Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions

MINI-REVIEW

Fungal microsomes in a biotransformation perspective:protein nature of membrane-associated reactions

Kateřina Svobodová & Hana Mikesková &

Denisa Petráčková

Received: 24 July 2013 /Revised: 16 October 2013 /Accepted: 17 October 2013 /Published online: 5 November 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Microsomal fraction of fungal cells grabs the atten-tion of many researchers for it contains enzymes that play arole in biotechnologically relevant processes. Microsomal en-zymes, namely, CYP450s, were shown to metabolize a widerange of xenobiotic compounds, including PAHs, PCBs, di-oxins, and endocrine disruptors, and take part in other fungalbiotransformation reactions. However, little is known about thenature and regulation of these membrane-associated reactions.Advanced proteomic and post-genomic techniques make itpossible to identify larger numbers of microsomal proteinsand thus add to a deeper study of fungal intracellular processes.In this work, proteins that were identified through a shotgunproteomic approach in fungal microsomes under various cul-ture conditions are reviewed. However, further research is stillneeded to fully understand the role of microsomes in fungalbiodegradation and biotransformation reactions.

Keywords Fungal microsomes . Cytochrome P450 .

Biodegradation .Microsomal proteins . Proteomics

Introduction

The biodegradation potential of white rot and other filamen-tous fungi has been extensively studied during the last

decades. The studies were focused mainly on fungal extracel-lular oxidative enzymes, their ability to oxidize various per-sistent organic compounds, and the elucidation of degradationpathways. Based on the results of inhibitor studies and me-tabolite identification, however, involvement of fungal cyto-chrome P450 (CYP450) system and other intracellular en-zymes in the biodegradation of organopollutants was sug-gested (Mougin et al. 1996; Mougin et al. 1997; Covinoet al. 2010; Prieto et al. 2011; Čvančarová et al. 2012;Křesinová et al. 2012). Filamentous fungi have been thenshown to possess a high diversity of CYP450 systems with abroad substrate activity (Vatsyayan et al. 2008; Lah et al.2008). Their relation to fungal metabolism of xenobioticchemicals has been reviewed previously (Črešnar and Petrič2011; Peng et al. 2008). It underlined the need of betterunderstanding of fungal intracellular processes and opened anew line for fungal biodegradation research.

In this work, findings supporting degradation ability andbiotransformation potential of fungal microsomal enzymes aresummarized. Fungal microsomes are equivalent to subcellularmembrane fractions that are obtained from homogenized fun-gal mycelium by differential centrifugation as described byCinti et al. (1972) for rat microsomes or through ultracentri-fugation steps (Machida and Saito 1993; Mougin et al. 1997).The purity of the microsomal preparations can be checked byenzymatic marker assays as described in Jauregui et al.(2003). Endoplasmic reticulum marker NADH-cytochrome creductase has been determined as a marker activity for micro-somal fractions.

To clarify the nature of microsomal processes, two types ofstudies have been nowadays carried out in fungi: narrowlyfocused works on functions of individual enzymes andproteome-wide studies. For example, mechanisms involvedin the recognition of aromatic compounds by the fungalCYP450 were studied in the model fungus Phanerochaetechrysosporium (Syed et al. 2011). Contrary to that, a largenumber of microsomal proteins were identified in Aspergillusniger (DeOliveira et al. 2010; DeOliveira et al. 2011) using

K. Svobodová (*) :H. MikeskováLaboratory of Environmental Biotechnology, Institute ofMicrobiology ASCR, v.v.i., Videnska 1083,14220 Prague, Czech Republice-mail: [email protected]

H. MikeskováInstitute of Chemical Technology Prague,Faculty of Food and Biochemical Technology, Technická 5,160 28 Prague 6, Czech Republic

D. PetráčkováLaboratory of Cell Signalization, Institute of Microbiology ASCR,v.v.i., Videnska 1083, 14220 Prague, Czech Republic

Appl Microbiol Biotechnol (2013) 97:10263–10273DOI 10.1007/s00253-013-5347-2

advanced proteomic techniques. Both approaches could helpto enhance the understanding of fungal intracellular processes.In this work, changes in membrane enzymes during biodeg-radation reactions and identification of microsomal proteinsare discussed to give an insight into the nature and regulationof microsomal biodegradation and biotransformation process-es. Advanced mapping of microsomal proteomes by high-throughput proteomics is especially highlighted as a usefultool for microsomal protein analyses.

Biodegradation potential of fungal microsomes

The implication of microsomal enzymes in xenobiotic detox-ification and degradation is summarized in this chapter. Ashort review is given in Table 1.

