase in essential biosynthetic pathways or play a critical role in sensing, metabolic regulation or DNA‐repair.
Abstract 12-oxophytodienoate reductase 3 (OPR3) is a key enzyme in the biosynthesis of jasmonoyl- L -isoleucine, the receptor-active form of jasmonic acid and crucial signaling molecule in plant ...defense. OPR3 was initially crystallized as a self-inhibitory dimer, implying that homodimerization regulates enzymatic activity in response to biotic and abiotic stresses. Since a sulfate ion is bound to Y364, mimicking a phosphorylated tyrosine, it was suggested that dimer formation might be controlled by reversible phosphorylation of Y364 in vivo. To investigate OPR3 homodimerization and its potential physiological role in more detail, we performed analytical gel filtration and dynamic light scattering on wild-type OPR3 and three variants (R283D, R283E, and Y364P). The experiments revealed a rapid and highly sensitive monomer–dimer equilibrium for all OPR3 constructs. We crystallized all constructs with and without sulfate to examine its effect on the dimerization process and whether reversible phosphorylation of Y364 triggers homodimerization in vivo. All OPR3 constructs crystallized in their monomeric and dimeric forms independent of the presence of sulfate. Even variant Y364P, lacking the putative phosphorylation site, was crystallized as a self-inhibitory homodimer, indicating that Y364 is not required for dimerization. Generally, the homodimer is relatively weak, and our results raise doubts about its physiological role in regulating jasmonate biosynthesis.
Bioluminescence refers to the production of light by living organisms. Bioluminescent bacteria with a variety of bioluminescence emission characteristics have been identified in Vibrionaceae, ...Shewanellaceae and Enterobacteriaceae. Bioluminescent bacteria are mainly found in marine habitats and they are either free-floating, sessile or have specialized to live in symbiosis with other marine organisms. On the molecular level, bioluminescence is enabled by a cascade of chemical reactions catalyzed by enzymes encoded by the lux operon with the gene order luxCDABEG. The luxA and luxB genes encode the α- and β- subunits, respectively, of the enzyme luciferase producing the light emitting species. LuxC, luxD and luxE constitute the fatty acid reductase complex, responsible for the synthesis of the long-chain aldehyde substrate and luxG encodes a flavin reductase. In bacteria, the heterodimeric luciferase catalyzes the monooxygenation of long-chain aliphatic aldehydes to the corresponding acids utilizing reduced FMN and molecular oxygen. The energy released as a photon results from an excited state flavin-4a-hydroxide, emitting light centered around 490 nm. Advances in the mechanistic understanding of bacterial bioluminescence have been spurred by the structural characterization of protein encoded by the lux operon. However, the number of available crystal structures is limited to LuxAB (Vibrio harveyi), LuxD (Vibrio harveyi) and LuxF (Photobacterium leiognathi). Based on the crystal structure of LuxD and homology models of LuxC and LuxE, we provide a hypothetical model of the overall structure of the LuxCDE fatty acid reductase complex that is in line with biochemical observations.
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In view of the unprecedented rate of current climate change, plants are exposed to an avalanche of adverse environmental conditions that will challenge their ability to cope with abiotic and biotic ...stresses. These changes are bound to affect crop plants as well, and thus, have the potential to jeopardize food security on a global scale. Hence, it will be critical to understand the molecular defence and adaptation mechanisms that enable plants to thrive in an increasingly hostile environment. In this Subject Collection, The FEBS Journal presents a collection of reviews and original articles dealing with aspects of plant defence systems and mechanisms of pathogenicity.
In view of the unprecedented rate of current climate change, plants are exposed to an avalanche of adverse environmental conditions that challenge their ability to cope with abiotic and biotic stresses. These changes affect crop plants as well, and thus have the potential to jeopardize food security on a global scale. Hence, it is critical to understand the molecular defense and adaptation mechanisms that enable plants to thrive in an increasingly hostile environment. In this Subject Collection, The FEBS Journal presents a collection of reviews and original articles dealing with aspects of plant defense systems as well as mechanisms of pathogenicity. Image source: Shutterstock (item ID: 1263201358).
Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the ...biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family.
Berberine bridge enzyme-like proteins are a multigene family in plants.
Members of the berberine bridge enzyme-like family were identified as monolignol oxidoreductases.
Berberine bridge enzyme-like enzymes play a role in monolignol metabolism and lignin formation.
Our results indicate a novel and unexpected role of berberine bridge enzyme-like enzymes in plant biochemistry and physiology.
The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in ...its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.
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•The RNA ligase RTCB is susceptible to copper-dependent oxidative inactivation•The flavoprotein PYROXD1 uses NAD(P)+ to safeguard mammalian RTCB against oxidation•Depletion of PYROXD1 impairs pre-tRNA splicing and the unfolded protein response•Myopathy-causing genetic variants of PYROXD1 fail to preserve the activity of RTCB
How did enzymes from Earth’s ancient anaerobic history, such as the RNA ligase RTCB, adapt to modern, aerobic environments? Asanovic et al. revealed an unexpected solution: RTCB co-evolved with a dedicated oxidoreductase, PYROXD1, which is linked to myopathies in humans, and paradoxically uses the principal prooxidative cofactor of the cell, NAD(P)+, as the “private” antioxidative protector of RTCB.
Black and white are opposites as are oxidation and reduction. Performing an oxidation, for example, of a sec-alcohol and a reduction of the corresponding ketone in the same vessel without separation ...of the reagents seems to be an impossible task. Here we show that oxidative cofactor recycling of NADP+ and reductive regeneration of NADH can be performed simultaneously in the same compartment without significant interference. Regeneration cycles can be run in opposing directions beside each other enabling one-pot transformation of racemic alcohols to one enantiomer via concurrent enantioselective oxidation and asymmetric reduction employing defined alcohol dehydrogenases with opposite stereo- and cofactor-preference. Thus, by careful selection of appropriate enzymes, NADH recycling can be performed in the presence of NADP+ recycling to achieve overall, for example, deracemisation of sec-alcohols or stereoinversion representing a possible concept for a “green” equivalent to the chemical-intensive Mitsunobu inversion.
The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a ...non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.
Fungi produce a plethora of natural products exhibiting a fascinating diversity of chemical structures with an enormous potential for medical applications. Despite the importance of understanding the ...scope of natural products and their biosynthetic pathways, a systematic analysis of the involved enzymes has not been undertaken. In our previous studies, we examined the flavoprotein encoding gene pool in archaea, eubacteria, the yeast
Saccharomyces cerevisiae
,
Arabidopsis thaliana
, and
Homo sapiens
. In the present survey, we have selected the model fungus
Neurospora crassa
as a starting point to investigate the flavoproteomes in the fungal kingdom. Our analysis showed that
N. crassa
harbors 201 flavoprotein-encoding genes amounting to 2% of the total protein-encoding genome. The majority of these flavoproteins (133) could be assigned to primary metabolism, termed the “core flavoproteome”, with the remainder of flavoproteins (68) serving in, as yet unidentified, reactions. The latter group of “accessory flavoproteins” is dominated by monooxygenases, berberine bridge enzyme-like enzymes, and glucose-methanol-choline-oxidoreductases. Although the exact biochemical role of most of these enzymes remains undetermined, we propose that they are involved in activities closely associated with fungi, such as the degradation of lignocellulose, the biosynthesis of natural products, and the detoxification of harmful compounds in the environment. Based on this assumption, we have analyzed the accessory flavoproteomes in the fungal kingdom using the MycoCosm database. This revealed large differences among fungal divisions, with Ascomycota, Basidiomycota, and Mucoromycota featuring the highest average number of genes encoding accessory flavoproteins. Moreover, a more detailed analysis showed a massive accumulation of accessory flavoproteins in Sordariomycetes, Agaricomycetes, and Glomeromycotina. In our view, this indicates that these fungal classes are proliferative producers of natural products and also interesting sources for flavoproteins with potentially useful catalytic properties in biocatalytic applications.
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