MINIREVIEW The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism Inge N A Van Bogaert1, Sara Groeneboer2, Karen Saerens1 and Wim Soetaert1 Department of Biochemical and Microbial Technology, Laboratory of Industrial Biotechnology and Biocatalysis, Ghent University, Ghent, Belgium Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, Belgium Keywords alkanes; bacteria; biosurfactants; cytochrome P450 monooxygenase; dicarboxylic acids; fatty acids; hydroxylation; oxylipins; polyketides; yeast Correspondence I N A Van Bogaert, Department of Biochemical and Microbial Technology, Laboratory of Industrial Biotechnology and Biocatalysis, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium Fax: +32 264 62 31 Tel: +32 264 60 34 E-mail: inge.vanbogaert@ugent.be Website: http://www.inbio.be Cytochrome P450 monooxygenases (P450s) are a diverse collection of enzymes acting on various endogenous and xenobiotic molecules Most of them catalyse hydroxylation reactions and one group of possible substrates are fatty acids and their related structures In this minireview, the significance of P450s in microbial fatty acid conversion is described Bacteria and yeasts possess various P450 systems involved in alkane and fatty acid degradation, and often several enzymes with different activities and specificities are retrieved in one organism Furthermore, P450s take part in the formation of fatty acid-based secondary metabolites Finally, there are a substantial number of microbial P450s displaying activity towards fatty acids, but to which no biological role could be assigned despite the often quite intense research (Received 22 June 2010, revised 19 August 2010, accepted 16 September 2010) doi:10.1111/j.1742-4658.2010.07949.x Introduction P450 classification and nomenclature Cytochrome P450s form a vast and divergent family of enzymes They are heme–thiolate proteins, bearing, in a hydrophobic pocket, a protoporphyrin IX linked to the apoprotein by a bond between the heme iron centre and the sulfur atom of a conserved cysteinyl residue In a typical reaction catalysed by a P450, molecular oxygen binds to the heme iron for activation before transfer to the substrate Carbon monoxide can also bind, leading to a reduced P450 producing a characteristic CO-binding difference spectrum with an absorbance maximum at 450 nm This P450–CO complex is inactive and has given the name to P450 (pigment absorbing at 450 nm) [1] Inhibition by CO and reversion of this inhibition by 450 nm light are characteristic for most reactions catalysed by P450s New genes are annotated as a P450 based on the presence of typical conserved domains involved in heme binding and proton transfer [2] P450s have been categorized in families and subfamilies They belong to the same family when they share ‡ 40% amino acid identity and they belong to the same subfamily when they share ‡ 55% amino acid identity [3] For example, Abbreviations GPo1, alkane hydroxylase; P450, cytochrome P450 monooxygenase; psi factor, precocious sexual inducers 206 FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS I N A Van Bogaert et al CYP94A1 from the plant Vicia sativa is the first member of subfamily A of family 94 (there is more about the activity of this enzyme in the accompanying minireview by Pinot & Beisson [4]) Over 11 000 P450 genes are included in this internationally used nomenclature system, and probably an extensive number of nonclassified genes are present in the emerging pile of genome sequencing data Besides being vast, the P450 superfamily is also highly divergent For example, in plant P450s, CYP94 identity may be as low as 16% with CYP51 and even only 12% with CYP74 Divergence is further illustrated by the huge number of families (977) and subfamilies (2519) Phylogenetic studies have suggested that the unicellular ancestor of plants had at least three distinct P450 branches [2] The first, sharing a common ancestor with animal P450s involved in xenobiotic metabolism, gave rise to Group A, catalysing typical plants reactions (i.e synthesis of lignin, flavonoids, etc.) Group non-A is composed of two branches The first originates from a common ancestor with animal CYP4 and fungal CYP52 fatty acid hydroxylases; plant fatty acid hydroxylases of the families CYP86 and CYP94 originate from this branch The second branch of Group non-A shares a common ancestor with animal, fungal and bacterial sterol oxidases (CYP51) This branch gave rise to the plant obtusifoliol 14 demethylase (CYP51) and brassinolide hydroxylases (CYP85 and CYP90 families) Microbial P450s and fatty acids Fatty acids are simple, yet indispensable, molecules to any living cell Incorporated in phospholipids, they make up the major part of the plasma membrane and besides structural roles, they also function as a carbon or energy source Furthermore, modified fatty acids are building blocks for other complex molecules or act as signalling molecules to trigger physiological changes All these roles and processes require specific enzymes, and cytochrome P450 monooxygenases (P450s) make up a significant part of them P450s are heme–thiolate proteins involved in the hydroxylation of a wide range of endogenous and xenobiotic compounds They are present in every eukaryotic organism and in a substantial number of prokaryotes More than 3800 microbial P450s are known to date [5] and we estimate that  10–17% of them display activity towards fatty acids or related structures On the one hand, these activities are linked to the degradation of fatty acids and alkanes; metabolization of these latter compounds is inherently coupled to fatty acid degradation because the conversion of alkanes to fatty acids is The role of P450 in microbial fatty acid metabolism an essential step in the alkane assimilation process On the other hand, P450s are also involved in the synthesis of special fatty acid-based molecules such as secondary metabolites or signal molecules Although the P450s described in this review all act on fatty acid substrates, this is not reflected in their overall similarity; according to Nelson’s classification system based on amino acid identity they belong to various families [5] Besides the involvement in different physiological functions, P450s also differ in the position of the hydroxylation; this may occur close to the carboxyl group, giving rise to a- or b-hydroxylated fatty acids (mediated by CYP152), in-chain (e.g CYP1006) or at the terminal or subterminal ending (e.g CYP52) Several classes of P450s involved in either metabolization or biosynthesis processes and with different regiospecificities are discussed Whenever possible, the internationally used nomenclature is