A bis(μ-oxo)diiron(IV,IV) complex as a model for intermediate Q in the methane monooxygenase reaction cycle has been prepared. The precursor complex with a Fe
Fe
(μ-O)
core was fully characterized ...by X-ray crystallography and other spectroscopic analyses and was converted to the Fe
(μ-O)
complex via electrochemical oxidation at 1000 mV (vs Ag/Ag
) in acetone at 193 K. The UV-vis spectral features, Mössbauer parameters (Δ
= 2.079 mm/s and δ = -0.027 mm/s), and EXAFS analysis (Fe-O/N = 1.73/1.96 Å and Fe···Fe = 2.76 Å) support the structure of the low-spin (
= 1, for each Fe) Fe
(μ-O)
core. The rate constants of the hydrogen abstraction reaction from 9,10-dihydroanthracene at 243 K suggest the high reactivity of these synthetic bis(μ-oxo)diiron complexes supported by simple N4 tripodal ligand.
Because it is an outstanding antioxidant with wide applications, biotechnological production of astaxanthin has attracted increasing research interest. However, the astaxanthin titer achieved to date ...is still rather low, attributed to the poor efficiency of β-carotene ketolation and hydroxylation, as well as the adverse effect of astaxanthin accumulation on cell growth. To address these problems, we constructed an efficient astaxanthin-producing Saccharomyces cerevisiae strain by combining protein engineering and dynamic metabolic regulation. First, superior mutants of β-carotene ketolase and β-carotene hydroxylase were obtained by directed coevolution to accelerate the conversion of β-carotene to astaxanthin. Subsequently, the Gal4M9-based temperature-responsive regulation system was introduced to separate astaxanthin production from cell growth. Finally, 235 mg/L of (3S,3′S)-astaxanthin was produced by two-stage, high-density fermentation. This study demonstrates the power of combining directed coevolution and temperature-responsive regulation in astaxanthin biosynthesis and may provide methodological reference for biotechnological production of other value-added chemicals.
Biocatalysts that perform C-H hydroxylation exhibit exceptional substrate specificity and site-selectivity, often through the use of high valent oxidants to activate these inert bonds. Rieske ...oxygenases are examples of enzymes with the ability to perform precise mono- or dioxygenation reactions on a variety of substrates. Understanding the structural features of Rieske oxygenases responsible for control over selectivity is essential to enable the development of this class of enzymes for biocatalytic applications. Decades of research has illuminated the critical features common to Rieske oxygenases, however, structural information for enzymes that functionalize diverse scaffolds is limited. Here, we report the structures of two Rieske monooxygenases involved in the biosynthesis of paralytic shellfish toxins (PSTs), SxtT and GxtA, adding to the short list of structurally characterized Rieske oxygenases. Based on these structures, substrate-bound structures, and mutagenesis experiments, we implicate specific residues in substrate positioning and the divergent reaction selectivity observed in these two enzymes.
Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the ...biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A.
Chlorophyllide
a
oxygenase (CAO) is responsible for converting chlorophyll
a
to chlorophyll
b
in a two-step oxygenation reaction. CAO belongs to the family of Rieske-mononuclear iron oxygenases. ...Although the structure and reaction mechanism of other Rieske monooxygenases have been described, a member of plant Rieske non-heme iron-dependent monooxygenase has not been structurally characterized. The enzymes in this family usually form a trimeric structure and electrons are transferred between the non-heme iron site and the Rieske center of the adjoining subunits. CAO is supposed to form a similar structural arrangement. However, in Mamiellales such as
Micromonas
and
Ostreococcus
, CAO is encoded by two genes where non-heme iron site and Rieske cluster localize on the distinct polypeptides. It is not clear if they can form a similar structural organization to achieve the enzymatic activity. In this study, the tertiary structures of CAO from the model plant
Arabidopsis thaliana
and the Prasinophyte
Micromonas pusilla
were predicted by deep learning-based methods, followed by energy minimization and subsequent stereochemical quality assessment of the predicted models. Furthermore, the chlorophyll
a
binding cavity and the interaction of ferredoxin, which is the electron donor, on the surface of
Micromonas
CAO were predicted. The electron transfer pathway was predicted in
Micromonas
CAO and the overall structure of the CAO active site was conserved even though it forms a heterodimeric complex. The structures presented in this study will serve as a basis for understanding the reaction mechanism and regulation of the plant monooxygenase family to which CAO belongs.
Since their discovery in the 1960s, the family of Fe(II)/2-oxoglutarate-dependent oxygenases has undergone a tremendous expansion to include enzymes catalyzing a vast diversity of biologically ...important reactions. Recent examples highlight roles in controlling chromatin modification, transcription, mRNA demethylation, and mRNA splicing. Others generate modifications in tRNA, translation factors, ribosomes, and other proteins. Thus, oxygenases affect all components of molecular biology’s central dogma, in which information flows from DNA to RNA to proteins. These enzymes also function in biosynthesis and catabolism of cellular metabolites, including antibiotics and signaling molecules. Due to their critical importance, ongoing efforts have targeted family members for the development of specific therapeutics. This review provides a general overview of recently characterized oxygenase reactions and their key biological roles.
Structural studies of 2OG-dependent oxygenases provide keen insights into the mechanisms of these enzymes, including recent visualization of the key ferryl intermediate by use of a stable structural mimic.
2OG-dependent oxygenases are increasingly associated with genetic disorders and cancers, stimulating intense efforts to develop specific therapeutic agents directed at these enzymes.
2OG-dependent hydroxylases participate in multiple steps in transcriptional and translational processes.
The list of oxygenases involved in cellular biosynthesis and catabolism continues to expand rapidly, especially in pathways for antimicrobial compounds.
The types of chemical reactions catalyzed by these oxygenases continues to diversify, and industrial applications of these activities are growing.
•Peroxygenases are very promising catalysts for preparative oxyfunctionalization chemistry.•The number of interesting applications and novel catalysts is growing rapidly.•The current toolbox of ...peroxygenases still suffers from insufficient selectivity.•Novel enzymes from natural and/or man-made diversity are urgently needed.
Peroxygenases are promising catalysts for preparative oxyfunctionalization chemistry as they combine the versatility of P450 monooxygenases with simplicity of cofactor-independent enzymes. Though many interesting applications have been reported, today ‘we have only scratched the surface’ and significant efforts are necessary to solve issues related to selectivity of the wild type enzymes and low product titers. For this, further elucidation of the vast natural diversity as well as protein and reaction engineering approaches are discussed.
Protein fold recognition using sequence profile searches frequently allows prediction of the structure and biochemical mechanisms of proteins with an important biological function but unknown ...biochemical activity. Here we describe such predictions resulting from an analysis of the 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenases, a class of enzymes that are widespread in eukaryotes and bacteria and catalyze a variety of reactions typically involving the oxidation of an organic substrate using a dioxygen molecule.
We employ sequence profile analysis to show that the DNA repair protein AlkB, the extracellular matrix protein leprecan, the disease-resistance-related protein EGL-9 and several uncharacterized proteins define novel families of enzymes of the 2OG-Fe(II) oxygenase superfamily. The identification of AlkB as a member of the 2OG-Fe(II) oxygenase superfamily suggests that this protein catalyzes oxidative detoxification of alkylated bases. More distant homologs of AlkB were detected in eukaryotes and in plant RNA viruses, leading to the hypothesis that these proteins might be involved in RNA demethylation. The EGL-9 protein from Caenorhabditis elegans is necessary for normal muscle function and its inactivation results in resistance against paralysis induced by the Pseudomonas aeruginosa toxin. EGL-9 and leprecan are predicted to be novel protein hydroxylases that might be involved in the generation of substrates for protein glycosylation.
Here, using sequence profile searches, we show that several previously undetected protein families contain 2OG-Fe(II) oxygenase fold. This allows us to predict the catalytic activity for a wide range of biologically important, but biochemically uncharacterized proteins from eukaryotes and bacteria.
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► Structural features of 2OG oxygenases involved in substrate recognition are analyzed. ► Crystallographic studies reveal the versatility of the jelly roll fold in substrate ...binding. ► Defined structural regions that interact with substrate(s) are biased by fold topology. ► The utility of the enzyme–substrate structures for engineering and selective inhibition are discussed.
2-Oxoglutarate (2OG) and ferrous iron dependent oxygenases catalyze two-electron oxidations of a range of small and large molecule substrates, including proteins/peptides/amino acids, nucleic acids/bases, and lipids, as well as natural products including antibiotics and signaling molecules. 2OG oxygenases employ variations of a core double-stranded β-helix (DSBH; a.k.a. jelly-roll, cupin or jumonji C (JmjC)) fold to enable binding of Fe(II) and 2OG in a subfamily conserved manner. The topology of the DSBH limits regions directly involved in substrate binding: commonly the first, second and eighth strands, loops between the second/third and fourth/fifth DSBH strands, and the N-terminal and C-terminal regions are involved in primary substrate, co-substrate and cofactor binding. Insights into substrate recognition by 2OG oxygenases will help to enable selective inhibition and bioengineering studies.
Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, ...for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO
); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.
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