Development of Biocatalysts Based on Cytochrome P450s
Cytochrome P450s and Decoy Molecules
Cytochrome P450s (P450s) are ubiquitous enzymes that comprise a superfamily of heme-containing monooxygenases and involved in oxidative metabolism, detoxification, and synthesis of steroids. P450s have been regarded as attractive candidates for oxidation catalysts because of their high catalytic activity for direct oxygen insertion into unactivated C-H bonds. In general, bacterial cytochrome P450s (P450s) possess high catalytic activity and high substrate specificity at the same time. To utilize bacterial P450s as a biocatalyst for the oxidation of various substrates, we have to alter their substrate specificity. Although site-directed and random mutagenesis have been regarded as one of the most powerful approach to change the substrate specificity of P450s, we recently have demonstrated that the substrate specificity of hydrogen peroxide-dependent P450s (P450BSβ and P450SPα) can be altered just by adding a decoy molecule, which has a structural similarity to their natural substrate. In this reaction system, the decoy molecule induced substrate misrecognition and non-natural substrates were oxidized by P450BSβ and P450SPα without replacing any amino acid residues. More recently, we have expanded this substrate misrecognition system to include P450BM3. Utilizing perfluorinated carboxylic acids as a decoy molecule toward P450BM3, we have succeeded in hydroxylation of propane and butane. Interestingly, the catalytic activity of P450BM3 are dependent on the alkyl chain length of perfluorinated carboxylic acid and perfluorinated decanoic acid gave the highest rate of product formation for propane hydroxylation among perfluorinated carboxylic acids examined. These results indicate that the substrate specificity can be changed by adding decoy molecules and the catalytic activity can be controlled by the choice of decoy molecule.
Hydrogen Peroxide-dependent P450s and Decoy Molecules
P450BSβ is one of hydrogen peroxide-dependent P450s that utilizes hydrogen peroxide as an oxidant and efficiently catalyzes site specific hydroxylation of fatty acids. The crystal structure of a palmitic acid-bound form of P450BSβ reveals that the carboxylate of palmitic acid interacts with Arg-242 located near the heme showing that the general acid-base function for facile generation of the active species is provided by the carboxyl group of the bound fatty acid. This unique reaction mechanism contributes to its high substrate specificity and P450BSβ never oxidizes substrates other than long-alkyl-chain fatty acids.
P450BSβ never oxidizes the substrates other than fatty acids having long alkyl chain. However, we demonstrated that P450BSβ is able to oxidize a wide variety of non-natural substrates in the presence of molecules having a carboxylate group with a series of short-alkyl chains. The reaction never proceeded without these molecules, and thus P450BSβ starts to catalyze oxidation of the non-natural substrates with the help of these short-alkyl carboxylic acids. In the case of short alkyl-chain carboxylic acids, their alkyl tails would be too short to reach to the hydrophobic channel of P450BSβ, resulting in the loose fixation of the short alkyl chains and failure of the hydroxylation. We, thus, have referred the short-alkyl carboxylic acids as ‘‘decoy molecules’’.
One-electron oxidation of guaiacol was catalyzed by P450BSβ at a high reaction rate in the presence of carboxylic acids with short alkyl-chain and heptanoic acid gave the maximum rate, 3,750 turnover/min/protein, among carboxylic acids examined. On the contrary, no appreciable reaction was observed neither in the absence of carboxylic acid nor in the presence of myristic acid under the same conditions. These results clearly indicate that the decoy molecule turns on the catalytic oxidation cycle of the P450BSβ reaction. P450BSβ may misrecognizes short alkyl-chain carboxylic acids as the substrate owing to its structural similarity and catalyzes the oxidation of nonfatty acids. The results imply the decoy molecule keeps the P450BSβ catalytic cycle always ‘‘on,’’ while the binding of long alkyl chain in the active site turns ‘‘on’’ the oxidation cycle. We also demonstrated that monooxygenations of non-natural substrates, such as epoxidation of styrene and hydroxylation of cumene and ethylbenzene, were catalyzed by P450BSβ in the presence of decoy molecules. That the catalytic activities are dependent on the alkyl-chain structure of the decoy molecule indicates the catalytic activities must be further improved by the design of the decoy molecule.
We also demonstrated the stereoselective styrene epoxidation catalyzed by P450SPα with carboxylic acids as decoy molecules. The crystal structure of a (R)-ibuprofen bound-ochrome P450SPα at 1.9Å resolution reveals that the carboxylate group of (R)-ibuprofen serves as an acid-base catalyst to initiate the epoxidation. The stereoselectivity of styrene epoxidation was largely affected by the chirality of ibuprofen and (R)-ibuprofen enhanced the (S)-styrene oxide formation. The docking simulation of styrene binding in the active site of (R)-ibuprofen-bound form suggests that the orientation of the vinyl group of styrene in the active site agrees with the (S)-styrene oxide formation. This unique strategy using the chiral-substrate-analogues allows us to alter the enantioselectivity of enzymes without any mutagenesis and thus contributes to development of novel asymmetric biocatalysts.
The crystal structure analysis of P450SPα with palmitic acid
ochrome P450SPα(CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H2O2-dependent P450. P450SPα hydroxylates fatty acids with high α-regioselectivity. Herein we report the crystal structure of P450SPα with palmitic acid as a substrate at a resolution of 1.65 Å. The structure revealed that the Cα of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective α-hydroxylation of fatty acid observed in P450SPα. Mutations at the active site and the F-G loop of P450SPα did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of ochrome P450BSβ (CYP152A1), which shows β-regioselectivity. This implies that the high regioselectivity of P450SPα is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450BSβ.
Gaseous Alkane Hydroxylation by P450BM3 in the presence of perfluorinated carboxylic acids as decoy molecules
Catalytic hydroxylation of inert C-H bonds under mild conditions has been a major challenge in synthetic chemistry. In particular, hydroxylation of gaseous alkanes has become an increasingly important approach to produce liquid fuel or chemical precursors from natural gas. Biocatalysts offer alternatives to conventional chemical processes for alkane hydroxylations. P450BM3 (CYP102A1) isolated from Bacillus megaterium is one of the most promising enzymes owing the highest monooxygenase activity among P450s reported thus far. The catalytic cycle of P450BM3 is initiated by its substrate (fatty acid) binding to the heme cavity of P450BM3 followed by reductive activation of molecular oxygen to generate a highly active oxidant species, oxoferryl(IV)porphyrin π cation radical, the so-called Compound I. Because the substrate binding is crucial to initiate the catalytic cycle of P450BM3, substrates whose structures are largely different from that of fatty acid cannot be hydroxylated by P450BM3. P450BM3 does not catalyze the hydroxylation of gaseous alkanes, because gaseous alkanes cannot bind to the heme active site of P450BM3. We, however, assume that Compound I of P450BM3 could be formed by adding a “dummy substrate” whose structure is similar to fatty acid. Furthermore, if gaseous alkane molecules could penetrate into the active site of P450BM3 in the presence of such a dummy substrate, gaseous alkane molecules could be hydroxylated. Herein, we report that the addition of perfluoro carboxylic acids (PFs) to wild-type P450BM3 as dummy substrates results in C−H bond hydroxylation of gaseous alkanes to the corresponding alcohols. The hydroxylation reactions of gaseous alkane molecules (propane and butane) and cyclohexane were catalyzed by P450BM3 in the presence of a series of PFs. For the propane hydroxylation, PFC10 gave the highest rate of product formation (67 min-1) and the highest coupling efficiency (18%) among PFs examined. Interestingly, it was noted that smaller alkanes tend to prefer longer alkyl chain PFs for an efficient reaction.