applied [3] Fatty acid metabolism Alkane degradation Microbial populations can break down almost every natural organic compound Even alkanes, which from a chemical point of view are almost inert molecules, can be degraded and utilized as a carbon source by both bacteria and fungi Traditional aerobic alkane assimilation is initiated by terminal hydroxylation In the subsequent oxidation steps, the corresponding primary alcohol is converted via an aldehyde to a fatty acid, which will enter b-oxidation [6] Depending on the particular microorganism, initial oxidation can be governed by several unrelated alkane hydroxylases Although microbial alkane degradation was first demonstrated about a century ago [7], research on this topic was only really boosted in the 1950s and 1960s when the production of single cell protein based on paraffin or alkanes became a hot topic The first alkane hydroxylase (GPo1) was found in the early 1960s in the soil bacteria Pseudomonas oleovorans (later renamed Pseudomonas putida) and was shown to be an integral membrane-bound nonheme di-iron monooxygenase [8] More recently, related genes have been isolated from a broad range of bacteria [9] Whereas various yeasts were also well established as single cell protein producers, it took another few decades before their corresponding alkane hydroxylase enzymes were identified as cytochrome P450 monooxygenases, unrelated to the bacterial GPo1 system [10] To date, all yeast alkane hydroxylases belong to the CYP52 family (membrane-bound class II) This family contains several enzymes with demonstrated activity towards FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS 207 The role of P450 in microbial fatty acid metabolism I N A Van Bogaert et al alkanes and ⁄ or fatty acids, but also harbours numerous genes which have not been explored (yet) or are proven to be pseudogenes Well-studied members of the CYP52 family are the enzymes of the alkane-metabolizing yeasts Candida tropicalis, Candida maltosa and Yarrowia lipolytica C tropicalis has at least 18 genes belonging to this family Craft et al [11] evaluated 10 of them by quantitative competitive reverse transcription-PCR; this highly specific technique is required because of the high identity between the genes Five genes were clearly induced by octadecane (CYP52A12, -13, -14, -17 and -18), whereas no transcription was detected for CYP52A15, -16, -19, -20 and D2 under those and other conditions Also, C maltosa possesses several so called P450alk genes: alk1 to alk8 Four genes are considered to be the primary P450alk genes: alk1, alk2, alk3 and alk5, corresponding to CYP52A3, -A5, A4 and -A9, respectively Quadruple mutants were unable to grow on alkanes as a sole carbon source, but complementation by one of the four genes restored growth on hexadecane [12] The corresponding enzymes differ in their substrate chain-length specificity, giving a possible reason for the multitude of P450alk genes often present in one organism Although the induction of P450alk genes in response to n-alkanes or fatty acids is a common feature among alkane-assimilating yeasts, the underlying molecular mechanisms remain largely unknown C maltosa alk2 turned out to be inducible by alkanes, as well as by the peroxisome proliferator clofibrate The respective cis-acting elements in the alk2 promoter region were identified and there are indications of similar motives in other C maltosa alk promoters [13] Y lipolytica is another well-studied alkanotrophic yeast with 12 P450alk genes in its genome Six of the eight tested genes showed induction by alkanes Among them, alk1 (CYP52F1) displayed the highest expression and although single disruptions in the other genes did not result in yeasts unable to metabolize alkanes, Dalk1 mutants are unable to grow on decane In addition, a Dalk1Dalk2 double mutant cannot grow on hexadecane either Therefore, it is suggested that the primary P450alk gene alk1 is required for assimilation of decane and dodecane, whereas alk2 (CYP52F2) is involved in the assimilation of molecules longer than dodecane The other alk genes possibly act on even longer alkanes or other types of carbon chains [14] Recently, Ohta’s group shed light on the transcriptional induction of the alk1 gene by alkanes [15]; in the presence of alkanes, Yas1p and Yas2p, two basic helix–loop–helix proteins form heterodimers and bind to the cis-acting alkane-responsive element in the alk1 promoter The protein complex also binds to 208 other promoters of genes interfering in alkane degradation such as the acetoacetyl-CoA thiolase gene and the yas1 gene itself This latter binding creates a positive autoregulatory feedback which results in a quick and profound transcriptional response to alkanes Furthermore, a third regulatory protein was identified The repressor Yas3p binds specifically to Yas2p when no alkanes are present, but when the yeast is exposed to alkanes, Yas3p is translocated from the nucleus to the endoplasmatic reticulum Conserved motives of the alkane-responsive element sequence were retrieved in the P450alk promoters of C maltosa, C tropicalis and Debaryomyces hansenii, suggesting a common mechanism for alkane-responsive induction Although Cardini and Jurtshuk [16] provided strong indications for the involvement of a bacterial P450 in the hydroxylation of octane in Rhodococcus rhodochrous, the role of bacterial P450s in alkane degradation in addition to the well-established alkane hydroxylase system has long been underestimated The first bacterial P450alk was cloned from Acinetobacter calcoaceticus in 2001, this class I P450 was assigned to the new family CYP153 [17] Recently, similar enzymes were found in other alkane-utilizing species such as Sphingomonas sp HXN200, Mycobacterium sp HXN1500 and Alcanivorax borkumensis Van Beilen et al [18] were able to demonstrate the functionality of seven of eleven genes (CYP153A6, -A7, A11, A13, A14 and -D1) by functional expression in a DGPo1 P putida strain and its restored ability to grow on alkanes Most alkanotrophes attack various or mixed substrates with different and often length-specific enzymes, reflected in the variation among yeast CYP52 genes and the bacterial alkane hydroxylase system A similar trend can be observed for the bacterial P450alk enzymes For example, Sphingomonas sp HXN200 possesses five CYP153 genes, three of which show activity towards C5–C10 alkanes, whereas no affinity for these substrates was observed for the other two genes, suggesting that these genes are either pseudogenes or act on different substrates such as long-chain alkanes Other organisms possess several types of enzymes; Al borkumensis contains two alkane hydroxylases and two CYP153s [19] and whereas the first alkane hydroxylase is essential for the degradation of C6, no clear role could be assigned to the second, but double knockouts resulted in deficient growth on C8–C16 Because this organism is, thanks to its efficient and broad-spectrum hydrocarbon-degrading capacities, a dominant microbe in oilpolluted waters, the CYP153 enzymes are postulated to cover the rest of the alkane length range In general, one associates alkane-degrading organisms with oil-polluted environments, but in fact FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS I N A Van Bogaert et al alkane-degrading enzymes are found in many more organisms than the ones strictly appearing in oilrelated niches Indeed, alkanes are persistent molecules and this property makes them the perfect components for natural barriers such as plant and insect cuticles The major component of plant cuticles is cutin, a polymer consisting of omega hydroxy fatty acids crosslinked by ester and epoxide bonds, which is impregnated and covered with waxes (more information on the constitution and biosynthesis of plant envelopes can be found in the accompanying minireview by Pinot & Beisson [4]) These cuticular and epicuticular waxes are a mixture of long-chain alkanes (C16–C30) and related structures For example, in the rice blast fungus Magnoporthe oryzae, a putative alkane-degrading cytochrome P450 (MGG_05908.5 or CYP584A2) is upregulated upon the first stages of infection, together with other genes regarding the utilization of nonconventional carbon sources [20] These findings suggest that, on the one hand, alkane degradation is required for breaking the plant’s defence but, on the other hand, alkanes serve as nutritional input during the initial colonization steps Insect cuticle consists of protein and chitin, and is covered by a highly resistant lipid layer: the epicuticle This epiculticle is composed of hydrocarbons, wax esters, fatty alcohols and fatty acids Hydrocarbons are the prevalent component and include alkanes, alkenes and methyl-branched chains in various ratios, depending on the insect species In general, alkanes make up the biggest part of the hydrocarbon moiety and their chain length ranges between C21 and C35, with a particular preference for odd-numbered chains [21] The entomopathogenic fungus Metarhizium anisopliae infects a broad range of insects by direct penetration of the host cuticle and hence can be exploited as a biological control agent of pests cDNA microarray analysis of the fungus grown on cuticular extracts revealed a clear upregulation of an alkane-inducible cytochrome P450 (AJ273607) The role of P450 in microbial fatty acid metabolism during the first hours of incubation [22] According to amino acid similarity, this gene should be classified in the CYP52 family Several other expressed sequence tag (EST) or microarray cDNA analysis regarding entomopathogenic infection concealed involvement of P450s supposed to hydroxylate alkanes and ⁄ or fatty acids Fatty acid degradation – omega oxidation Although cytochrome P450s not intervene in the degradation of fatty acids in the b-oxidation cycle itself, they take part in the steps preceding b-oxidation (Fig 1) As mentioned in the previous section, alkanes can be converted to common fatty acids by initial cytochrome P450 interference However, the same enzymes responsible for terminal alkane oxidation often also mediate subterminal oxidation The corresponding secondary alcohols are oxidized to ketones and a Baeyer–Villiger monooxygenase converts them to esters, which are cleaved to give rise to a primary alcohol and a common fatty acid Furthermore, common fatty acids can be terminally oxidized by a cytochrome P450 monooxygenase and the resulting hydroxy-fatty acid is further converted to a dicarboxylic acid, which then enters b-oxidation This so-called x-oxidation occurs both in bacteria and yeasts, and yet is far more documented for this latter group because medium- and long-chain dicarboxylic acids are commercially produced by yeast fermentations to serve as building blocks of, for example, perfume, polymers, high-quality lubricants or macrolide antibiotics In this respect, the previously discussed fungal CYP52 enzymes are versatile enzymes; several isoforms exhibit different activities and specificities towards alkanes, as well as towards fatty acids or related structures An individual CYP52 not only demonstrates a distinct substrate specificity regarding chain lengths, Fig Assimilation of alkanes and fatty acids The (putative) involvement of cytochrome P450 monooxygenases is indicated by grey arrows FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS 209 The role of P450 in microbial fatty acid metabolism I N A Van Bogaert et al but in addition has preferences for either alkanes or fatty acids, and similarly, compared with the chain lengths, alkane ⁄ fatty acid specificities of the various enzymes within species are often overlapping The C maltosa P450Cm1 (ALK1 or CYP52A3), for example, prefers alkanes, whereas P450Cm2 (ALK3 or CYP52A4) shows highest affinity towards fatty acids Nevertheless, both enzymes are able to hydroxylate both substrates However, the C maltosa CYP52A10 and CYP52A11 enzymes are only able to convert fatty acids [23,24] It is not completely clear which gene(s) exactly mediate dicarboxylic acid formation, but Kogure et al [25] demonstrated that the alk5 gene (CYP52A9) was highly induced when comparing a C maltosa dicarboxylic acid overproducing mutant with a reference strain Interestingly, P450alk5 is indeed the isozyme with the strongest tendency to x-hydroxylate fatty acids By contrast, in vitro experiments after heterologous expression demonstrated that the highly active CYP52A3, although alkane preferring, not only converts hexadecane to its primary product 1-hexadecanol, but also further oxidizes this component to hexadecanal, hexadecanoic acid, 1,16hexadecandiol, 16-hydroxyhexadecanoic acid and even 1,16-hexadecanedioic acid, in this way bypassing the two enzymes normally involved in x-oxidation [26] The authors did not verify this phenomenon for other fatty acid-oxidizing C maltosa isozymes, but one can postulate that CYP52A9 generates a similar oxidation cascade This assumption is supported by data from another dicarboxylic acid-producing strain Just like C maltosa, C tropicalis mutant strains are used in industrial dicarboxylic acid production, the strains are among others blocked in b-oxidation by inactivation of the POX genes, resulting in higher dicarboxylic acid yields Upon exposure to oleic acid, CYP52A13 and CYP52A17 are strongly and consistently induced