Several biodegradation studies suggested the involvementof intracellular enzymes in the biodegradation reactions. Thework of Masaphy et al. (1996a) indicated that CYP450-mediated benzo[a]pyrene hydroxylase activity in both micro-somal and soluble fractions of the white rot fungus P.chrysosporium could play a role in the xenobiotic transfor-mation by this fungus. The biotransformation of an insecticidelindane and herbicide atrazine by the liquid cultures ofP. chrysosporium has been drastically reduced by1-aminobenzotriazole (a CYP450 inactivator) (Mougin et al.1996; Mougin et al. 1997). Conversely, phenobarbital (a P450inducer) did not significantly increase lindane breakdown.Various inhibition studies also affirmed the implication ofP. chrysosporium CYP450s (PcCYPs) in the degradation ofnonylphenol (Subramanian and Yadav 2009) and pentachlo-rophenol (PCP) (Ning and Wang 2012), hydroxylation ofxenobiotics (Hiratsuka et al. 2005; Teramoto et al. 2004a,b),and oxidation of the chlorinated pesticide endosulfan(Kullman and Matsumura 1996). To study substrate specific-ity of individual PcCYPs, 120 yeast clones expressing indi-vidual CYP450s were screened for transformation of dioxins(Kasai et al. 2010). Out of 40 positive clones, a microsomalPcCYP designated as PcCYP11a3 showed the highest activ-ity. It catalyses the hydroxylation of 2,3- dichlorodibenzo-p-dioxin and has the highest activity towards polychlorinateddioxins among the known CYP450s derived from microor-ganisms. Recently, a genome-wide gene induction strategyrevealed multiple PcCYPs responsive to individual classesof xenobiotics (Syed et al. 2010). CYP5136A3 then showeda common responsiveness and catalytic versatility towardsendocrine-disrupting alkylphenols and polycyclic aromatichydrocarbons (PAHs; Syed et al. 2011). Metabolic pathwaysof PAHs by fungal P450 monooxygenases were alreadyreviewed in the work of Peng et al. (2008).

Next to P. chrysosporium , the potential of microsomalfractions to metabolize xenobiotics was studied in other fungi,too. Cytosolic and microsomal fractions of Cunninghamella

elegans were assayed for activities of cytochrome P450monooxygenase, aryl sulfotransferase, glutathione S -transfer-ase, UDP-glucuronosyltransferase, and N -acetyltansferaseand connected with the physiological versatility of the fungusin the metabolism of xenobiotics (Zhang et al. 1996). Eilerset al. (1999) showed that the metabolism of 2,4,6-trinitrotol-uene (TNT) in Bjerkandera adusta may include CYP450-dependent reactions.

Bezalel et al. (1997) examined the enzymatic mechanismsinvolved in the degradation of phenanthrene by Pleurotusostreatus . CYP450 activity was detected in both cytosolicand microsomal fractions of the fungus; however, it wasinhibited differently by the CYP450 inhibitors 1-aminobenzotriazole, SKF-525A (proadifen), and carbon mon-oxide. The experiments indicated the involvement of cyto-chrome P450 monooxygenase and epoxide hydrolase in theinitial oxidation of phenanthrene to form phenanthrenetrans-9,10-dihydrodiol.

The extracellular and microsomal fractions of P. ostreatus7989 were tested for in vitro degradation of five pesticides(Jauregui et al. 2003). No enzymatic modification of any ofthe pesticides was detected with ligninolytic enzymes(ligninperoxidase, manganese peroxidase, laccase) in the ex-tracellular fraction, while the microsomal fraction was able totransform three pesticides. The structure of degradation prod-ucts, supported by specific inhibition experiments and thestringent requirement for NADPH during the in vitro assays,suggested the involvement of a CYP450 (Jauregui et al. 2003).

Another set of in vitro experiments with P. ostreatus wascarried out to track the degradation mechanisms involved inthe degradation of the synthetic hormone 17 alpha-ethinylestradiol (EE2) (Křesinová et al. 2012). The white rotis able to completely remove EE2 from a liquid complex ormineral medium within 3 or 14 days, respectively. The resultsdocumented the involvement of various simultaneous mech-anisms in the EE2 degradation by P. ostreatus , including boththe ligninolytic system and the eukaryotic machinery ofCYP450s. EE2 was degraded by the isolated laccase of P.ostreatus , by the intracellular microsomal fraction, and alsoby a laccase-like activity associated with fungal mycelium.The degradation was completely suppressed in the presence ofCYP450 inhibitors, piperonylbutoxide and carbon monoxide,indicating the role of this monooxygenase in the degradationprocess.

CYP450 was also detected in the microsomal fraction ofIrpex lacteus (Cajthaml et al. 2008). Several novel intermedi-ates of PAHs degradation, probably connected with the par-ticipation of CYP450 in their biodegradation, were detected inthis study. Nevertheless, using PAHs as substrates, noCYP450 activity was detected in microsomal or cytosolicfractions regardless of the culture conditions (Cajthaml et al.2008). Covino et al. (2010) studied in vivo and in vitro PAHsdegradation by Lentinus tigrinus CBS 577.79. The

10264 Appl Microbiol Biotechnol (2013) 97:10263–10273

Table 1 Implication of microsomal enzymes in xenobiotic degradation

Organism/enzyme Pollutant Note References

P. chrysosporium/microsomal andcytosolic fractions

Benzo(a)pyrene CYP450 and CYP 450-mediated benzo(a)pyrenehydroxylase were detected in microsomal fractions;benzo(a)pyrene hydroxylation was NADPHdependent and inhibited by CO

Masaphy et al. (1996a)

P. pulmonarius /mycelial fractions

Atrazine Increase in CYP450 concentration during atrazinedegradation; piperonyl butoxid inhibited atrazinetransformation by fungal mycelium

Masaphy et al. (1996b)

P. chrysosporium liquidcultures/mycelialfractions

Lindane, atrazine Microsomal CYP450 was detected in themycelial fractions; 1-aminobenzotriazolereduced pesticide metabolism

Mougin et al.(1996,1997)

P. chrysosporiumliquid cultures

Nonylphenol 100 % degradation of 100 ppm nonylphenolby fungal cultures; degradation inhibited bypiperonyl butoxide, a CYP450 inhibitor

Subramanian andYadav (2009)

P. chrysosporium /microsomal fractions

PCP PCP oxidation by microsomal fractions of thefungus; carbon monoxide difference spectraindicated induction of CYP450 by PCP

Ning andWang (2012)


Biphenyl, dibenzofuran,diphenyl ether

Involvement of CYP450s in degradation–hydroxylation reactions on the aromaticring was inhibited by piperonyl butoxide

Hiratsuka et al. (2005)


Endosulfan The fungus utilizes both oxidative and hydrolyticpathways for metabolism of endosulfan;piperonyl butoxide inhibited the oxidationof endosulfane and enhanced its hydrolysis

Kullmann andMatsamura (1996)


Nitroaromatic compounds(4-nitrotoluene,4-nitrobenzoic acid,4-nitrophenol)

Fungal formation of 4-nitrobenzyl alcohol and1,2-dimethoxy-4-nitrobenzene was inhibitedby piperonyl butoxide, a CYP450 inhibitor

Teramoto et al.(2004a,2004b)

P.chrysosporiumCYP450s (PcCYPs)

Dioxins Screening of individual PcCYPs for transformationof dioxins; microsomal PcCYP11a3 has thehighest activity and catalyzes hydroxylationof 2,3-dichlorodibenzo-p-dioxin

Kasai et al. (2010)

P. chrysosporium /cytochromeP450 monooxygenases

PAHs Identification and functional characterizationof PAH-degrading CYP450 monooxygenases;identification of 6 PAH-responsive P450genes (Pc-pah1-Pc-pah6)

Syed et al. (2010)

P.chrysosporium /CYP5136A3

PAHs, endocrine-disruptingalkylphenols

CYP5136A3, cytochrome P450 monooxygenaseshowed common responsiveness and catalyticversatility towards endocrine-disruptingalkylphenols and PAHs

Syed et al. (2011)

C . elegans /cytosolic andmicrosomal fractions

PAHs, pharmaceuticaldrugs

Microsomal fractions contained cytochrome P450monooxygenase activities for aromatic hydroxylationand N-demethylation of cyclobenzaprine

Zhang et al. (1996)

B . adusta /microsomal fractions TNT Microsomal fraction of cell grown in thepresence of TNTwas found to contain CYP450;in cells grown without TNT, no microsomalCYP450 could be found; piperonyl butoxidediminished TNT mineralization; TNT metaboliteswere identified as aminodinitrotoluenes

Eilers et al. (1999)

P. ostreatus/mycelial extracts Phenanthrene Cytochrome P450 monooxygenase and epoxidehydrolase were involved in the initial oxidationof phenanthrene to form phenanthrenetrans-9,10-dihydrodiol

Bezalel et al. (1997)

P. ostreatus/microsomalfractions

Five pesticides—trichlorfon,phosmet, terbufos, azinphos-methyl, malathion

The microsomal fraction was able to transform threepesticides (phosmet, terbufos, azinphos-methyl)

Jauregui et al. (2003)

P. ostreatus/laccase,microsomal fractions

17 alpha-ethinylestradiol (EE2) EE2 was degraded by both isolated laccaseand microsomal fractions containingCYP450; EE2 degradation was suppressedby piperonyl butoxide and CO

Křesinová et at. (2012)

I . lacteus liquid cultures PAHs PAHs removal by liquid fungal cultures ina complex medium; CYP450 detected inmicrosomal fractions of the fungus

Cajthaml et al. (2008)

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identification of degradation products showed the presence ofseveral PAH derivatives, such as quinones, dicarboxylated,and ring fission derivatives, presumably derived from theaction of lignin-modifying enzymes. On the other hand, thepresence of hydroxylated derivatives of anthrone and phenan-threne 9,10- dihydrodiol suggested the possible involvement

of CYP450 epoxide hydrolase system, documenting the in-volvement of various simultaneous degradation mechanismssimilar to P. ostreatus .

Prieto et al. (2011) studied degradation of antibiotics by thewhite rot fungus Trametes versicolor. More than 90 % ofciprofloxacin (CIPRO) and norfloxacin (NOR) were degraded

Table 1 (continued)

Organism/enzyme Pollutant Note References

L . tigrinus liquid cultures/CYP450-epoxidehydrolase system

PAHs PAH degradation superior in shaken cultures(up to 97 %) compared to static cultures(<50 %); the presence of hydroxylatedderivatives suggested the involvementof CYP450-epoxide hydrolase system

Covino et al. (2010)

T. versicolor liquid cultures Ciprofloxacin andnorfloxacin

>90 % degradation in 7 days; degradationwas inhibited by 1-aminobenzotriazole

Prieto et al. (2011)

Fusarium moniliforme /cell extracts

Propylbenzene Hydroxylation of propylbenzene neededmolecular oxygen and NADPH, FAD, andFMN as coenzymes; it was inhibited by CO

Uzura et al. (2001)

T. trogii , T. hirsuta ,P. chrysosporium,T. versicolor, T. palustrisliquid cultures

Dibenzyl sulfide 1-Aminobenzotriazole eliminateddibenzyl sulfoxide oxidation

Van Hammeet al. (2003)

Phlebia brevisporaliquid cultures

Dieldrin Transformation included 9-hydroxylation;a potential involvement of microsomalmonooxygenases was suggested

Kamei et al. (2010)

P. ostreatus, I . lacteus,B . adusta, D . squalens,P. chrysosporium, P.magnoliae , P. cinnabarinus ,T. versicolor

PCBs - Delor 103 Degradation by liquid fungal cultures; theinvolvement of intracellular enzymes(CYP450, aryl-alcohol dehydrogenase,aryl-aldehyde dehydrogenase) in thedegradation was suggested

Čvančarováet al. (2012)

A . terreus /cytochrome P450monooxygenases

Alkanes, alkane derivatives,alcohols, aromaticcompounds, organicsolvents, steroids

In vitro degradation by microsomal fractions;inhibition by taxifolin; determination ofCYP450 substrate specificity

Vatsyayanet al. (2008)

R . nigricans /NADPH-cytochrome P450reductase

Progesterone NADPH-cytochrome P450 reductase isinvolved in hydroxylation of progesteroneat 11alpha position; CPR was isolatedfrom induced mycelia and characterized

Makovec andBreksvar (2002)

P. chrysosporium liquid cultures/microsomal fractions

Benzoic acid CYP450-mediate degradation of benzoic acid;induction of CYP450 by benzoic acid

Ning et al. (2010b)

P. chrysosporium liquidcultures/microsomalfractions

Phenanthrene Transformation of phenanthrene tophenanthrene trans-9,10-dihydrodiolwas inhibited by piperonyl butoxide

Ning et al. (2010a)

P.chrysospporiumCYP450s (PcCYPs)

Anthracene 12 cytochrome P450 monooxygenases involvedin anthracene metabolism were identifiedby transcriptomic profiling; 14 PcCYPspecies catalyze stepwise conversionof anthracene to anthraquinon viaintermediate formation of anthrone

Chigu et al. (2010)

Trichoderma harzianumCYP450

n-Alkanes A microsomal, n-alkane-inducible CYP450was identified; CYP450-dependent conversionof alkanes to fatty acids allowing theirincorporation into lipids was suggested

Del Carratoreet al. (2011)

P.chrysosporium /PcCYP1f Benzoic acid Recombinant PcCYP1f catalyzed hydroxylationof benzoic acid to 4-hydroxybenzoic acid;PcCYP1f was induced at a transcriptionallevel by benzoic acid

Matsuzaki andWariishi (2005)

P.chrysosporium /CYP63A3 PAHs, alkanes, oxygenatedmono aromatics

The expression of CYP63A3 was inducedby various xenobiotics

Doddapaneniet al. (2005)


after 7 days. Inhibition of CIPRO and NOR degradation bythe CYP450 inhibitor 1-aminobenzotriazole suggested thatthe CYP450 system also played a role in the degradation ofthe two antibiotics. Moreover, transformation products ofCIPRO and NOR were monitored in this study. CYP450-mediated reaction mechanisms were also proposed for xenobi-otic transformation in several other fungi (Uzura et al. 2001; VanHamme et al. 2003; Kamei et al. 2010; Čvančarová et al. 2012).

