(Cytochrome P450 Complement) of Aspergillus Nidulans

Fungal Genetics and Biology 46 (2009) S53–S61

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Fungal Genetics and Biology

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The CYPome (Cytochrome P450 complement) of Aspergillus nidulans

Diane E. Kelly a,*, Nada Kraševec b, Jonathan Mullins a, David R. Nelson c a Institute of Life Sciences, 6 School of Medicine, Swansea University, Singleton Park, Swansea SA2, 8PP, UK b National Institute of Chemistry, POB 660 SI-1001 Ljubljana, Slovenia c Department of Molecular Sciences, University of Tennessee, Memphis, TN, USA article info abstract

Article history: The cytochromes P450 (CYPs) are found in all biological kingdoms and genome sequencing projects con- Received 9 May 2008 tinue to reveal an ever increasing number. The principle aim of this paper is to identify the complete Accepted 19 August 2008 CYPome of Aspergillus nidulans from the genome sequence version AN.3 deposited at the Broad institute, Available online 12 September 2008 assign the appropriate CYP nomenclature and deﬁne function where possible. The completed analysis revealed a total of 111 CYP genes, 3 of which were previously unknown and 8 pseudogenes, representing Index descriptor: 89 CYP families, 21 of which are unique. We have identiﬁed 28 potential gene clusters associated with Cytochrome P450 one or more CYP genes and discussed those with putative PKS and NRPS associated function. The chro- CYPs mosomal location of the genes, predicted cellular location of the proteins and possible function(s) are Haem-containing mono-oxygenases Aspergillus nidulans discussed. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction son.utmem.edu/CytochromeP450.html), but now to version AN.3 deposited at the Broad Institute (http://www.broad.mit.edu/anno- Filamentous fungi produce a vast array of secondary metabo- tations/fungi/aspergillus). We describe the chromosomal location lites and an excellent review (Hoffmeister and Keller, 2007) has of the genes, predicted cellular location of the proteins and focus identified the major structural classes of these compounds, many on defining function for as many of the CYPs found as possible, of which may be harnessed as useful bioactive compounds, but while seeking clues to function for the remainder. which have arisen so that the organism in question may occupy a particular ecological niche. A consequence of this evolution is 1.1. Sequence motifs as a tool to identify CYPs that specific biosynthetic pathways may only exist in certain fungi and not in others. Closer inspection of such pathways has revealed The nomenclature for CYPs is based upon amino acid identity; that many of these compounds are tailored (modified) by the ac- 40% identity and above place a CYP in the same family, more than tion of a particular family of enzymes known as cytochromes 55% identity places them in the same subfamily (Nebert et al., P450 (CYPs). 1987; Nelson et al., 1996). Families are designated a CYP number CYPs are found throughout all the biological kingdoms and are a based on those reserved for different taxonomic groups (http:// superfamily of haem-containing monooxygenases, interest in drnelson.utmem.edu/CytochromeP450.html), for fungi these are which has resulted in extensive research papers and review arti- CYP51-CYP69, CYP501-CYP699 and CYP5001-CYP6999. Detection cles, (Ortiz de Montellano et al., 1995; Ortiz de Montellano et al., of CYP genes in a genome is greatly facilitated by the presence of 2005). Genome sequencing projects continue to reveal the ever consensus amino acid sequences (Fig. 1): FXXGXXXCXG, the haem increasing number of these CYPs (presently > 8000, http://drnel- binding domain containing the axial Cys ligand to the haem (high- son.utmem.edu/CytochromeP450.html, numbering 1672 from lighted). EXXR motif found in the K – helix and the PER(W) domain. 35 fungal genomes, (Intikhab et al., 2007), with 1384 already as- Modifications in the haem binding region were found in CYP5120B1 signed a CYP name http://drnelson.utmem.edu/Cyto- (AN5335.3), this unusually has no conserved Cys downstream of chromeP450.html, although the functions of the vast majority are the normal EXXR and PER(W) motifs, in common with A. terreus, still unknown. The primary aim of this work was to identify the CYP5121A1 Neosartorya fischeri, CYP5121A2 A. fumigatus and complete CYPome of Aspergillus nidulans and match these with CYP5121B1 from A. clavatus alignments, none of the five fungal se- the standardized nomenclature assigned by Nelson, D. R. originally quences had a Cys in this region. Instead the Cys is found at the to version AN.2 of the genome sequence (http://drnel- location of the conserved Thr in the I-helix oxygen binding pocket and may represent the Cys bound to the haem iron in an inverted * Corresponding author. P450 structure. Modifications in the haem binding domain are E-mail address: [email protected] (D.E. Kelly). more usually found in CYPs with catalytic activity, often not