Again, enzymatic tests after heterologous expression revealed the enzyme’s abilities to synthesize dicarboxylic acids CYP52A17 shows the greatest overoxidizing capacities not only regarding substrate chain length, but also concerning activity; the conversion of oleic acid to its diacid occurs twice as quickly as the formation of x-hydroxy oleic acid [27] Despite the in vitro evidence, there is no clear answer to the question of which role this P450 oxidation cascade plays in vivo One can assume that the prevalence of different enzyme systems contributes to the yeast capacities to grow efficiently on a broad chain-length range of alkanes and fatty acids (e.g C7–C40 for C maltosa) Another advantage of the P450 bypass is the circumvention of H2O2 formation by the fatty alcohol oxidase However, the overoxidation cascade requires 210 three molecules of NADPH, potentially creating metabolic limitations In classical x-oxidation, only one NADPH molecule is used, whereas the alcohol dehydrogenase delivers one reducing equivalent (NADH) Members of the CYP52 family are supposed to be all linked to alkane and ⁄ or fatty acid hydroxylation, yet these assumptions are made based on the amino acid sequence and some enzymes might be involved in unrelated biological processes CaAlk8, for example, is the only CYP52 member originating from C albicans (CYP52A21) Although it has been shown that the enzyme terminally and subterminally hydroxylates lauric, myristic and palmitic acid [28], and is involved in alkane degradation, Panwar et al [29] suggested that CYP52A21 takes part in conferring multidrug resistance to the opportunistic pathogen C albicans Disruption of CYP52A21 in the wild-type strain did not lead to a drug-sensitive strain, probably attributed to the presence of several other multidrug resistance mechanisms Nevertheless, the role of CYP52A21 in multidrug resistance was demonstrated by overexpression in Saccharomyces cerevisiae and in a hypersensitive C albicans host, rendering the latter resistant to fluconazole, itraconazole and 4-nitroquinoline oxide In addition, experiments with C albicans microsomes indicate that resistance is caused by CYP52A21-mediated drug modification Besides the yeast species discussed above, Corynebacterium sp is also known as a producer of dicarboxylic acids [30] P450s are probably involved, but the exact pathway remains unrevealed By contrast, a specific P450 could be put forward as a candidate for x-oxidation in the cyanobacteria Anabaena variabilis Although CYP110 is induced by alkanes such as hexadecane, CYP110 does not participate in alkane degradation; findings which are supported by the fact that alkanes are toxic for Anabaena variabilis Based on the sequence similarity and binding affinity experiments with fatty acids, it was suggested that CYP110 is related to fatty acid x-oxidation of saturated and (poly)unsaturated fatty acids and subsequent formation of dicarboxylic acids which then undergo b-oxidation [31] The alkane inducibility of the cyp110 mRNA was used to design a hexadecane biosensor system [32] Biosynthesis of a- and b-hydroxylated fatty acids Not only are hydroxylated fatty acids intermediates in alkane and fatty acid metabolization, they can also be useful components as such a-Hydroxylated long-chain fatty acids, for example, are important constituents of sphingolipids These lipids are essential components of FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS I N A Van Bogaert et al mammalian cell membranes, but can also be found in some bacterial and fungal cell membranes Interestingly, bacterial sphingolipid synthesis predominantly occurs in anaerobic species One such anaerobe is Sphingomonas paucimobilis Its sphingolipids are rich in a-hydroxymyristic acid and the enzyme responsible for the conversion of myristic acid has been identified as a member of the P450 superfamily: P450SPa or CYP152B1 [33] However, the enzyme utilizes H2O2 instead of O2 and does not require reducing equivalents, comparable with the peroxide shunt reaction for common P450s [34] Indeed, P450SPa lacks the critical residues compulsory for the fulfilment of the typical monooxygenation reaction; in the distal I helix the Asp ⁄ Glu–Thr proton delivery system is substituted by Arg–Pro and, albeit the heme-binding cysteine was retrieved, the preceding consensus motive involved in electron transfer was modified [35,36] Furthermore, it sounds quite logical that anaerobic organisms try to circumvent the use of molecular oxygen The enzyme is highly specific towards fatty acids; no alkanes, fatty alcohols or fatty aldehydes are hydroxylated In addition to myristic acid, the enzyme shows activity to slightly shorter or longer saturated fatty acids and arachidonic acid [37] Based on a database similarity search with the P450SPa gene, Matsunaga’s group were able to identify another fatty acid hydroxylase in Bacillus subtillis However, this P450Bsb or CYP152A1 enzyme is less regiospecific; myristic acid is converted to a mixture of a- and b-hydroxymyristic acid, with a slightly higher amount of the b-hydroxylated product [38] A few years ago, P450CLA (CYP152A2) from the anaerobe Clostridium acetobutylicum was characterized: like P450Bsb, this enzyme performs both a- and b-hydroxylations of saturated and unsaturated fatty acids, but in this particular case the a-position is preferred [39] Although there is a link between the occurrence of fatty acid a-hydroxylation and sphingolipids, some questions remain: what is the biological role of P450Bsb in the non-sphingolipid-producing B subtillis and why is a mixture of a- and b-forms produced? It was suggested that peroxide-utilizing P450s might be involved in the oxygen-detoxification system of Clostidium acetobutylicum [39] Although anaerobic, Clostridium species can tolerate microoxic conditions (< 5% O2) Besides the classical oxygen detoxification systems, heme oxygenases, oxidases and lipid peroxidase scavenging enzymes are involved in the establishment of the anoxic microenvironment [40] Furthermore, P450SPa turned out to be capable of hydroxylating phytanic acid (3,7,11,15-tetramethyl hexadecanoic acid), a degradation product of chlorophyll The role of P450 in microbial fatty acid metabolism [41] This branched fatty acid cannot undergo b-oxidation because of methylation at the b-position In humans, metabolization occurs by an initial a-oxidation step, resulting in the removal of one carbon instead of two [42] The subsequent pristanic acid will be entirely degraded by b-oxidation Oxidation of phytanoyl-CoA is mediated by phytanoyl-CoA dioxygenase, an iron requiring non-heme oxidoreductase Homologue enzymes can be retrieved in a wide variety of bacteria, questioning the role of P450 in bacterial a-oxidation of phytanic acid Fatty acid hydroxylating P450s involved in secondary metabolite synthesis Biosurfactants Biosurfactants are surface-active compounds capable of reducing interfacial tension between liquids thanks to their amphiphilic properties Amphiphilic molecules consist of a hydrophilic and a hydrophobic moiety that interacts with the phase boundary in heterogeneous systems, allowing them to, for example, act as a detergent, wetting agent or emulsifier of oil ⁄ water mixtures In general, common fatty acids or b-hydroxy fatty acids originating from b-oxidation act as the hydrophobic part However, in some particular cases, P450 hydroxylated fatty acids make up the hydrophobic tail One such example are the cellobiose lipids produced by several yeast-like fungi (Fig 2A) Ustilagic acids from the plant-pathogen Ustilago maydis contain 15,16-dihydroxypalmitic acid or 2,15,16-trihydroxypalmitic acid Teichmann and co-workers [43] elucidated the biosynthetic gene cluster harbouring two P450 genes Upon disruption of the first P450 gene, cyp1, no cellobiose lipids could be detected CYP1 catalyses the conversion of palmitic acid to juniperic acid and this terminal hydroxylation is essential for the covalent binding of the hydroxylated fatty acid to the cellobiose moiety By contrast, Dcyp2 mutant strains retain their capacities to secrete cellobiose lipids Yet, these molecules lack the typical hydroxylation at the subterminal position These findings prove that CYP1-dependent x-hydroxylation does not depend on prior subterminal hydroxylation and that both enzymes are highly selective for either the terminal or subterminal position Surprisingly, CYP1 and CYP2 share only 15% amino acid identity and despite their activity towards the terminus of fatty acids, they are not classified into the CYP52 family but, based on their amino acid sequence, are assigned CYP5025A1 and CYP5030A1, respectively No P450 activity is linked to a-hydroxylation; this FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS 211 The role of P450 in microbial fatty acid metabolism I N A Van Bogaert et al on the role of CYP52E1 and -E2 [45] C bombicola, another sophorolipid-producing yeast closely related to C apicola, harbours at least eight cyp52 genes One of these, CYP52M1, was highly induced upon sophorolipid production, suggesting its participation in the sophorolipid biosynthesis pathway [46] A Polyketides B Fig Structure of (A) cellobiose lipids produced by Ustilago maydis; n = or (B) A common sophorolipid molecule in the acidic form R=H or COCH3 reaction is governed by AHD1, a non-heme diiron oxido-reductase cyp1 homologues were retrieved in two other cellobiose lipids producing organisms, Pseuodozyma flocullosa and Pseuodozyma fusiformata [44] One was not able to provide direct evidence for cyp1 contribution to the biosurfactant synthesis, but a positive correlation between cyp1 expression and flocculosin synthesis was demonstrated Sophorolipids are another type of hydroxyfatty acid containing biosurfactants They consist of a x- or x-1hydroxylated fatty acid etherified via its hydroxylgroup to a sophorose unit (Fig 2B) The fatty acid can be palmitic, palmitoleic, stearic, oleic or linoleic acid Typical sophorolipid-producing organisms are the yeasts Candida bombicola and C apicola P450 involvement in sophorolipid biosynthesis was suggested from a simultaneous increase of cellular P450 content in C apicola Two cyp52 genes were cloned from C apicola (CYP52E1 and CYP52E2) Yet, Southern hybridization results indicated the existence of additional cyp52 sequences, making it hard to draw conclusions 212 Polyketides are a structurally very diverse family of secondary metabolites with different biological activities occurring in bacteria, plants and animals They are synthesized by polymerization of acetyl and propionyl in a similar process to fatty acid synthesis and can undergo extensive derivatization; in many cases, macrolidic structures are formed which are further modified by, for example, several hydroxylation steps (Figs and 4) These hydroxylation steps are very often mediated by cytochrome P450 monooxygenases Discussing biosynthesis of all microbial polyketides would take us too far from the scope of this review, but we focus on two well-described polyketides of which the backbone structure displays fatty acid similarity Fumonisin is a mycotoxin produced by several Fusarium species, among others Fusarium verticilloides and Fusarium proliferatum, both widespread plantpathogens infecting maize and other grains, rendering this mycotoxin a common contaminant of corn Fumonisin is hepatotoxic and nephrotoxic, but its acute toxicity is low The long-term effect of low concentrations is less clear, but fumonisin is suggested to be carcinogenic The 17 fumonisin biosynthetic genes are located in a gene cluster and three of them are P450 enzymes The FUM6 protein (CYP505B1) intervenes in one of the first steps in fumonisin synthesis by hydroxylation of the polyketide-amino acid at C-14 and C-15 (Fig 3) Fum6 deletion mutants of F verticilloides are unable to produce fumonisin-like compounds because the hydroxylgroups are required for the esterification of tricarballylic moieties downstream of the biosynthesis pathway FUM6 is a self-sufficient P450 containing a NADPH-dependent reductase domain and belongs to the same family (CYP505) as the first discovered self-sufficient eukaryotic P450: P450foxy from F oxysporum The second cytochrome P450 monooxygenase, FUM2 (CYP65AH1), most likely catalyses fumonisin C-10 hydroxylation, whereas the third, FUM15 (CYP617F1), is suggested to be responsible for the synthesis of low levels of a fumonisin isoform [47] Well-studied examples of bacterial polyketides are the antifungal components typically produced by soil actinomycetes Nystatins produced by Streptomyces FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS I N A Van Bogaert et al The role of P450 in microbial fatty acid metabolism Fig Part of the biosynthetic pathway of fumonisin in Fusarium sp R1 = H or OH (hydroxylation is rare and is supposed to be governed by Fum15p), R2 = H or OH Steps with P450 involvement are marked with an arrow noursei are commercialized as an antibiotic to treat Candida sp and Cryptococcus sp infections (Fig 4A) The nystatin backbone is composed of a 38-membered macrolactone ring which can be further modified by cytochrome P450 enzymes; NysN (CYP105H1) oxidizes the methyl group at C-16 and NysL (CYP161A1) performs a hydroxylation at the C-10 position DnysL mutants produce 10-deoxynystatin, but