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The reactions of enzymes and cofactors with gaseous molecules such as dioxygen (O2) are challenging to study and remain among the most contentious subjects in biochemistry. To date, it is largely enigmatic how enzymes control and fine-tune their reactions with O2, as exemplified by the ubiquitous flavin-dependent enzymes that commonly facilitate redox chemistry such as the oxygenation of organic substrates. Here we employ O2-pressurized X-ray crystallography and quantum mechanical calculations to reveal how the precise positioning of O2 within a flavoenzyme's active site enables the regiospecific formation of a covalent flavin-oxygen adduct and oxygenating species (i.e., the flavin-N5-oxide) by mimicking a critical transition state. This study unambiguously demonstrates how enzymes may control the O2 functionalization of an organic cofactor as prerequisite for oxidative catalysis. Our work thus illustrates how O2 reactivity can be harnessed in an enzymatic environment and provides crucial knowledge for future rational design of O2-reactive enzymes.

Flavoproteins catalyse a diversity of fundamental redox reactions and are one of the most studied enzyme families (12). As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate (1,3,4). Here we report that the bacterial flavoenzyme EncM (5,6) catalyses the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics and isotope labelling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unexpected stable flavin-oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization., The antibiotic enterocin (Fig. 1, compound 1) is producedby various streptomycete bacteria (7) and contains a unique tricyclic caged core. Nearly 40 years ago, isotope labelling studies suggested the involvement [...]

Catabolism may give rise to toxic intermediates that compromise cell vitality, such as epoxide formation in the recently elucidated and apparently universal bacterial coenzyme A (CoA)-dependent degradation of phenylacetic acid (1). This compound is central to the catabolism of a variety of aromatics, such as phenylalanine, lignin-related compounds or environmental contaminants (2,3). The key phenylacetyl-CoA monooxygenase (epoxidase) of the pathway, PaaABCE (1,4,5), is also connected to the production of various primary and secondary metabolites (6-9), as well as to the virulence of certain pathogens (1,10,11). However, the enzyme complex has so far not been investigated in detail. Here we characterize the bacterial multicomponent monooxygenase PaaABCE that, surprisingly, not only transforms phenylacetyl-CoA into its ring-1,2-epoxide, but also mediates the NADPH-dependent removal of the epoxide oxygen, regenerating phenylacetyl-CoA with formation of water. We provide evidence for a catalytic di-iron centre that is probably the key to the unprecedented deoxygenation of an organic compound by an oxygenase. Presumably, the bifunctionality is vital to avoid toxic intracellular epoxide levels if the subsequent catabolic steps are impeded. Our data suggest that detoxification is assisted by two thioesterases (Paal and PaaY) forming non-reactive breakdown products. Hence, PaaABCE may harbour an intrinsic escape mechanism from its own toxic product and represents the archetype of a bifunctional oxygenase/deoxygenase. Analogous reactions may possibly be catalysed by other di-iron epoxidases., PaaABCE catalyses a crucial step in bacterial degradation of phenylacetic acid (paa), namely, the epoxidation of phenylacetyl (pa)-CoA (Fig. 1, Supplementary Fig. 1). Its encoding genes are present in ~16% [...]

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic C[O.sub.2] fixation in various Crenarchaea, as studied in some detail in Metaliosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.

Thiol‐containing nucleophiles such as cysteine react spontaneously with the citric acid cycle intermediate fumarate to form S‐(2‐succino)‐adducts. In Bacillus subtilis, a salvaging pathway encoded by the yxe operon has recently been identified for the detoxification and exploitation of these compounds as sulfur sources. This route involves acetylation of S‐(2‐succino)cysteine to N‐acetyl‐2‐succinocysteine, which is presumably converted to oxaloacetate and N‐acetylcysteine, before a final deacetylation step affords cysteine. The critical oxidative cleavage of the C–S bond of N‐acetyl‐S‐(2‐succino)cysteine was proposed to depend on the predicted flavoprotein monooxygenase YxeK. Here, we characterize YxeK and verify its role in S‐(2‐succino)‐adduct detoxification and sulfur metabolism. Detailed biochemical and mechanistic investigation of YxeK including 18O‐isotope‐labeling experiments, homology modeling, substrate specificity tests, site‐directed mutagenesis, and (pre‐)steady‐state kinetics provides insight into the enzyme's mechanism of action, which may involve a noncanonical flavin‐N5‐peroxide species for C–S bond oxygenolysis. [ABSTRACT FROM AUTHOR]

As a rare feature among organic cofactors, reduced flavins (Fl red ) can efficiently react with dioxygen (O 2 ). As a consequence, many flavin-dependent enzymes may serve as either oxidases that use O 2 as an electron acceptor or as monooxygenases that transfer one oxygen atom derived from O 2 to an organic substrate. For the latter functionality, covalent flavin: oxygen adducts are formed that function as oxygenating species. Remarkably, despite intensive research, many open questions remain how flavoenzymes control the reaction with O 2 . Here, we describe O 2 -pressurized protein crystallography in detail as a structural approach to gain insight into the interactions between the protein scaffold, the flavin cofactor and O 2 . This may allow to further our understanding of how flavoenzymes can steer the formation of different oxygenating species and thus provide missing puzzle pieces for rational flavoenzyme design., (© 2019 Elsevier Inc. All rights reserved.)

Flavoenzymes often function as oxygenases and have been extensively studied for many decades. Commonly, oxygenation reactions are mediated by a transient C4a-peroxyflavin formed from reaction of reduced flavin with O 2 . EncM, however, employs a previously unrecognized flavin-oxygenating species, the flavin-N5-oxide, which is key to a complex oxidative Favorskii-type rearrangement and cyclization cascade in the biosynthesis of the bacterial polyketide antibiotic enterocin produced by the marine bacterium Streptomyces maritimus. Here, the methodology and key experiments are described that led to the discovery of this novel flavin redox species and granted insight into one of the most astounding single-enzyme-catalyzed reaction cascades in natural product biosynthesis., (© 2018 Elsevier Inc. All rights reserved.)

Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome.