The cytochromes P450 (P450s) are a superfamily of oxidative haemoproteins that are capable of catalysing a vast range of oxidative transformations, including the oxidation of unactivated alkanes, often with high stereo- and regio-selectivity. Fatty acid hydroxylation by P450s is widespread across both bacteria and higher organisms, with the sites of oxidation and specificity of oxidation varying from system to system. Several key examples are discussed in the present article, with the focus on P450BioI (CYP107H1), a biosynthetic P450 found in the biotin operon of Bacillus subtilis. The biosynthetic function of P450BioI is the formation of pimelic acid, a biotin precursor, via a multiple-step oxidative cleavage of long-chain fatty acids. P450BioI is a member of an important subgroup of P450s that accept their substrates not free in solution, but rather presented by a separate carrier protein. Structural characterization of the P450BioI–ACP (acyl-carrier protein) complex has recently been performed, which has revealed the basis for the oxidation of the centre of the fatty acid chain. The P450BioI–ACP structure is the first such P450–carrier protein complex to be characterized structurally, with important implications for other biosynthetically intriguing P450–carrier protein complexes.
Cytochrome P450s (P450s) are powerful oxidative haemoproteins that are found in many different species, including mammals, plants, insects and bacteria . The main function of these enzymes is, through a cysteine-ligated haem moiety, to activate molecular oxygen as an iron-oxo (ferryl) species. This particular species is a highly powerful oxidant and is able to return to a stable resting state through oxygen donation to a substrate, in a process that often occurs with high degrees of regio- and stereo-selectivity . Differences in the peptide backbone confer different substrate specificities on the various P450s and are able to subtly vary the oxidation chemistry that they carry out. The biological roles of P450s are as varied as the reactions that they catalyse; in eukaryotes, they are involved in a variety of biosynthetic conversions (steroid biosynthesis), cell signalling (arachidonic acid signalling pathway) and biodegradative transformations (xenobiotic metabolism). Prokaryotic P450s have been found to catalyse a plethora of biodegradative and biosynthetic oxidation reactions, the latter often affording molecules of medicinal significance .
The P450s are a growing superfamily of enzymes, not only purely in the number of identified P450s, but also in the chemistry that they catalyse, the substrates that they bind and the manner in which they effect oxygen activation . This can be seen in the increasing number of different types of redox partners possible for P450s, the identification of P450s capable of using peroxides as oxidants via the peroxide shunt pathway and in the use of CPs (carrier proteins) as scaffolds for oxidation of substrates . An excellent example of P450 diversity can be found in the oxidation of fatty acids, which, although appearing to be deceptively simple substrates, serve to highlight the biosynthetic ingenuity of Nature .
Notable fatty-acid-hydroxylating P450s
A large number of P450s have been reported to catalyse the oxidation of fatty acids in vitro, including examples from bacteria, mammals and plants (for example, see [6–9]). Fatty acids are often used as one of a small number of potential substrates for the initial characterization of P450s, especially where the biological function is unclear, which can often lead to reports of P450 activity with fatty acids as a substrate where the true nature of the biologically relevant oxidation is unknown. A small selection of P450s showing activity at different parts of the fatty acid chain is discussed in the present paper, focusing on those where some type of structural or biochemical rationale exists for the selectivity of oxidation observed (Figure 1).
Sites of oxidation reported for (a) free fatty acids or (b) fatty acyl–ACP by specific cytochrome P450s indicated by filled circles (hydroxylation) or a broken line (carbon–carbon bond cleavage).