A broad substrate P450monooxygenase activity was foundin the cells of Aspergillus terreus (Vatsyayan et al. 2008). Forthe first time in this study, evidence was brought for a shift inCYP450 activity localization during biodegradation. TheP450 monooxygenase activity was localized in the cytosolof n -hexadecane-grown cells, while it was apparently distrib-uted in light mitochondrial and microsomal fraction ofglucose-grown cells. The substrate specificities of CYP450present in all the locations, however, were similar irrespectiveof the substrates used for the growth. Apart from cytosolicCYP450s, the microsomal enzymes may also cooperate withother intracellular enzymes in xenobiotic metabolism. Epox-ide hydrolase, glutathione S -transferase, methyl transferase,aryl-alcohol dehydrogenase, and aryl-aldehyde dehydroge-nase activities are discussed in this view in some works(Bezalel et al. 1997; Čvančarová et al. 2012; Křesinová et al.2012). Kulmann and Matsumura (1996) suggested that P.chrysosporium utilizes two divergent pathways for metabo-lism of pesticide endosulfan, one hydrolytic and the otheroxidative that is catalysed by CYP450.

Microsomal enzymes-mediated biotransformation

Apart from biodegradation, microsomal proteins are also con-nected with other biotechnologically relevant reactions infungi. For example, OrdA enzyme, a microsomal enzyme ofAspergillus parasiticus , was shown to be involved in aflatoxinbiosynthesis by this fungus (Zeng et al. 2011; Yabe et al.2012). Another step in the aflatoxin biosynthesis has beenalready previously shown to be catalysed by a P450monooxygenase encoded by the cypA gene (Ehrlich et al.2004). Microsomal fractions of Pleurotus sapidus were usedfor the conversion of alpha-pinene to verbenols, verbenone,and minor volatile flavors (Krings et al. 2009). A highlystereospecif ic monoterpenol dehydrogenase anddioxygenases were proposed to catalyse the bioconversionof terpene substrates in the addition to previously assumedCYP450 enzymes (Krings et al. 2009). The microbial bio-transformation of readily available terpenoids, like verbenone,into more valuable compounds has economic potential in theperfumery, food, and pharmaceutical industries. Similarly,two strains ofAspergillus and P. digitatum have been recentlyreported to hydroxylate verbenone to 10-hydroxyverbenone(Yildirim 2011).

Further, fungal biotransformation models are also consid-ered to be complementary sources for the preparation ofhuman drug metabolites that are, in many cases, critical forfurther pharmacokinetics, pharmacologic, and toxic evalua-tion of the remedy (Yang et al. 2012; Hilario et al. 2012). Anantihistamine, cyproheptadine hydrochloride, was extensivelytransformed by the zygomycete C . elegans via aromatichydroxylation metabolic pathways (Zhang et al. 1997).CYP450 was detected in the microsomal fractions of thefungus and assumed to play a role in cyproheptadine hydro-chloride metabolism. Next to bacterial CYP450 enzymes(Otey et al. 2006) and fungal peroxygenases (Poraj-Kobielskaet al. 2011), fungal CYP450s could represent a potentialapproach for human drug metabolite preparation.

An alternative genetic approach to the production of poly-unsaturated fatty acids (PUFA) may target another group ofmicrosomal enzymes, fatty acid desaturases. A gene for amicrosomal delta12-fatty acid desaturase was recently isolatedfrom a marine alga, Pinguiochrysis pyriformis , and expressedin yeasts and thraustochytrids that are known to accumulatePUFA in their lipid droplets (Matsuda et al. 2011). With theincreasing demand of obtaining PUFAs from alternativesources, the genes and enzymes involved in the biosynthesisof PUFAwith nutraceutical potentials have been studied alsoin fungi (Zhang et al. 2013; Huang et al. 2011; Sakuradaniet al. 2008). Tan et al. (2011) analyzed delta 6 desaturase anddelta 6 elongase from Conidiobolus obscurus . A novel fattyacid elongase with wide substrate specificity was also identi-fied in an arachidonic acid-producing fungus Mortierellaalpina 1S-4 (Sakuradani et al. 2009). However, the molecularmechanism for functions of these enzymes is still unclear. Oldmutant and immunochemical studies described only the in-volvement of cytochrome-b5 in fatty acid desaturation byyeast microsomes (Ohba et al. 1979; Tamura et al. 1976).

