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1.
The hydroxylation of alkane molecules, especially at terminal positions, is a challenging reaction. Enzymes that catalyze this reaction could be used to produce high-value compounds from aliphatic and alkyl-substituted substrates. However, until a few years ago, all known alkane hydroxylating enzymes were membrane-bound, and difficult to use. Recently, three bacterial P450 enzymes of the (soluble) CYP101 and CYP102 families were engineered to hydroxylate alkanes, but even after extensive efforts hydroxylation was mainly at sub-terminal positions. More recently, a new soluble P450 family (CYP153) was identified and characterized, which activates the terminal position of alkanes and alkyl-substituted compounds with very high regio-selectivity. The use of CYP153s in biotechnological applications is now being explored. 相似文献
2.
van Beilen JB Funhoff EG van Loon A Just A Kaysser L Bouza M Holtackers R Röthlisberger M Li Z Witholt B 《Applied and environmental microbiology》2006,72(1):59-65
Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases. 相似文献
3.
Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases 总被引:7,自引:2,他引:5
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Jan B. van Beilen Enrico G. Funhoff Alexander van Loon Andrea Just Leo Kaysser Manuel Bouza Ren Holtackers Martina Rthlisberger Zhi Li Bernard Witholt 《Applied microbiology》2006,72(1):59-65
Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases. 相似文献
4.
Funhoff EG Bauer U García-Rubio I Witholt B van Beilen JB 《Journal of bacteriology》2006,188(14):5220-5227
The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida GPo1 AlkB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes (C6 to C11) to 1-alkanols with a maximal turnover number of 70 min(-1) and has a regiospecificity of > or =95% for the terminal carbon atom position. Spectroscopic binding studies showed that C6-to-C11 aliphatic alkanes bind in the active site with Kd values varying from approximately 20 nM to 3.7 microM. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation. 相似文献
5.
Daniel J. Koch Mike M. Chen Jan B. van Beilen Frances H. Arnold 《Applied and environmental microbiology》2009,75(2):337-344
Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.Microbial utilization and degradation of alkanes was discovered almost a century ago (27). Since then, several enzyme families capable of hydroxylating alkanes to alkanols, the first step in alkane degradation, have been identified and categorized based on their preferred substrates (30). The soluble and particulate methane monooxygenases (sMMO and pMMO) and the related propane monooxygenase and butane monooxygenase (BMO) are specialized on gaseous small-chain alkanes (C1 to C4), while medium-chain (C5 to C16) alkane hydroxylation seems to be the domain of the CYP153 and AlkB enzyme families.Conversion of C1 to C4 alkanes to alkanols is of particular interest for producing liquid fuels or chemical precursors from natural gas. The MMO-like enzymes that catalyze this reaction in nature, however, exhibit limited stability or poor heterologous expression (30) and have not been suitable for use in a recombinant host that can be engineered to optimize substrate or cofactor delivery. Alkane monooxygenases often cometabolize a wider range of alkanes than those which support growth (12). We wished to determine whether it is possible to engineer a medium-chain alkane monooxygenase to hydroxylate small alkanes, thereby circumventing difficulties associated with engineering MMO-like enzymes as well as investigating the fundamental question of whether enzymes unrelated to MMO can support growth on small alkanes.The most intensively studied medium-chain alkane hydroxylases are the AlkB enzymes (2, 20, 29), especially AlkB from Pseudomonas putida GPo1 (13, 28, 32, 35). While most members of the AlkB family act on C10 or longer alkanes, some accept alkanes as small as C5 (30). A recent study (12) indicated that AlkB from P. putida GPo1 may also be involved in propane and butane assimilation. AlkB selectively oxidizes at the terminal carbon to produce the 1-alkanols. No systematic protein engineering studies have been conducted on this di-iron integral membrane enzyme, although selection and site-directed mutagenesis efforts identified one amino acid residue