and apdB (AN8408.3, CYP685A1) involved in aspyridone biosynthesis. An alternative approach is to predict function based on sequence similarity to those proteins of known function. However, Fig. 1. Consensus amino acid sequences in P450 from A. nidulans. Data were created it is difficult and in most instances impossible, to predict the spe- using WebLogo (http://weblogo.berkeley.edu/)(Crooks et al., 2004; Schneider and cific functions of the A. nidulans CYPs simply from their sequence Stephens, 1990). a - Oxygen binding and activation; b and c – ERR triad and d – Heme binding. similarities particularly for the novel families. It is known that a single amino acid change can significantly alter the metabolic capabilities of a CYP (Lindeberg and Negishi, 1989; Wen et al., requiring oxygen, such as prostacyclin synthases (CYP8A, Li et al., 2005). However, for some families there is sufficient knowledge 2008) and allene oxide synthases (CYP74, Song et al., 1993) and to be able to assign a putative function that may then be the sub- may indicate a novel catalytic activity in this instance. Two further ject for further experiments. CYPs, CYP62C1 (AN6414.3) and CYP66A1 (AN2607.3) possess the The fungal sterol pathway is known to require two CYP activi- invariant Cys, but residues upstream are replaced either by fewer ties. The first CYP51 is the only P450 that has an orthologue (show- amino acids in the signature GXXCXG, or by an alternative residue ing 22-23% sequence ID between kingdoms) found in all kingdoms HXXXCXG respectively. It has been postulated that the haem bind- of life from bacteria, lower eukaryotes through fungi, plants and ing signature requires only the invariant Cys (Rupasinghe et al., animals (Yoshida et al., 2000). This is thought to be the most an- 2006). cient of CYPs (Aoyama et al., 1996; Nelson, 1999) and is a sterol 14a-demethylase involved in the biosynthesis of cholesterol in 1.2. Gene assignation animals, phytosterols in plants and ergosterol in fungi. CYP51 genes have been cloned from a number of fungi including Saccharomyces A complete list of all the A.nidulans CYPs is given in Table 1. The cerevisiae, Candida albicans and A. fumigatus (Bard et al., 1993; Mel- total gene count is 119, including 8 pseudogenes (with in-frame lado et al., 2001) and some of the proteins have been characterised, stop codons, frameshifts or deletions), representing 89 CYP families such that 41 conserved residues have been identified (Lepesheva (see Fig. 3 for their phylogenetic relationships), 21 of which are un- and Waterman, 2004). Unlike in S. cerevisiae where only one copy ique to A. nidulans and representing approximately 1% of the of this essential gene (ERG11) is present, two CYP51 sequences are genome. present in A. nidulans CYP51F1 (AN8283.3, cyp51B) and CYP51F2 Closer inspection of protein sequences showed there is substan- (AN1901.3, cyp51A). tial variation in sequence length across the CYPs, ranging from 286 Hu et al. (2007) have demonstrated that both CYP51F1 and F2 to 750 residues. All sequences were further analysed to determine orthologues in A. fumigatus can act in a compensatory manner in incorrect exon calling, incorrect start or stop sequences and 41 se- the ergosterol pathway, i.e. neither is essential individually, but a quences were identified with different gene structure requiring double knockout is lethal. They postulate that CYP51F2 (CYP51A) manual correction. Corrected proteins, after exclusion of pseudo- may encode the major 14a-demethylase activity required for genes and a fusion protein contained between 417– 607 residues, growth based on accumulation of multiple missense mutations with an