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In the direct hydroxylation of C–H bonds by cytochrome P450s, their regioselectivities between primary and secondary carbons depend strongly on their C–H bond-dissociation energies. Thus, selective hydroxylation at the terminal position is a challenging task because of its low reactivity compared with internal positions. On the other hand, CYP153A33 can hydroxylate long fatty acids with high regioselectivity at the terminal position. For highly selective hydroxylation of gaseous alkanes at the terminal position, we herein combined CYP153A33 with substrate analog decoy molecules to alter the substrate specificity of the enzyme. Perfluoroacyl amino acids can effectively activate CYP153A33 and facilitate the regioselective hydroxylation of propane at the terminal position to afford 1-propanol (1-propanol/2-propanol = 80/20). In addition, we found that the use of CYP153A33 with decoy molecules also enables the hydroxylation of ethane and methane.

Cytochrome P450BM3 is a haemoenzyme that catalyses the monooxygenation of unactivated C–H bonds. In the absence of an appropriate substrate, however, P450BM3 remains in an inactive state and catalysis does not proceed. Traditionally, mutagenesis has been the mainstay technique employed to overcome this limitation, opening P450BM3 up for non-native substrate hydroxylations. This highlight review will discuss a different approach, wherein native substrate mimics (decoy molecules) are employed to trick P450BM3 into mistakenly hydroxylating non-native substrates. This decoy molecule system has the advantage that the wild-type enzyme can be utilised, and laborious mutagenesis can be avoided. Major developments from the first discovery of decoy molecules to the present day will be briefly covered herein.Cytochrome P450BM3 is a haemoenzyme that catalyses the monooxygenation of unactivated C-H bonds with high substrate specificity. This highlight review will introduce a different approach to alter the substrate specificty of wild-type P450BM3, wherein native substrate mimics (decoy molecules) trick P450BM3 into mistakenly hydroxylating non-native substrates. Major developments from the first discovery of decoy molecules through to the present day will be briefly covered herein.

5,15-Diazaporphyrins are porphyrin analogues with imine-type sp2-hybridized nitrogen atoms at the meso-positions. Even though these compounds are more electron-deficient than regular porphyrins, the use of iron diazaporphyrins as catalysts has not been reported. Herein, we disclose the synthesis, structure, and electronic properties of iron(III) 5,15-diazaporphyrins. We evaluate their structures and electronic natures by X-ray analysis and electrochemical analyses. We also demonstrate that chloroiron(III) 5,15-diazaporphyrins exhibit high catalytic activity in the direct oxidation of alkanes due to their intrinsic electron-deficient nature. On the basis of stoichiometric reactions of iron(III) diazaporphyrin with iodosylbenzene as an oxidant, it was possible to demonstrate the existence of an iodosylbenzene–iron diazaporphyrin adduct reaction intermediate that serves as a reservoir to generate oxo-iron species.

To survive in the iron-devoid environment of their host, pathogenic bacteria have devised multifarious cunning tactics such as evolving intricate heme transport systems to pirate extracellular heme. Yet, the potential of heme transport systems as antimicrobial targets has not been explored. Herein we developed a strategy to deliver antimicrobials by exploiting the extracellular heme acquisition system protein A (HasA) of Pseudomonas aeruginosa. We demonstrated that, analogous to heme uptake, HasA can specifically traffic an antimicrobial, gallium phthalocyanine (GaPc), into the intracellular space of P. aeruginosaviathe interaction of HasA with its outer membrane receptor HasR. HasA enables water-insoluble GaPc to be mistakenly acquired by P. aeruginosa, permitting its sterilization (>99.99%) by irradiation with near-infrared (NIR) light, irrespective of antibiotic resistance. Our findings substantiate that bacterial heme uptake viaprotein–protein recognition is an attractive target for antimicrobials, enabling specific and effective sterilization.