that sterically determines long-chain alkane degradation (35).The most recent addition to the known biological alkane-hydroxylating repertoire is the CYP153 family of heme-containing cytochrome P450 monooxygenases. Although their activity was detected as early as 1981 (1), the first CYP153 was characterized only in 2001 (16). Additional CYP153 enzymes were identified and studied more recently (9, 10, 31). These soluble class II-type three-component P450 enzymes and the AlkB enzymes are the main actors in medium-chain-length alkane hydroxylation by the cultivated bacteria analyzed to date (31). CYP153 monooxygenases have been the subject of biochemical studies (9, 16, 19), and their substrate range has been explored (10, 14). Known substrates include C5 to C11 alkanes. The best-characterized member, CYP153A6, hydroxylates its preferred substrate octane predominantly (>95%) at the terminal position (9).Recent studies have shown that high activities on small alkanes can be obtained by engineering bacterial P450 enzymes such as P450cam (CYP101; camphor hydroxylase) and P450 BM3 (CYP102A; a fatty acid hydroxylase) (8, 36). The resulting enzymes, however, hydroxylate propane and higher alkanes primarily at the more energetically favorable subterminal positions; highly selective terminal hydroxylation is difficult to achieve by engineering a subterminal hydroxylase (22). We wished to determine whether a small-alkane terminal hydroxylase could be obtained instead by directed evolution of a longer-chain alkane hydroxylase that exhibits this desirable regioselectivity. For this study, we chose to engineer AlkB from P. putida GPo1 and CYP153A6 from Mycobacterium sp. strain HXN-1500 (9, 33) to enhance activity on butane. Because terminal alkane hydroxylation is the first step of alkane catabolism in P. putida GPo1, we reasoned that it should be possible to establish an in vivo evolution system that uses growth on small alkanes to select for enzyme variants exhibiting the desired activities.The recombinant host Pseudomonas putida GPo12(pGEc47ΔB) was engineered specifically for complementation studies with terminal alkane hydroxylases and was used previously to characterize members of the AlkB and CYP153 families (26, 31). This strain is a derivative of the natural isolate P. putida GPo1 lacking its endogenous OCT plasmid (octane assimilation) (5) but containing cosmid pGEc47ΔB, which carries all genes comprising the alk machinery necessary for alkane utilization, with the exception of a deleted alkB gene (34). We show that this host can be complemented by a plasmid-encoded library of alkane hydroxylases and that growth of the mixed culture on butane leads to enrichment of novel butane-oxidizing terminal hydroxylases. 相似文献
6.
7.
Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported. 相似文献
8.
Benjamin H. Gregson Gergana Metodieva Metodi V. Metodiev Boyd A. McKew 《Environmental microbiology》2019,21(7):2347-2359
Alcanivorax borkumensis SK2T is an important obligate hydrocarbonoclastic bacterium (OHCB) that can dominate microbial communities following marine oil spills. It possesses the ability to degrade branched alkanes which provides it a competitive advantage over many other marine alkane degraders that can only degrade linear alkanes. We used LC–MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on linear (n-C14) or branched (pristane) alkanes. During growth on n-C14, A. borkumensis expressed a complete pathway for the terminal oxidation of n-alkanes to their corresponding acyl-CoA derivatives including AlkB and AlmA, two CYP153 cytochrome P450s, an alcohol dehydrogenase and an aldehyde dehydrogenase. In contrast, during growth on pristane, an alternative alkane degradation pathway was expressed including a different cytochrome P450, an alcohol oxidase and an alcohol dehydrogenase. A. borkumensis also expressed a different set of enzymes for β-oxidation of the resultant fatty acids depending on the growth substrate utilized. This study significantly enhances our understanding of the fundamental physiology of A. borkumensis SK2T by identifying the key enzymes expressed and involved in terminal oxidation of both linear and branched alkanes. It has also highlights the differential expression of sets of β-oxidation proteins to overcome steric hinderance from branched substrates. 相似文献
9.