Oxidation towards the methyl terminus: P450BM3 (CYP102A1)
The most widely studied fatty-acid-metabolizing P450 is P450BM3 (CYP102A1), isolated from Bacillus megaterium (a number of other P450s sharing similar features with P450BM3 have been identified more recently in the CYP102 subfamily) . P450BM3 is one of a small subset of soluble P450s that are found to be expressed as a fusion protein, where the required redox partners are combined within a single polypeptide chain (the same as is observed with mammalian P450s) . Turnover of fatty acids of C12–C18 chain length has been reported with oxidation occurring at the sub-terminal methylene carbons of the fatty acid chain . P450BM3 is able to catalyse very rapid substrate turnover with a high degree of coupling (representing the efficiency of oxidation with regard to electrons received) and, as such, has been a target for many mechanistic and directed evolution studies [12–14]. The selectivity of oxidation of P450BM3 normally covers three to five sub-terminal methylene carbons, with oxidation varying in distribution across these positions depending upon fatty acid chain length . The stereochemical purity of the oxidations is high and favours R-hydroxylation, with the stereoselectivity decreasing with increasing distance from the methyl group [15–17]. Selectivity of oxidation can be improved with the incorporation of functionality within the fatty acid chain; methyl-branched iso- and anteio-fatty acids show vastly improved regiochemical selectivity over their unbranched counterparts . In addition, the oxidation of unsaturated fatty acids can result in either hydroxylation or epoxidation, depending upon the position of the double bond(s) in relation to the terminal methyl carbon .
Oxidation towards the carboxylate group: P450BSβ (CYP152A1)
P450BSβ (CYP152A1), isolated from Bacillus subtilis, is a fatty acid hydroxylase with specificity for the carboxylic acid end of the fatty acid (the α- and β-carbons adjacent to the carboxy group) . Unusually, oxidation is supported not through the transfer of electrons from redox partner proteins, but rather through the use of hydrogen peroxide as an oxidant, making this enzyme a peroxygenase rather than a mono-oxygenase. This mechanism makes use of the so-called ‘peroxide shunt’ route in forming the active ferryl species and may contribute to the high rate of turnover observed for enzymes such as P450BSβ . The exact function of the enzyme within B. subtilis is unknown, although the degree of biochemical and structural characterization indicates that fatty acids are likely to be substrates in vivo . The use of the inexpensive oxidant hydrogen peroxide also makes this enzyme an attractive target for industrial biocatalysis.
Oxidation of the methyl terminus: CYP4A1 and CYP52A21
The selective oxidation of the methyl terminus of fatty acids is a more challenging transformation than in-chain methylene oxidation, as the oxidation is more energetically demanding and the centre to be oxidized lies directly next to more easily oxidized methylene carbons . The mammalian CYP4 subfamily is able to perform this oxidation with impressive degrees of selectivity obtained for oxidation of the terminal carbon atom . A mechanistic explanation for this process involves a constricted channel controlling access of the substrate methyl terminus to the active-site haem. Calibration of this site using terminally halogenated fatty acids has revealed that, whereas chlorine and bromine atoms can be oxidized, iodine atoms are not oxidized, implying an access tunnel with a diameter between 3.9 and 4.3 Å (1 Å=0.1 nm) for CYP4A1 . The fungal P450 CYP52A21, another fatty acid terminal methyl hydroxylase, has also been investigated using this technique with similar results, albeit that the iodine terminally substituted fatty acid is oxidized to a minor extent in this system .
Oxidation at the centre of the chain: P450BioI (CYP107H1)
Cytochrome P450BioI (CYP107H1) is a biosynthetic P450 found in the biotin operon of Bacillus subtilis . The role of P450BioI is the biosynthesis of pimelic acid, a C7 diacid, formed via the oxidation of a long-chain fatty acid . Initial postulates for the mechanism of P450BioI included ω-(methyl)-hydroxylation of fatty acids followed by chain-shortening reactions [27,28] or via direct in-chain cleavage of the fatty acid . Turnover data for long-chain fatty acids indicated that pimelic acid was indeed a direct product of P450BioI-mediated fatty acid oxidation, albeit at low levels . The vast majority of oxidation observed formed fatty acids hydroxylated towards the methyl terminus of the fatty acid chain, with a similar type of product distribution to P450BM3 in terms of both regiochemistry and stereochemistry . Importantly, no ω-hydroxylated product was found in any turnovers of long-chain fatty acids, which argued against the first postulate for the mechanism of pimelic acid formation. The identities of the products of oxidation were confirmed by analysis with chemically synthesized standards, thus removing any potential ambiguity in the assignment of the positions oxidized in the fatty acid chain. Incubation of synthetic fatty acid intermediates bearing oxidation at the centre of the fatty acid chain also indicated that an in-chain cleavage mechanism was consistent with alcohol/vic-diol intermediates that then undergo cleavage to two aldehydes .