Bacterial cytochrome P450s (P450s) are at the focus of attention as potential biocatalysts for applications in green synthetic chemistry, as they possess high activity for the hydroxylation of inert substrate C–H bonds. The high activity of bacterial P450s, such as P450BM3, is chiefly due to their high substrate specificity, and consequently, the catalytic activity of P450BM3 toward non-native substrates is very low, limiting the utility of bacterial P450s as biocatalysts. To enable oxidation of non-native substrates by P450BM3 without any mutagenesis, we have developed a series of “decoy molecules”, inert dummy substrates, with structures that resemble those of the native substrates. Decoy molecules fool P450BM3 into generating the active species, so-called Compound I, enabling the catalytic oxidation of non-native substrates other than fatty acids. Perfluorinated carboxylic acids (PFCs) serve as decoy molecules to initiate the activation of molecular oxygen in the same manner as long-alkyl-chain fatty acids, due to their structural similarity, and induce the generation of Compound I, but, unlike the native substrates, PFCs are not oxidizable by Compound I, allowing the hydroxylation of non-native substrates, such as gaseous alkanes and benzene. The catalytic activity for non-native substrate hydroxylation was significantly enhanced by employing second generation decoy molecules, PFCs modified with amino acids (PFC-amino acids). Cocrystals of P450BM3 with PFC9-Trp revealed clear electron density in the fatty-acid-binding channel that was readily assigned to PFC9-Trp. The alkyl chain terminus of PFC9-Trp does not reach the active site owing to multiple hydrogen bonding interactions between the carboxyl and carbonyl groups of PFC9-Trp and amino acids located at the entrance of the substrate binding channel of P450BM3 that fix it in place. The remaining space above the heme after binding of PFC9-Trp can be utilized to accommodate non-native substrates. Further developments revealed that third generation decoy molecules, N-acyl amino acids, such as pelargonoyl-l-phenylalanine (C9-Phe), can serve as decoy molecules, indicating that the rationale “fluorination is required for decoy molecule function” can be safely discarded. Diverse carboxylic acids including dipeptides could now be exploited as building blocks, and a library of decoy molecules possessing diverse structures was prepared. Among the third-generation decoy molecules examined N-enanthyl-l-proline modified with l-phenylalanine (C7-Pro-Phe) afforded the maximum turnover rate for benzene hydroxylation. The structural diversity of third-generation decoy molecules was also utilized to control the stereoselectivity of hydroxylation for the benzylic hydroxylation of Indane, showing that decoy molecules can alter stereoselectivity. As both the catalytic activity and enantioselectivity are dependent upon the structure of the decoy molecules, their design allows us to regulate reactions catalyzed by wild-type enzymes. Furthermore, decoy molecules can also activate intracellular P450BM3, allowing the use of E. coliexpressing wild-type P450BM3 as an efficient whole-cell bioreactor. It should be noted that Mn-substituted full-length P450BM3 (Mn-P450BM3) is also active for the hydroxylation of propane in which the regioselectivity diverged from that of Fe-P450BM3. The results summarized in this Account represent good examples of how the reactive properties of P450BM3 can be controlled for the monooxygenation of non-native substrates in vitro as well as in vivo to expand the potential of P450BM3.

Cytochrome P450 peroxygenases belonging to the CYP152 family catalyse the oxidation of fatty acids using H2O2. CYP152N1 isolated from Exiguobacteriumsp. AT1b exclusively catalyses the α-selective hydroxylation of myristic acid at physiological H2O2concentration. However, a series of shorter-alkyl-chain fatty acids such as tridecanoic acid were produced from myristic acid by increasing the concentration of H2O2(1–10 mM). The yield of tridecanoic acid from myristic acid reached 17%. An 18O-labeled oxidant study suggested that CYP152N1 catalysed the overoxidation of α-hydroxymyristic acid to form α-ketomyristic acid, which in turn was spontaneously decomposed by H2O2to yield tridecanoic acid. Crystal structure analysis of CYP152N1 revealed its high similarity to other CYP152 family enzymes, such as CYP152A1 and CYP152B1. MD simulations of α-hydroxymyristic acid accommodated in CYP152N1 proposed a possible pre-oxidation conformation of α-hydroxymyristic acid for the decarboxylation reaction.