Maier T Förster HH Asperger O Hahn U 《Biochemical and biophysical research communications》2001,286(3):652-658
CYP153 a cytochrome P450 from Acinetobacter sp. EB104 catalyzes the hydroxylation of unsubstituted n-alkanes. We have decided to use the CYP153 system as a model for mechanistic studies on regioselective n-alkane oxidation and the interaction of hydrophobic substrates with soluble enzymes. Here the molecular cloning of the CYP153 gene is reported. Single specific primer PCR was applied to yield the whole gene sequence via chromosomal walks. CYP153 consists of 497 amino acids (M(r) = 56 kDa) and thus represents an unusually long bacterial P450, containing all P450 typical structural elements. It constitutes the new P450 family CYP153. The prolonged N-terminus of about 90 amino acids does not contain a so far known membrane-anchoring sequence but a 28-amino acid long amphipathic helix. The relevance of the remarkably long N-terminus and of other sequence motives like the hydrophobic F-G loop is discussed with respect to substrate binding and recognition. 相似文献
10.
11.
J. Alonso‐Gutiérrez M. Teramoto A. Yamazoe S. Harayama A. Figueras B. Novoa 《Journal of applied microbiology》2011,111(4):800-810
Aims: Investigation of the alkane‐degrading properties of Dietzia sp. H0B, one of the isolated Corynebacterineae strains that became dominant after the Prestige oil spill. Methods and Results: Using molecular and chemical analyses, the alkane‐degrading properties of strain Dietzia sp. H0B were analysed. This Grampositive isolate was able to grow on n‐alkanes ranging from C12 to C38 and branched alkanes (pristane and phytane). 8‐Hexadecene was detected as an intermediate of hexadecane degradation by Dietzia H0B, suggesting a novel alkane‐degrading pathway in this strain. Three putative alkane hydroxylase genes (one alkB homologue and two CYP153 gene homologues of cytochrome P450 family) were PCR‐amplified from Dietzia H0B and differed from previously known hydroxylase genes, which might be related to the novel degrading activity observed on Dietzia H0B. The alkane degradation activity and the alkB and CYP153 gene expression were observed constitutively regardless of the presence of the substrate, suggesting additional, novel pathways for alkane degradation. Conclusions: The results from this study suggest novel alkane‐degrading pathways in Dietzia H0B and a genetic background coding for two different putative oil‐degrading enzymes, which is mostly unexplored and worth to be subject of further functional analysis. Significance and Impact of the Study: This study increases the scarce information available about the genetic background of alkane degradation in genus Dietzia and suggests new pathways and novel expression mechanisms of alkane degradation. 相似文献
12.
Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450. 相似文献
13.
Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range. 相似文献
14.
Broad substrate Cytochrome P450 monooxygenase activity in the cells of Aspergillus terreus MTCC 6324
Cytochrome P450 (CYP) monooxygenase activities with different category of substrates namely, alkanes, alkane derivatives, alcohols, aromatic compounds, organic solvents, and steroids were detected in the cells of Aspergillus terreus. High CYP specific activity was observed when methanol (5.6+/-0.017 U mg(-1)), acetone (7.76+/-0.02 U mg(-1)), dimethylsulphoxide (DMSO) (9.70+/-0.005 U mg(-1)), n-hexadecane (4.39+/-0.02 U mg(-1)), or n-octadecane (4.23+/-0.01 U mg(-1)) were used as substrates. Significant CYP specific activity was also detected when naphthalene (3.80+/-0.002 U mg(-1)) was used as substrate. The CYP catalysis of n-hexadecane had followed both terminal and sub terminal oxidations. The activity was localized in the cytosol of n-hexadecane grown cells, while, it was apparently distributed in light mitochondrial fraction and microsomal fraction of glucose grown cells. The substrate specificities of CYP present in all the locations were similar irrespective of the substrates used for the growth. Heme staining of the microsomal fraction containing CYP and other proteins in SDS-PAGE showed single heme protein band with corresponding molecular weight of 110 kDa. 相似文献
15.