Enzymes in fungal microsomes

Biodegradation reactions carried out by microsomal fractionsof fungi and some of the fungal biotransformations are fre-quently connected with fungal CYP450 systems. FungalCYP450s catalyze the monooxygenation of a variety of hy-drophobic substrates and are key enzymes in fungal primaryand secondary metabolisms. By the action of CYP450s, lipo-philic compounds are metabolized to more hydrophilic deri-vates by introducing an oxygen atom originating from molec-ular oxygen. Typical fungal microsomal CYP450 systems aremembrane-bound enzymes and consis t of P450monooxygenases that primarily obtain electrons from theP450 reductases. Beside that, electron transfer from NADHto P450 monooxygenase via cytochrome b5-containing redoxpathways is also known (Hannemann et al. 2007; Črešnar andPetrič 2011; Ichinose and Wariishi 2012). Most eukaryotic


membrane-bound CYP450s are likely to have an N-terminalTMD sequence that acts as a membrane anchor. The TMD-associated subcellular localization to membranes should beimportant for protein–protein interactions of P450monooxygenases with themembrane-anchored redox partners(Nazir et al. 2010).

In membrane systems like in microsomes, a limitingamount of P450 reductase may be effectively limiting aP450 reaction. As a result of that, a biphasic reduction ofCYP450 is usually observed in microsomes (Guengerich2001). Filamentous fungi with numerous CYP450s oftenpossess multiple microsomal redox partners, cytchromeP450 reductases, which may also influence the specificity ofP450 monooxygenase-mediated reactions. In the plant-pathogenic ascomycete Cochliobolus lunatus , two P450 re-ductase paralogues, CPR1 and CPR2, supported CYP450activity, but with different product specificities during degra-dation of phenolic plant compounds (Lah et al. 2011). It wasconcluded that CPR1 is important in primary metabolism,whereas CPR2 plays a role in xenobiotic detoxification.Recently, whole genome sequence analyses have revealedlarge-scale divergences in basidiomycetous CYP450s, whichimplies that basidiomycetes have diversified monooxygenasefunctions to acquire metabolic adaptations such as xenobioticdegradation (reviewed in Ichinose (2013)).

A high diversity of fungal CYP450 enzymes is wellreflected in genes encoding CYP450s. Fungal CytochromeP450 Database archives CYP450 genes in the genomes of 70fungal species (Park et al. 2008). In P. chrysosporium ,CYP450-encoding genes were found to be differentially ex-pressible, reflecting their functional diversity (Doddapaneniand Yadav 2005). Despite being members of tandem geneclusters, the genes are independently regulated and inducibleby various xenobiotics. The genes encoding P450monooxygenases CYP63A1, A2, A3, and PcCYP1f wereshown to be inducible at a transcriptional level by certainaliphatic hydrocarbons, oxygenated mono aromatics and low-er molecular weight PAHs (Doddapaneni et al. 2005;Matsuzaki and Wariishi 2005). In the case of CYP63A1 andPcCYP1f, up-regulation of protein production in response tobenzoic acid was observed using two-dimensional electropho-resis (Matsuzaki et al. 2008). The regulation of expression ofthe family of P450 monooxygenases, the CYP63 family, in P.chrysosporium was also studied by Subramanian and Yadav(2008) upon induction with 42 different xenobitics. TheCYP450 genes Pff311b and Pff4a showed high levels ofinduction in P. chrysosporium cultures degradingnonylphenol (Subramanian and Yadav 2009). More recently,an induction of microsomal CYP450s by phenanthrene,benzoic acid, chlorbenzoic acids, and n -hexane was indicatedby carbon monoxide difference spectra analysis during thebiodegradation studies (Ning et al. 2010a,b). Twelve P.chrysosporium P450 monooxygenases were up-regulated at

a level of transcription in response to exogenous addition ofanthracene (Chigu et al. 2010). Syed et al. (2010) identifiedsix PAH-responsive genes encoding P450 monooxygenasesin P. chrysosporium that were capable of PAHs oxidation.One of them, CYP5136A3, showed a common responsive-ness and oxidizing capability towards PAHs and endocrine-disrupting alkylphenols (Syed et al. 2011), demonstrating thecatalytic versatility of fungal microsomal CYP450s.

Similarly to P. chrysosporium , diverse CYP450 enzyme-encoding genes and xenobiotic-responsive CYP450 enzymeswere observed also in other filamentous fungi, likeAspergillusoryzae (Nazir et al. 2010), A . niger (Van den Brink et al.1996), Rhizopus nigricans (Kunic et al. 2001), andTrichoderma harzianum (Del Carratore et al. 2011). In thecase of R . nigricans , the first strong indication that the bio-logical role of CYP450(11alpha) induction is in detoxificationof steroids was brought by Breskvar et al. (1995) who studiedthe toxic effects of steroids on fungal growth. NADPH-cytochrome P450 reductase from R . nigricans (Makovecand Breskvar 1998) and progesterone-induced microsomalfungal monoxygenase system (Makovec and Breskvar 2000)were isolated and characterized later. Compared to that, recentfungal genome analyses projects have enabled the annotationof many novel CYP450s, many of which are with novelcatalytic functions.