Pimelic acid production by P450BioI
P450BioI binds an ACP (acyl-carrier protein)-bound substrate
Acyl–ACP had originally been identified as a potential substrate for P450BioI when the overexpression of P450BioI in Escherichia coli afforded moderate amounts of ACP-bound P450BioI complex in addition to free P450BioI . Turnover of the isolated P450BioI–acyl–ACP complex was supported under the same conditions as unbound fatty acid turnover, with the sole product ACP-bound pimelic acid (identified following base cleavage from the ACP thiol and MS analysis). This gave further support to the hypothesis that P450BioI oxidized fatty acids via an in-chain mechanism, and also that the possible substrates were likely to be ACP-bound, rather than unbound, fatty acids .
The P450BioI–ACP complex can be reconstituted
To gain further insights into the P450BioI–ACP complex, it was important to be able to recreate the complex with specific fatty acids in vitro. Reconstitution of the P450BioI–ACP complex in vitro was successfully achieved via the incubation of P450BioI with various acyl–ACPs, which had been prepared in a two-step process from apo-ACP. This process made use of E. coli ACP synthase to form the holo-ACP using CoA, followed by acyl–ACP synthase that loaded various fatty acids in the presence of ATP. Formation of the acyl–ACPs was determined by MS and denaturing PAGE analysis, with the purification of the complex performed using a two-step affinity purification protocol taking advantage of the presence of different affinity tags on each protein .
Structural analysis of the P450BioI–ACP complex
The structure of the P450BioI–ACP complex was determined, using single anomalous diffraction of seleneomethionine labelled crystals, for tetradecanoic acid, hexadec-(9Z)-enoic acid and octadec-(9Z)-enoic acid fatty acyl–ACPs (PDB codes 3EJB, 3EJD and 3EJE respectively) . P450BioI displays the canonical P450 fold, possessing primarily α-helical secondary structure, with some important alterations in the so-called SRSs (substrate-recognition sites) when compared with similar P450 structures . These include differences in the β-1 sheet (SRS 5), the loop between helices B and B2 (SRS 1) and the loop region between the F- and G-helices (SRSs 2 and 3), which together form the entry tunnel for the fatty acid and the attached prosthetic linker of ACP to access the active site (Figure 2).
Overall structure of the P450BioI–ACP complex
Substrate orientation and binding in P450Biol and comparable P450s
Fatty acid substrates are bound in the active site of P450BioI in a U-conformation, with the region of closest approach to the haem being the C-7 and C-8 carbons of the fatty acid chain [average carbon atom–haem iron distances for C-7 (4.3±0.3 Å) and for C-8 (4.7±0.5 Å)]. This unusual conformation is enforced by a series of large hydrophobic residues that line the binding pocket (Figure 3B). Ile169 is particularly notable, as this residue provides a pivot point directly above the haem iron, forcing the fatty acid to adopt a conformation appropriate for oxidation. Accommodation for the methyl termini of fatty acid chains is provided in a large hydrophobic cavity predominantly formed by residues of the F-, G- and I-helices. The size of this cavity is such that the methyl termini of fatty acids greater than 16 carbons in length are no longer visible in the electron density due to conformational mobility, with this structural feature allowing ACP-bound fatty acids of various lengths to be utilized as substrates by P450BioI .
The prosthetic phosphopantetheine linker of ACP, to which the fatty acid is bound, plays a crucial role in orienting the fatty acid appropriately for oxidation. The amide nitrogen and carbonyl oxygen atoms form hydrogen bonds with various P450BioI side chain or backbone atoms, whereas the phosphate and alcohol moieties are involved in numerous water-mediated hydrogen-bonding interactions with P450BioI. The presence of the linker allows the fatty acid to be positioned such that the two atoms above the haem are the required C-7 and C-8 carbons by forming the particular hydrogen-bonding pattern that it adopts. Such interactions are crucial given that the highly hydrophobic pocket surrounding the haem has no hydrogen-bonding interactions with the fatty acid carboxyl oxygen (Figure 3A).