Kubota M Nodate M Yasumoto-Hirose M Uchiyama T Kagami O Shizuri Y Misawa N 《Bioscience, biotechnology, and biochemistry》2005,69(12):2421-2430
The cytochrome P450 CYP153 family is thought to mediate the terminal hydroxylation reactions of n-alkanes. We isolated 16 new P450 CYP153A genes (central region) from various environments such as petroleum-contaminated soil and groundwater, as well as one from the n-alkane-degrading bacterium Alcanivorax borkumensis SK2 (designated P450balk). The sequences of the new P450 genes were extended by PCR to generate full-length chimeric P450 genes, using the N- and C-terminal domains of P450balk. A differential CO-reduced P450 spectral analysis indicated that 8 P450 genes among the 16 chimeric genes were expressed in Escherichia coli to generate a soluble and functional enzyme. The several functional chimeric P450s and P450balk were further fused to the reductase domain of the self-sufficient P450 monooxygenase (P450RhF) at the C-terminus. E. coli cells expressing these self-sufficient P450 chimeric genes converted n-alkanes, cyclohexane, 1-octene, n-butylbenzene, and 4-phenyl-1-butene into 1-alkanols, cyclohexanol, 1,2-epoxyoctane, 1-phenyl-4-butanol, and 2-phenethyl-oxirane, respectively. 相似文献
16.
We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation. 相似文献
17.
Kim D Cryle MJ De Voss JJ Ortiz de Montellano PR 《Archives of biochemistry and biophysics》2007,464(2):213-220
Candida albicans contains 10 putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6x(His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, omega-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (omega-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d(3)-dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the omega- to the (omega-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The regioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYP4A family of enzymes, does not involve covalent binding of the heme to the protein. 相似文献
18.
A novel bacterial strain, DQ12-45-1b, was isolated from the production water of a deep subterranean oil-reservoir. Morphological, physiological and phylogenetic analyses indicated that the strain belonged to the genus Dietzia with both alkB (coding for alkane monooxygenase) and CYP153 (coding for P450 alkane hydroxylase of the cytochrome CYP153 family) genes and their induction detected. It was capable of utilizing a wide range of n-alkanes (C6-C40), aromatic compounds and crude oil as the sole carbon sources for growth. In addition, it preferentially degraded short-chain hydrocarbons (?C25) in the early cultivation phase and accumulated hydrocarbons with chain-lengths from C23 to C27 during later cultivation stage with crude oil as the sole carbon source. This is the first study to report the different behaviors of a bacterial species toward crude oil degradation as well as a species of Dietzia degrading a wide range of hydrocarbons. 相似文献
19.
Bell SG Hoskins N Xu F Caprotti D Rao Z Wong LL 《Biochemical and biophysical research communications》2006,342(1):191-196
Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically diverse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to approximately 2.0A have been obtained. 相似文献
20.
Do mammalian cytochrome P450s show multiple ligand access pathways and ligand channelling?
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Understanding substrate binding and product release in cytochrome P450 (CYP) enzymes is important for explaining their key role in drug metabolism, toxicity, xenobiotic degradation and biosynthesis. Here, molecular simulations of substrate and product exit from the buried active site of a mammalian P450, the microsomal CYP2C5, identified a dominant exit channel, termed pathway (pw) 2c. Previous simulations with soluble bacterial P450s showed a different dominant egress channel, pw2a. Combining these, we propose two mechanisms in CYP2C5: (i) a one-way route by which lipophilic substrates access the enzyme from the membrane by pw2a and hydroxylated products egress along pw2c; and (ii) a two-way route for access and egress, along pw2c, for soluble compounds. The proposed differences in substrate access and product egress routes between membrane-bound mammalian P450s and soluble bacterial P450s highlight the adaptability of the P450 fold to the requirements of differing cellular locations and substrate specificity profiles. 相似文献