despite the absence of the hydroxyl group, the product retains its antifungal activity [48] Streptomyces nodosus synthesizes amphotericin; in addition to its antifungal properties, this antibiotic is also active against human immunodeficiency virus, Leishmania parasites and prion diseases Amphothericin has a 38-membered macrolacton backbone similar to the one of nystatin (Fig 4B) and also this biosynthetic cluster contains two P450 genes, amphL and amphN or CYP161A3 and CYP105H4 respectively AmphL mediates C-8 hydroxylation, whereas AmphN oxidizes the C-16 methyl group [49] The biosynthetic gene clusters for a number of polyketides have been elucidated (amphotericin, nystatin, candicidin, pimaricin and rimocidin) All these clusters contain a P450 gene homologue to amphN and NysN associated to the C-16 oxidation These polyketide-specific P450 sequences can be used for screening purposes This strategy led to the identification of a nystatin-like gene cluster in Pseudonocardia autrophica containing the typical nppL and nppN genes [50] and the isolation of putative polyketide producing actinomycetes [51] Oxylipins Fatty acids have an established role as building blocks of membranes and triacylglycerols, acting as a structural component or energy reservoir Besides their classical roles, fatty acid derivates act as signalling molecules with great physiological significance One such example are oxylipins, molecules originating from oxidized unsaturated fatty acids They are widespread in aerobic organisms such as plants, animals and fungi, but also occur in certain bacteria Also the well-described mammal prostaglandins and leukotrienes belong to the oxylipin family Although oxylipin synthesis in mammals and plants is well documented, far less information can be found on microbial oxylipins and reports on the cloning of responsible genes are scarce Oxylipin-forming enzymes are structurally very diverse; in plants they belong to an atypical cytochrome P450 subfamily, whereas most other lipoxygenases are non-heme ironcontaining proteins FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS 213 The role of P450 in microbial fatty acid metabolism NysL A H3C HO I N A Van Bogaert et al OH OH O O CH3 OH OH OH OH OH OH O O H3C H3C HO H2N AmphL B H3C HO O OH OH O OH O O CH3 OH OH OH OH OH O OH O H3C H3C O HO OH H2N O Oxylipins of fungal species are believed to be involved in signalling, but more research is required to assign specific functions Aspergillus nidulans is a model organism for the understanding of fungal development because it has a defined sexual and asexual development cycle Oxylipins regulate the balance between both cycles Furthermore, they regulate secondary metabolism and are in this way important for plant–host colonization and mycotoxin production [52] The involved oxylipins are called precocious sexual inducers (psi-factors) and are derived from unsaturated C18 fatty acids One such psi factor-producing oxygenases or Ppo enzyme is PpoA, a bifunctional protein with a fatty acid heme dioxygenase ⁄ peroxidase domain in its N-terminal region and a P450 heme–thiolate domain in its C-terminal region The enzyme first oxidizes linoleic acid to (8R)-hydroxyperoxyoctadecadienoic acid and transfers this product in the second reaction step to 5,8-dihydroxyoctadecadienoic acid by means of the P450 domain PpoA acts in vitro on unsaturated C16 and C20 fatty acids as well, and was assigned as CYP6001A1 [53] Several CYP6001 homologues can be retrieved among fungi such as other Aspergillus sp., Neurospora sp., Fusarium sp and Ustilago maydis, although their function is not confirmed in these latter species [54,55] Another enzyme taking part in oxylipin biosynthesis is PpoC (CYP1006C1) Like PpoA, two different heme-containing regions are present, but in contrast to PpoA the P450 heme–thiloate domain is degenerated; the conserved cysteine residue known as the fifth heme iron ligand is replaced by a glycine or phenylalanine in 214 NysN AmphN Fig Structure of (A) nystatin produced by Streptomyces noursei, (B) amphotericin produced by Streptomyces nodosus P450mediated hydroxylation or oxidations are marked with an arrow A nidulans or A fumigatus PpoC, respectively This is reflected in the enzymes’ activity; whereas PpoA further converts (8R)-hydroxyperoxyoctadecadienoic acid to 5,8-dihydroxyoctadecadienoic acid, PpoC only performs the first reaction step [56] Oxylipins are also present in bacteria and might take part in stress responses and host–pathogen interactions Most bacterial lipoxygenases are non-heme iron proteins, but few plant CYP74-like proteins can be retrieved, for example, in the rhizobacterium Methylobacterium nodulans This bifunctional protein possesses an N-terminal peroxidase region and a C-terminal CYP74-like P450 region Because Methylobacterium nodulans is a root-nodule-forming and nitrogen-fixing symbiont of Crotalaria (plants belonging to the Fabaceae family), it is plausible that this bacterial lipoxygenase originated from horizontal gene transfer [57] Fatty acid-acting P450s with unclear biological function Self-sufficient P450 The majority of the P450 monooxygenases obtain the necessary electrons for oxygen cleavage and substrate hydroxylation via one or two redox partners There are several P450 redox systems which can be classified into different groups according to the components involved (reviewed in [58]) Most eukaryotic microsomal P450s – among them the previously discussed CYP52 family – use the class II redox system They form a small electron transfer chain together with the NADPH cytochrome P450 reductase Both enzymes FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS I N A Van Bogaert et al are N-terminally anchored to the endoplasmatic reticulum and derived structures, and the body of the protein is located in the cytoplasmatic space [59] Cytochrome P450 reductase is a flavoprotein containing the flavin cofactors FAD and FMN It transfers the hydride ion of NADPH to the lower redox potential FAD FAD then transfers single electrons to FMN, which in turn reduces the cytochrome P450 monooxygenase heme centre as required to activate molecular oxygen [60] By contrast, most prokaryotic P450s are cytosolic and communicate with two separate redox partners: a NAD(P)H-binding and FADcontaining reductase and a ferrodoxin or flavodoxin that transfers the electrons from the reductase to the P450 heme These redox systems are referred to as class I Yet, in the 1980s, a catalytically self-sufficient 119 kDa protein was characterized and purified from Bacillus megaterium: BM-3 or CYP102A1, now positioned in the class VIII redox family This large protein is a gene fusion product between a P450 and a cytochrome P450 reductase, rendering electron transfer extremely efficient and resulting in a catalytic activity of 4600 nmol fatty acid per nmol P450 per minute, whereas most class II systems have catalytic activities of two or even three orders of magnitude lower [61] The self-sufficient and soluble properties simplify the enzyme’s overexpression and purification, and turn it into an ideal model for spectroscopic and structural studies The derived models for the heme–substrate interactions indeed gave substantial input for the understanding of mammalian P450 systems [62,63] Despite the available data on the enzyme’s structure and in vitro substrates, its proper biological role and natural substrate remain to be revealed BM-3 hydroxylates fatty acids with a chain length between 12 and 18 carbon atoms at the x-1, x-2 or x-3 position and has highest affinity towards pentadecanoic and palmitic acid (Km of lm) Unsaturated fatty acids are even better substrates and besides the typical x-1, x-2 or x-3 hydroxylation, additional epoxidation of double bonds can occur Turnover rates of > 15 000 have been reported for arachidonic acid (C20:4) hydroxylation Saturated fatty acid amides and fatty alcohols are hydroxylated as well, yet at lower efficiencies (reviewed in [64]) BM-3 not only displays structural similarity with mammalian P450s, it also shows an induction profile very similar to mammalian CYP4A enzymes These P450s are fatty acid x-hydroxylases which are induced by barbirturates and other peroxisomal proliferators Also in the 5¢-flanking region of the BM-3 gene a so-called Barbie-box is retrieved; motives occurring in all barbiturate-inducible genes The regulatory system The role of P450 in microbial fatty acid metabolism includes the positive transcription factor BM3P1, the autoregulated repressor Bm3R1 and several regulatory sites English et al [65] found that the branched fatty acid phytanic acid is not only an inducer of BM-3, but also a substrate that is converted to x-1 hydroxyphytanic acid B megaterium is a soil bacterium and the authors state that many plant-derived unsaturated fatty acids are extremely toxic to this bacteria; the induction of BM-3 by phytanic or other fatty acids may contribute to a metabolization or detoxification system However, it must be mentioned that phytanic acid is not toxic to B megaterium, although it is a major vegetative breakdown product occurring in the soil Furthermore, branched chain fatty acids make up 80% of the fatty acid content of the Bacillus sp membranes and when the hydroxylation of these substrates was studied in more detail, they were shown to be at least as good substrates as their straight chain analogues, having a higher regio- and stereospecific hydroxylation pattern Therefore, it is possible that BM-3 takes part in the oxidative degradation of branched chain fatty acids [66] Its high catalytic activity, elucidated protein structure and ease of expression and use in in vivo experiments have made BM-3 an attractive target for protein engineering with possible biotechnological applications Various mutants are described as being able to act on shorter fatty acids, polycyclic aromatic hydrocarbons and even gaseous alkanes [67–69] or displaying a shift in the hydroxylation pattern towards the terminal or internal positions [70,71] Meanwhile, the self-sufficient CYP102A family has been extended with > 10 members, mainly originating from soil bacteria CYP102A2 and -A3 from B subtillis, CYP102A5 from B cereus and CYP102A7 from B licheniformis have been characterized and in general hydroxylate the same substrates as BM-3, sometimes with even higher activities [72] This group of proteins even harbours a sequence of a noncultured soil bacterium obtained by screening a metagenome database [73] Again, the biological roles in the different organisms remain to be discovered, but it has been demonstrated that CYP102A2 and -A3 are nonessential genes and are not involved in the adaptive response concerning fatty acid detoxification [74] The same conclusion can be drawn for CYP102B1 CYP102B1 is a cofactor requiring arachidonic acid hydroxylating and epoxydizing P450 from Streptomyces coelicolor No differences concerning cell development or antibiotic production were observed when comparing Dcyp102b1 strains with wild-type strains, but the lipid profiles of both strains were quite different, suggesting the involvement of CYP102B1 in lipid biochemical pathways FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS 215 The role of P450 in microbial fatty acid metabolism I N A Van Bogaert et al Unfortunately, it has not yet been possible to identify CYP102B1-mediated products because of the complex lipid fingerprint of Streptomyces coelicolor [75] In the 1990s, a membrane-bound eukaryotic BM-3 counterpart was found in the fungus Fusarium oxysporum The enzyme was first called P450foxy, but is nowadays also referred to as CYP505A1 P450foxy resembles BM-3 in its regiospecificity (x-1, x-2 or x-3) and catalytic turnover, but differs slightly in fatty acid preferences: saturated fatty acids are favoured over unsaturated ones and the highest turnover is observed for lauric acid [76] Homologous sequences have been found in other fungi such as Aspergillus, Neurospora and Fusarium species, and the previously discussed polyketide hydroxylases also belong to the CYP505 family Cofactor requiring P450s Some P450s acting on fatty acids are hidden between related proteins acting on totally different substrates CYP106A1 or BM-1 for B megaterium, for example, hydroxylates fatty acids, but information about the CYP106 family is dominated by the steroid-hydroxylating capacities of CYP106A2 or BM-2 Another example is CYP105D5 from Streptomyces coelicolor, belonging to a large family merely constituted of actinomycetes enzymes involved in various biological processes such as vitamin D3 hydroxylation, degradation of xenobiotics and synthesis of polyketide antibiotics Most enzymes can handle a broad range of substrates, although CYP105D5 activity is restricted to fatty acids only x-1, x-2, x-3 and x-4 hydroxylation products are formed, with the x-1 compound being most prominent [77] The same trend can be observed in the bacterial CYP107 family: whereas most P450s intervene in xenobiotic degradation, CYP107H1 from B subtilis is active towards myristic and palmitic acid The x-1, x-2 and x-3 hydroxylation of myristic acid is believed to be required for the generation of pimelic acid equivalents for biotin biosynthesis Pimelic acid is formed by P450-mediated in-chain cleavage via alcohol and threo-diol intermediates [78] Another