The ACP molecule forms protein–protein interactions with P450BioI, with the majority of interactions occurring through the interaction of acidic residues of ACP with basic residues of P450BioI, or the interaction of backbone amide nitrogen and carbonyl oxygen atoms (Figure 3C). The structure of the P450-bound ACP molecule is little altered from the structure of apo-ACP , with the exception of the post-translationally modified serine residue (Ser57ACP) that rotates to allow the linker and bound fatty acid to project into the core of P450BioI.
The binding of the ACP-bound fatty acid in P450BioI is clearly different to that observed in the crystal structures of the fatty acid hydroxylases P450BM3 and P450BSβ, although both of these enzymes also share (somewhat unsurprisingly) a largely hydrophobic active-site pocket (Figure 3D). The electrostatic interactions of hexadec-(9Z)-enoic acid and P450BM3 involve the carbonyl oxygen of the fatty acid and the phenol of Tyr51BM3 (PDB code 1FAG) . Addition of a β-glycine unit to the fatty acid carboxy group results in a much tighter interaction with P450BM3, which is mediated through additional interactions of the glycine carboxy group with the backbone amide nitrogens of residues Gln73BM3 and Ala74BM3 (PDB code 1JPZ) . The binding of fatty acids in P450BSβ occurs in essentially the opposite orientation to that seen in P450BM3, with the carboxylic acid moiety located very close to the haem iron (PDB code 1IZO) . Electrostatic interactions of the substrate with the P450 are achieved through the interaction of the guanidinium group of Arg242BSβ with the carboxy group of the fatty acid. Examining the conformations of the three fatty acid substrates in the overlaid P450s shows that the fatty acids in both P450BM3 and P450BSβ adopt an orientation that is essentially parallel to the haem, in contrast with P450BioI, which utilizes a distinct U-conformation.
Implications for other P450 systems
CP-bound substrates have been either indicated or postulated for a number of other P450s, the majority of which occur in biosynthetic operons of medicinally important natural products. The oxidation of CP-bound amino acid residues have been shown to occur in the biosynthesis of the antibiotic novobiocin, where the CP domain of the non-ribosomal peptide synthase NovH presents tyrosine for hydroxylation by P450NovI . Similar CP–P450 systems are present in the biosynthesis of hydroxylated amino acid precursors for several further aminocoumarin antibiotics [39–41], the nikkomycins  and also the vancomycin antibiotics . Another group of P450s acting upon CP-bound substrates are found in the biosyntheses of the vancomycin- and teicoplanin-type glycopeptide antibiotics [44,45], where CP domains bearing the biosynthesized peptides undergo P450-mediated phenolic coupling of select aromatic side chains of the peptide amino acid residues [46,47]. The biosyntheses of these compounds require three and four of these phenolic coupling steps respectively, with the reactions occurring upon the peptide bound to the sixth or seventh CP domains of the non-ribosomal peptide synthases . The phenolic coupling catalysed by the first phenolic coupling P450 in the biosynthesis of vancomycin, P450OxyB, has been demonstrated to occur upon CP-bound substrate, with turnover and binding both facilitated by the presence of the CP as a scaffold for oxidation . The medicinal relevance of these compounds makes the characterization of these phenolic coupling P450–CP systems one of clear importance for the future.
Experimental Approaches to Protein–Protein Interactions: A Biochemical Society Focused Meeting held at University of Sheffield, Sheffield, U.K., 11–12 January 2010. Organized and Edited by Michael Sutcliffe (Manchester, U.K.) and Mike Williamson (Sheffield, U.K.).
I am indebted to Professor Dr Ilme Schlichting for her continual support, advice and encouragement.
This work is supported by the Human Frontier Science Program (Cross Disciplinary Fellowship).