With the advancement of molecular cloning and genomesequencing technologies, many novel fungal front-enddesaturases for the production of PUFA with nutraceuticalpotentials were also described, and the enzymes were func-tionally characterized (Zhang et al. 2013; Meesapyodsuk andQiu 2012; Tan et al. 2011; Zhang et al. 2007; Hongsthonget al. 2006). These enzymes belong to membrane-bound non-heme iron-containing oxygenases, catalyzing the formation ofa double bond in a hydrocarbon chain. Fungal front-enddesaturases are modular proteins containing a cytochromeb5-like domain at the N-terminus. The regions of two-membrane-spanning helices and C-terminus are probably im-portant for the substrate selectivity and high regioselectivity ofthe enzymes (Meesapyodsuk and Qiu 2012).

Despite the cited studies, our understanding of the individ-ual functional domains of front-end desaturases still remainslimited. Being membrane-bound, this type of desaturase isrecalcitrant to biochemical purification, and therefore there isalso no information available on the 3D structure ofdesaturases so far. Correspondingly to desaturases, the mem-brane location also makes structural modelling of microsomalCYP450 enzymes less successful compared to cytosolic oneswhen extending the known P450 structural paradigm for newenzymes (Hasemann et al. 1995). Unlike the CYP450s,however, very little is known about the regulation ofexpression of fungal front-end desaturases. All of thesefindings document lacks and difficulties in the workwith membrane enzymes.


Proteomic studies of microsomal proteins

Several individual proteins/enzymes have been isolated so farfrommicrosomal fractions of filamentous fungi for their func-tional characterization (Machida and Saito 1993; Makovecand Breskvar 1998; Maspahy et al. 1999; Makovec andBreskvar 2000; Yoshida et al. 2000; Faber et al. 2001) or theirmicrosomal localization has been proven (Husson et al. 1998).The biochemistry of microsomal cytochromes of fungi wasstudied in order to develop azole antifungal agents selectivefor fungi (reviewed in Yoshida (1988)). For this project,reconstituted enzyme systems consisting of purified yeastCYP450 enzymes were later developed and used for kineticanalysis of the enzymes (Aoyama et al. 1991).

With the use of advanced protein analyses, the amount ofknown and characterized microsomal proteins has dramatical-ly increased, which enabled functional studies of microsomesat the organelle level. Microsomal membrane fractions of P.chrysosporium were analyzed to study the nature and regula-tion of the membrane-associated components (Shary et al.2008). Tryptic digests of the microsomal proteins were ana-lyzed by shotgun liquid chromatography–tandem mass spec-trometry, and the results were compared against the predictedproteome of the fungus. The resulting data sets comprisedtypically 300 to 400 proteins in this study. Catalase, involvedin H2O2 metabolism, and a protein belonging to glucose–methanol–choline oxidoreductase superfamily were connect-ed with ligninolytic conditions. Microsomal preparations alsocontained six proteins that could have a transporter functionand six CYP450s out of 150 encoded in the genome (Sharyet al. 2008).

Shotgun proteomics was also applied to identify the micro-somal components involved in protein secretion by A . niger(DeOliveira et al. 2010). Better understanding of the proteinsecretion components could help to overcome the observedlimitations in protein secretion by filamentous fungi as sum-marized in the work of Gouka et al. (1997). Proteins from themicrosomal fungal fractions of A . niger were first separatedby SDS-PAGE and trypsin-digested. After that, proteins wereanalyzed by LC-MS/MS. Out of all detected proteins, 254were predicted to play direct roles in membrane traffic andprotein secretion (DeOliveira et al. 2010). Next, this studyclearly demonstrated that D-xylose induction led to 20S pro-teasome recruitment to the microsomal fraction and to anincrease in specific small GTPases known to be associatedwith polarized growth.

In total, 1,126 microsomal proteins were identified in A .niger microsomal protein composition resulting from culturesgrown in the conditions of amylolytic and xylanolytic enzymesecretion (DeOliveira et al. 2011). The proteins were groupedin the following categories: membrane traffic and proteinsecretion (23 %), mitochondrial (13 %), translation (12 %),metabolism and defense against reactive oxygen species

(12 %), cargo proteins (8 %), lipid biosynthesis (8 %), trans-porters (5 %), and others (14 %). Similarly to the previouswork of the group (DeOliveira et al. 2010), induction ofextracellular enzyme production resulted in specific changesin the secretory subproteome of A . niger. An association of20S core of the proteasome with secretory organelles was alsoobserved in both studies, suggesting that the recruitment of theproteasome may be a general feature of the shift to a secretionstate of the cell. Other microsomal proteins that were highlyexpressed included the ribosomal assembly protein, a smallGTPase RhoA, a plasma membrane H+-ATPase for cell po-larity PmaA, and a metabolic enzyme oxaloacetate acetylhydrolase.