so-called orphan P450 is the thermostable CYP119A1 from archaebacteria Sulfolobus acidocaldarius The strong hydrogen bonds and salt link networks, shortened loops and optimal aromatic stacking safeguard the enzyme activity up to 85 °C Despite extensive structural analysis, the physiological function of the enzyme is yet unclear Initially, styrene was put forward as a (poor) substrate, but recently a tight binding affinity was demonstrated towards lauric acid, resulting in the synthesis of mainly x-1 hydroxylated 216 lauric acid, suggesting a role in the lipid oxidative metabolism Interestingly, the percentage of the x-hydroxylated product increases from 2.5 to 12% when increasing the temperature from 24 to 80 °C [79] Phylogenetic relationship of the discussed enzymes Figure depicts the phylogenetic tree of the P450s described in this minireview Obviously, members of the same family cluster together The only exception is CYP584A2, which is located in the CYP52 cluster because of its high similarity with these molecules (30– 36% amino acid identity) P450s are classified in the same family if their amino acid identity is at least 40% In general, the criteria work quite well, but for some proteins with high similarity instead of identity, correct classification is not always that straightforward Furthermore, the self-sufficient P450s, the CYP102 and the CYP505 families, are found on a common branch, indicating a mutual ancestor One would expect a close phylogenetic relationship between the alkane-hydroxylating enzymes of bacteria (CYP153) and yeasts (CYP52) Nevertheless, these families are located quite distant from each other and only share low amino acid identities (8–10%; Table S1); convergent evolution has led to two types of enzymes that in the end could fulfil identical biological functions The proteins displaying the lowest similarity to the other P450s are those involved in oxylipin biosynthesis (2–6% amino acid identity) This can be explained in part by their bifunctional structure Conclusion In November 2009, 1015 bacterial and 2780 fungal P450s were listed on the cytochrome P450 homepage from Nelson [5], making up about one-third of the total P450 database The majority of these enzymes catalyse hydroxylation reactions and although various endogenous and xenobiotic compounds such as steroids and complex aromatic structures can act as substrates, a significant fraction of the P450s shows activity towards simple molecules such as fatty acids and alkanes The terminal methyl groups of such molecules are quite inert from a chemical point of view, yet P450s are able to activate this thermodynamically disfavoured position by hydroxylation It is suggested that x-hydroxylation is associated with a narrow substrate-access channel governing restricted sterical activity CYP52A21, for example, possesses a small access channel and FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS I N A Van Bogaert et al The role of P450 in microbial fatty acid metabolism Fig Phylogenetic tree of the P450s discussed in this minireview The tree was constructed using the Protein Maximum Likelihood (ProML) algorithm The marker bar denotes the integer branch length Ac ca, Acinetobacter calcoaceticus; Al bo, Alcanivorax borkumensis; An va, Anabaena variabilis; As fu, Aspergillus fumigatus; As ni, Aspergillus nidulans; Ba ce, Bacillus cereus; Ba li, B licheniformis; Ba me, B megaterium; Ba su, B subtillus; Ca al, Candida albicans; Ca ap, C apicola; Ca bo, C bombicola; Ca ma, C maltosa; Ca tr, C tropicalis, Cl ac, Clostridium acetobutylicum; Fu ox, Fusarium oxysporum; Fu ve, F verticilloides; Ma or, Magnoporthe oryzae; My, Mycobacterium sp.; No ar, Novosphingobium aromaticivorans; St co, Streptomyces coelicolor; St nod, S nodosus; St nou, S noursei; Su ac, Sulfolobus acidocaldarius; Sp, Sphingomonas sp.; Sp pa, Sphingomonas puacimobilis; Us ma, Ustilago maydis; Ya li, Yarrowia lipolytica predominantly hydroxylates the x-position [28] BM-3, however, is characterized by a large access channel and is unable to perform hydroxylations at the terminal ending However, one must keep in mind that the x ⁄ x-1 ratio can be influenced by other parameters such as the substrate itself and more specifically its absolute length, in vitro conditions and temperature as has been demonstrated for CYP119 This latter enzyme is referred to as an orphan enzyme; its native substrates and biochemical function remain unclear Yet, CYP119A1 is not the only P450 without biochemical connotation; in the growing pile of data generated by genome-sequencing projects numerous genes are annotated as putative P450s and although sequence similarity can give a clue about the enzymes function, this still can be surprisingly different as described for CYP105D1, CYP106A1 and CYP107H1 Even for comprehensively studied P450 systems such as BM-3, there are merely assumptions regarding its physiological role Most P450 activities are studied by heterologous expression This approach is indeed convenient to determine the enzyme’s potential activity, substrates and specificities because there is a high expression level and no background activity, but the approach fails to provide information on the enzyme’s physiological role and natural substrates Therefore, more direct experiments such as knockout studies and transcription analysis are required A common characteristic of P450s are the many gene duplication and conversion events The fatty acid and ⁄ or alkane-hydroxylating enzymes nicely illustrate this feature within one organism, isoenzymes with different substrate specificities and expression levels are FEBS Journal 278 (2011) 206–221 ª 2010 The Authors Journal compilation ª 2010 FEBS 217 The role of P450 in microbial fatty acid metabolism I N A Van Bogaert et al jointly able to degrade a whole range of substrates In the long-term, gene duplication and conversion progressing over different species result in a huge P450 diversity, in this way leading to various enzymes which in the end could fulfil related or even identical reactions (e.g the fungal CYP52 and bacterial CYP153 families) Throughout this review it has become clear that there are numerous poorly studied or even orphan P450s However, there are quite a lot of biochemical processes that require hydroxylation steps and that are not associated with a specific enzyme Hence, it is suggested that several of these hydroxylating roles are fulfiled by uncharacterized or yet to be discovered P450s Acknowledgements The authors wish to thank the Flemish Agency or Innovation by Science and Technology (IWT) for financial support (grants 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