Cytochrome P450s are a family of heme-containing enzymes that catalyze monooxygenation of inert substrates. Bacterial cytochrome P450s are great candidates as biocatalysts for synthetic applications because of their high hydroxylation activity and their high solubility in water. However, the substrate specificity of bacterial cytochrome P450s is generally high, and their low catalytic activities toward nonnative substrates tends to restrict their application as biocatalysts. We have developed a reaction system that utilizes dummy substrates with structures similar to those of natural substrates, as “decoy molecules”. The decoy molecules induce substrate misrecognition of bacterial P450s, leading to the generation of the active species and ultimately enabling them to catalyze the oxidation of nonnative substrates. The catalytic activity and the enantioselectivity are dependent on the structure of the decoy molecules, suggesting that the reactions can be controlled by variation in the designed structure of the decoy molecules.Cytochrome P450s are a family of heme-containing enzymes that catalyze monooxygenation of inert substrates. Bacterial cytochrome P450s are great candidates as biocatalysts for synthetic applications because of their high hydroxylation activity and their high solubility in water. However, the substrate specificity of bacterial cytochrome P450s is generally high, and their low catalytic activities toward nonnative substrates tends to restrict their applicable use as biocatalysts. We have developed a reaction system that utilize dummy substrates whose structures are similar to those of natural substrates, as “decoy molecules”. The decoy molecules induce substrate misrecognition of bacterial P450s, leading to the generation of the active species and ultimately enabling them to catalyze the oxidation of nonnative substrates. The catalytic activity and the enantioselectivity are dependent on the structure of the decoy molecules, suggesting that the reactions can be controlled by variation in the designed structure of the decoy molecules.

The hydroxylation of non-native substrates catalysed by wild-type P450BM3 is reported, wherein “decoy molecules”, i.e., native substrate mimics, controlled the stereoselectivity of hydroxylation reactions. We employed decoy molecules with diverse structures, resulting in either a significant improvement in enantioselectivity or clear inversion of stereoselectivity in the benzylic hydroxylation of alkylbenzenes and cycloalkylbenzenes. For example, supplementation of wild-type P450BM3 with 5-cyclohexylvaleric acid-l-phenylalanine (5CHVA-Phe) and Z-proline-l-phenylalanine yielded 53% (R) ee and 56% (S) ee for indane hydroxylation, respectively, although 16% (S) ee was still observed in the absence of any additives. Moreover, we performed a successful crystal structure analysis of 5CHVA-l-tryptophan-bound P450BM3 at 2.00 Å, which suggests that the changes in selectivity observed were caused by conformational changes in the enzyme induced by binding of the decoy molecules.

A substrate-binding-state mimic of H2O2-dependent cytochrome P450 that is able to catalyze monooxygenation of non-native substrates was constructed by one-point mutagenesis of P450SPα(CYP152B1). P450SPα, a long-alkyl-chain fatty acid hydroxylase, lacks any general acid–base residue around the heme. The carboxylate group of a fatty acid is thus indispensable for the generation of active species using H2O2. We prepared an A245E mutant to mimic a substrate-binding state by placing a carboxylate group at the active site. The active site structure of the A245E mutant is similar to that of the fatty-acid-bound state of P450SPαand catalyzes styrene oxidation at a rate of 280 min−1(kcat), whereas the wild-type enzyme does not show any catalytic activity. More importantly, the same mutation, i.e.the mutation of the highly conserved threonine in P450s to glutamic acid, was also effective in introducing peroxygenase activity into P450BM3, P450cam, and CYP119. These results indicate that a variety of peroxygenases based on P450s can be constructed by one-point mutagenesis.

It has long been believedthat the fatty acid hydroxylase wild‐type P450BM3 is unable to oxidize gaseous alkanes. However, the simple addition of a perfluorocarboxylic acid as a dummy substrate to initiate the P450BM3 catalytic cycle enabled the efficient hydroxylation of butane and propane (see picture).

Sie kann's doch!Lange wurde geglaubt, der Wildtyp der Fettsäure‐Hydroxylase P450BM3 sei nicht befähigt, gasförmige Alkane zu oxidieren. Die einfache Zugabe einer Perfluorcarbonsäure als Blindsubstrat zur Initiierung des P450BM3‐Katalysezyklus ermöglichte jedoch die effiziente Hydroxylierung von Butan und Propan (siehe Bild).

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