In addition to the previous works, the presence of signalsequences was predicted in the microsomal protein dataset(DeOliveira et al. 2011). It showed that only 25 % of A . nigermicrosomal proteins contained either a signal peptide or asignal anchor. In A . niger, approximately 10 % of the totalproteins (Braaksma et al. 2010) and 92 % of the secretedproteins (DeOliveira et al. 2011) are predicted to contain asignal sequence. Similarly to that, only 41 % of microsomalproteins isolated from K562 cells were assigned as membraneproteins based on the presence of transmembrane regions orpost-translational modifications that could account for mem-brane association (Ghosh et al. 2008). It indicates that theremay be a significant component of non-integral proteins with-out any signal information associated with fungal microsomes.

Likewise in other organisms (Jacobs et al. 2006; Kislingeret al. 2006; Štefanič et al. 2006; Ghosh et al. 2008), the citedstudies (Shary et al. 2008; DeOliveira et al. 2010; DeOliveiraet al. 2011) demonstrated the power of shotgun proteomicanalysis in the study of specific organelle fraction compositionin fungi. Proteomic studies can extend genome and tran-scriptome analyses of fungi and fungal processes like proteinsecretion. A weakness lies in the comparison of protein rela-tive amounts in the fungal secretomes and the microsomalproteomes based on the calculations of normalized spectralabundance factors of proteins (DeOliveira et al. 2010;DeOliveira et al. 2011). Because of the factor time and dueto the experimental setup of the works, the secreted proteinsaccumulated over a time period. The microsomal proteome,on the other hand, was the result of microsomes isolated in adefined moment in time.

In comparison to the shotgun proteomic approach, two-dimensional gel electrophoresis (2-DE)-based analyses ofsubcellular membrane organelles has a disadvantage in itslow performance in the separation and analysis of membraneproteins. Although proteins in cytoplasmic membranefractions of bacteria have been successfully analysedby 2-DE (e.g., in Zuobi-Hasona et al. 2005; Petráčkováet al. 2010), the proteomic analysis of the subcellular organ-elles containing highly hydrophobic membrane proteins re-mains a major challenge.


Different methods for sample preparation were tested so farfor 2-DE analyses of proteins of microsomal fractions ofplants, rat brain microsomes, and rat hepatic microsomes,mitochondria, and endoplasmic reticulum. In most studies,membrane samples are lysed in the presence of 1–4 %CHAPS (Thomas et al. 2013; Messina et al. 2010; Galevaand Altermann 2002; Koen and Hanzlik 2002). A combina-tion of 4 % CHAPS and 0.5 % Triton X-100 was successfullyapplied for sample lyses by Sandoval et al. (2013). In the workof Meisrimler and Luthje (2012), sample preparations byTCA/acetone and methanol/chloroform precipitation, withand without SDS pre-solubilization, were compared for mi-crosomal fractions of plant leaves and roots, showing thesuperiority of methanol/chloroform precipitation and off-gelfractionation of proteins. To improve the subsequent proteinidentification, a combination of 1-DE and 2-DE-based ap-proaches was suggested (Galeva and Altermann 2002; Koenet al. 2013).

With all the methodical progress, the application of 2-DEcould help in the identification of differentially regulatedproteins and thus would bring additional information to shot-gun analyses of microsomal proteomes. To our knowledge,however, no 2-DE analyses of fungal microsomal proteinshave been published up to now.

Conclusions

Except extracellular enzymes, the biodegradation reactions per-formed by many filamentous fungi are also assisted by intracel-lular enzyme machinery. Microsomal CYP450s were especiallyshown to metabolize a wide range of xenobiotic chemicals andwere demonstrated to be inducible by the compounds. In addi-tion to biodegradation, the intracellular membrane-bound en-zymes play their roles in other biotransformations, like aflatoxinsynthesis, bioconversion of verbenols, preparation of humandrug metabolites, and production of PUFA.

However, little is known about the regulation of expressionof microsomal proteins. A differential expression of moststudied enzymes, CYP450s has been extensively studied inthe model fungus P. chrysosporium only. Regulation mecha-nisms involved in the expression of membrane-bound fattyacid desaturases have been only hypothesized. Therefore,further research on the composition of microsomal compo-nents is needed to extend our understanding of the intracellu-lar processes during the biodegradation and biotransformationreactions in fungi.

As summarized here, the advanced protein analyses repre-sent a high-throughput method for the identification of largesets of proteins and thus enable the study of specific organellefraction composition and of the role of organelles in fungalprocesses. They can extend genome and transcriptome analy-ses of fungi.

Acknowledgments This work was supported by the projects DAADA/13/07824, TE01020218 of the Czech Technology Agency and theInstitutional Research Concept RVO: 61388971.

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Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions