Computation has guided the design of conformationally-strained dioxolane-fused trans-cyclooctene (d-TCO) derivatives that display excellent reactivity in the tetrazine ligation. A water soluble derivative of 3,6-dipyridyl-s-tetrazine reacts with d-TCO with a second order rate k2 366,000 (+/− 15,000) M−1s−1 at 25 °C in pure water. Furthermore, d-TCO derivatives can be prepared easily, are accessed through diastereoselective synthesis, and are typically crystalline bench-stable solids that are stable in aqueous solution, blood serum, or in the presence of thiols in buffered solution. GFP with a genetically encoded tetrazine-containing amino acid was site-specifically labelled in vivo by a d-TCO derivative. The fastest bioorthogonal reaction reported to date [k2 3,300,000 (+/− 40,000) M−1s−1 in H2O at 25 °C] is described herein with a cyclopropane-fused trans-cyclooctene. d-TCO derivatives display rates within an order of magnitude of these fastest trans-cyclooctene reagents, and also display enhanced stability and aqueous solubility.

Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion.

Chlorite dismutases (Clds) convert chlorite to O2 and Cl–, stabilizing heme in the presence of strong oxidants and forming the O=O bond with high efficiency. The enzyme from the pathogen Klebsiella pneumoniae (KpCld) represents a subfamily of Clds that share most of their active site structure with efficient O2-producing Clds, even though they have a truncated monomeric structure, exist as a dimer rather than a pentamer, and come from Gram-negative bacteria without a known need to degrade chlorite. We hypothesized that KpCld, like others in its subfamily, should be able to make O2 and may serve an in vivo antioxidant function. Here, it is demonstrated that it degrades chlorite with limited turnovers relative to the respiratory Clds, in part because of the loss of hypochlorous acid from the active site and destruction of the heme. The observation of hypochlorous acid, the expected leaving group accompanying transfer of an oxygen atom to the ferric heme, is consistent with the more open, solvent-exposed heme environment predicted by spectroscopic measurements and inferred from the crystal structures of related proteins. KpCld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type K. pneumoniae has a significant growth advantage in the presence of chlorate relative to a Δcld knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of KpCld may be detoxification of endogenously produced chlorite.

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism isO-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringolO-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover inPseudomonas putidaKT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromaticO-demethylases in the biological conversion of lignin-derived aromatic compounds.

Heme oxygenases (HOs) 3 are enzymes that oxidatively liberate iron from the heme tetrapyrrole (1-3). In the well characterized HOs from animals and many bacteria, the same heme molecule acts as both the O 2 -activating cofactor and substrate. Three successive monooxygenation steps yield Fe(II), CO, and biliverdin IX␣ as the end products of the reaction (Fig. 1) (2, 3). Animals use HOs to maintain cellular heme homeostasis as part of a constant cycle of heme synthesis and breakdown. The products report on the status of this cycle and serve as antioxidants and signaling agents (4, 5). Many bacteria also use HO homologs, both to control heme homeostasis and to liberate iron from host-derived heme (6, 7). Heme, found primarily in hemoglobin, can therefore be used as a rich nutritional source of iron. Because of the intriguing nature of the reaction, which uses heme as both cofactor and substrate (8 -10), as well as the acute biological importance of HO-mediated processes, HOs from several species have been exceptionally well characterized (2, 3).By the early 2000s, however, it was apparent that many important Gram-positive pathogens that degrade host heme did not possess an HO-encoding gene in their genomes. A new family of heme-degrading proteins known as IsdGs was subsequently discovered, with representatives found in bacteria from both Gram-positive and Gram-negative phyla (11). IsdG family proteins are evolutionarily and structurally distinct from the well studied HOs (12, 13), and they yield different end products. Instead of biliverdin IX␣ and CO, the IsdG protein from Mycobacterium tuberculosis (known as MhuD) generates triply-oxygenated linear tetrapyrroles called mycobilins (Fig. 1) (14, 15). A formyl group remains appended to pyrrole ring A or B at the site of macrocycle cleavage, and an oxo group is generated on the pyrrole ring on the opposite side. Notably, no C1 product is released (16).Although homologous to MhuD, the IsdG from Staphylococcus aureus degrades heme to yet a third set of products. The macrocycle is not cleaved at the ␣-meso-but rather at either the ␤-or ␦-meso-carbon. Oxo groups are generated on both the carbon backbone and the pyrrole rings at the cleavage site, generating tetrapyrrole products known as staphylobilins (Fig. 1) (17). It was recently shown that a C1 product is indeed released by the S. aureus IsdG; however, quite unexpectedly, the major C1 product was determined to be formaldehyde (CH 2 O) instead of CO (18). Unlike CO, formaldehyde may be undetectable by animal immune systems, offering a potential selective advantage for heme-feeding pathogens that use IsdG-type enzymes (5,19,20). Mechanistically, the observation that CH 2 O instead of CO implies that verdoheme, the green inter-* This work was supported in part by National Institutes of Health Grants GM090260 and 5P20RR02437 of the CoBRE Program (to J. L. D.) and Grant RO1 AI069233 (to E. P. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the resp...

Members of the antibiotic biosynthesis monooxygenase family catalyze O 2 -dependent oxidations and oxygenations in the absence of any metallo-or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O 2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater. As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pK a . The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O 2 -dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O 2 reactivity via markedly different means.

Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O -methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O- methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o -and p -vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o - and p -isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p -vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo . This work expands the known aromatic O- demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.

several classes of oxidases and oxygenases accelerate direct reactions between substrate and O 2 using only the protein environment. Nogalamycin monooxygenase (NMO) from Streptomyces nogalater is a cofactor-independent enzyme that catalyzes rate-limiting electron transfer between its substrate and O 2 . Here, using enzymekinetic, cyclic voltammetry, and mutagenesis methods, we demonstrate that NMO initially activates the substrate, lowering its pK a by 1.0 unit (⌬G* ‫؍‬ 1.4 kcal mol ؊1 ). We found that the oneelectron reduction potential, measured for the deprotonated substrate both inside and outside the protein environment, increases by 85 mV inside NMO, corresponding to a ⌬⌬G 0 of 2.0 kcal mol ؊1 (0.087 eV) and that the activation barrier, ⌬G ‡ , is lowered by 4.8 kcal mol ؊1 (0.21 eV). Applying the Marcus model, we observed that this suggests a sizable decrease of 28 kcal mol ؊1 (1.4 eV) in the reorganization energy (), which constitutes the major portion of the protein environment's effect in lowering the reaction barrier. A similar role for the protein has been proposed in several cofactor-dependent systems and may reflect a broader trend in O 2 -utilizing proteins. In summary, NMO's protein environment facilitates direct electron transfer, and NMO accelerates rate-limiting electron transfer by strongly lowering the reorganization energy.