average of 511 amino acids. linked to azole resistance and CYP51F1 may serve a redundant role During the course of this work, a comparative study for four fil- with CYP51F2 or an alternative function under particular growth amentous Ascomycetes by Deng et al., 2007 identified putative CYP conditions yet to be defined. sequences for A. nidulans. We have since determined that previ- A BLASTP search (Altschul et al., 1997) against the ERG5 yeast ously designated CYPs 539A4P (AN5478.3), 551A3P (AN5460.3), sequence (22-sterol desaturase) has revealed the second (single 630B3P (AN2191.3), 669A1P (AN0008.3) and 686A1P (AN8510.3) copy) CYP in the ergosterol pathway in A. nidulans (AN4042.3, were incorrect and are in fact pseudogenes. The completed analysis CYP61A1). revealed three new CYP sequences in the updated version AN.3 A third and final CYP found in S. cerevisae is involved in the bio- from Broad, these are AN10887.3, AN1703.3, AN8437.3 and named synthesis of dityrosine, a major component of the spore wall sur- CYP5095B1, CYP5128A1 and CYP5125A1 respectively. Table 1 illus- face (Briza et al., 1996), DIT2 or CYP56. A homologue AN2706.3 trates the linkage between all the A. nidulans CYPs, including pseu- (CYP56B1, 41% ID) is present in A. nidulans. Closer examination dogenes, their chromosomal location and AN.3 reference number shows that the adjacent upstream gene AN2705.3 has 35% ID with identified in this study. DIT1 from S. cerevisae. Further homologues are also present in A. fumigatus, A. orzyae and A. fisherianus and it therefore probable that 1.3. Protein function these genes code for the same function, i.e. that a dityrosine mac- romolecular network is also present in the Aspergillus genus. The natural substrates of fungal CYPs are largely unknown, but will include precursors of membrane sterols in the ergosterol bio- 1.4. CYP clusters synthesis pathway, involving CYP51 and CYP61 homologues (see below) and it can be expected that many of the CYPs will be in- Deng et al. (2007) defined a gene cluster as 4 or more CYPs pres- volved in processes such as biosynthesis of pigments, antioxidants, ent within a 100 kb sliding window of genome sequence and this defence compounds, toxin inactivation, virulence factors as well as revealed the presence of 3 gene clusters for A. nidulans. By sorting in structural components of the cell (e.g. pyroverdine. dityrosine CYPs into groups that have less than 7 genes between them (http:// biosynthesis, see below) and signalling compounds. drnelson.utmem.edu/CytochromeP450.html), an additional 10 To date the functions of 13 CYPs from A. nidulans have been de- gene clusters are observable, giving 13 in total. These are given in)- duced experimentally and these are given in Table 1. The relatively Fig. 2 9 having only two CYPs as near neighbours, 1 having 3 CYPs, slow progress of assigning function to specific CYPs poses a chal- 2 having 4 CYPs in a cluster and 1 has potentially 5 CYPs grouped. lenge, but progress has been made recently through an interesting The function of only two of these groups is known, that for sterig- approach by Bergmann et al., 2007. They have shown that by matocystin biosynthesis, (Fig. 2, cluster 5) (Brown et al., 1996) and inducing so-called ‘‘silent metabolic pathways” it was possible to aspyridone biosynthesis (Fig. 2 cluster 6) (BergmanN et al., 2007), uncover the function of a number of genes including in this in- the remaining groups have no function assigned and as such may stance two previously unknown CYPs, apdE (AN8411.3, CYP655A1) be amenable to the same experimental approach. However, for D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61 S55

Table 1 CYPome of A. nidulans

CYP AN.3 loc. gene CYP AN.3 loc. gene CYP AN.3 loc gene CYP680A1 AN0338.3 VIIIR CYP631B1 AN3225.3 VIR CYP59A1 AN7808.3 IVR stcSe,f CYP578C1 AN0459.3 VIIIR CYP649A1* AN3253.3 VIR CYP60A2 AN7818.3 IVR stcFe,f,g CYP5076A1 AN0606.3 VIIIR CYP648A1* AN3256.3 VIR CYP62A1 AN7824.3 IVR stcBe,f,g CYP684A1 AN10028.3 VIIIR CYP667A1 AN3272.3 VIR CYP548C1 AN7881.3 IIL CYP532A4 AN10259.3 VIIR CYP567E1 AN3275.3 VIR CYP682A1 AN7932.3 IIL CYP654A1 AN10389.3 VIR CYP567D1 AN3281.3 VIR CYP674A1* AN7969.3 IIL CYP5116B1 AN10435.3 IIR CYP659A1 AN3349.3 VIR CYP541B1 AN8004.3 IIL CYP540B4 AN10479.3 IIR CYP620D1 AN3394.3 VIR CYP504A1 AN8078.3 IIL phacAk CYP682E1 AN10573.3 IIIL ivoCa CYP623B2 AN3497.3 IIR CYP652A1* AN8139.3 IIL CYP619B1 AN10613.3 IIIL CYP681A1* AN3609.3 IIR CYP5073A1* AN8141.3 IIL CYP539B2 AN10617.3 IIIL CYP539D1 AN3917.3 IIR CYP653A1 AN8184.3 IIL CYP657A1 AN10647.3 V ahbBb CYP61A1 AN4042.3 IIR CYP647A1* AN8250.3 IIL CYP59C1 AN10704.3 VR CYP660A1 AN4117.3 IIR CYP51F1 AN8283.3 IIL cyp51Bi CYP619B2 AN10776.3 IL CYP675A1 AN4643.3 IIIL CYP682B1 AN8309.3 VL CYP65U1 AN10811.3 IL CYP5120B1 AN5335.3 VR CYP671A1 AN8338.3 VL CYP578B1 AN1087.3 VIIIR CYP58C1 AN5360.3 VR CYP530A3 AN8358.3 VL CYP5095B1 AN10887.3 IVL CYP673A1 AN5433.3 VR CYP685A1* AN8408.3 VL apdBl CYP53A3 AN10950.3 IVR bzuAc,d CYP5080D1 AN5553.3 VR CYP655A1 AN8411.3 VL apdEl CYP60B1 AN11013.3 IVR stcLe,f,j CYP531D2 AN5665.3 VR CYP5125A1 AN8437.3 VL CYP547C1 AN11142.3 VIIL CYP630B2 AN5837.3 IL CYP658A1* AN8438.3 VL CYP664A1* AN11192.3 VIL CYP52G1 AN6057.3 IL CYP68L1 AN8530.3 VL CYP687A1 AN11220.3 VIIIL CYP671B1 AN6101.3 IL CYP677A1 AN8615.3 IIIR CYP504B1 AN1397.3 VIIIR phacBh CYP656A1* AN6321.3 IL CYP537B1 AN8905.3 VIIL CYP503B1 AN1598.3 VIIR CYP552E1 AN6407.3 IL CYP540A2 AN8919.3 VIIL CYP620E1 AN1601.3 VIIR CYP62C1 AN6414.3 IL CYP676A1 AN8952.3 VIIL CYP5128A1 AN1703.3 VIIR CYP672A1 AN6434.3 IL CYP548D1 AN9007.3 VIIL CYP567C1 AN1737.3 VIIR CYP5077A1* AN6449.3 IL CYP683A1* AN9030.3 VIIL CYP666A1 AN1748.3 VIIR CYP678A1* AN6485.3 IL CYP662A1 AN9210.3 VIL CYP682D1 AN1794.3 VIIR CYP682C1 AN6787.3 IR CYP550B2 AN9214.3 VIL CYP617D1 AN1884.3 VIIR CYP505A8 AN6835.3 IR CYP540D1 AN9218.3 VIL CYP51F2 AN1901.3 VIIR cyp51Ai CYP670A1* AN7066.3 IVL CYP679A1* AN9225.3 VIL CYP552A2 AN2040.3 VIIR CYP52H1 AN7131.3 IVL CYP650B1* AN9248.3 VIIIL CYP646A1 AN2596.3 VIIR CYP5078A3 AN7359.3 IVL CYP650A1* AN9251.3 VIIIL CYP661A1 AN2607.3 VIIR CYP663A1 AN7399.3 IVL CYP651A1* AN9253.3 VIIIL CYP566C1 AN2610.3 VIIR CYP65T1 AN7522.3 IVR CYP535D1 AN9296.3 VIIIL CYP56B1 AN2706.3 VIR CYP5080B1 AN7772.3 IVR CYP58D1 AN9313.3 VIIIL CYP665A1* AN2727.3 VIR CYP573A3 AN7773.3 IVR CYP584E1 AN9384.3 VIIIL Pseudogenes CYP669A1P AN0008.3 VIIIR CYP630B3P AN2191.3 VIIR CYP539A4P AN5478.3 VR CYP65AC3P AN10101.3 VIIIR CYP504E4P AN2347.3 VIIR CYP686A1P* AN8510.3 VL CYP548G2P AN1300.3 VIIIR CYP551A3P AN5460.3 VR

* Unique families. a McCorkindale et al., 1983. b Lin and Momany, 2004. c Hynes, 1975. d Fraser et al., 2002. e Brown et al., 1996. f Keller et al., 1995. g Yu and Leonard, 1995. h Ferrer-Sevillano and Fernández-Cañón, 2007. i Mellado et al., 2001. j Kelkar et al., 1997. k Mingot et al., 1999. l Bergmann et al., 2007.

the majority of the genes 90%, the function is unknown and that (AN1899.3), homogentisate 1,2-dioxygenase (AN1897.3), fumaryl- these may well function individually, i.e. only one CYP in a path- acetoacetate hydrolase (AN1896.3), and maleylacetoacetate isom- way (e.g. DIT2 above), or as in the case of the sterol pathway erase (AN1895.3). In A. terreus, CYP51F2 (ATEG_05917) is four (CYP51 and CYP61) even though present on the same chromosome genes from the four aromatic amino acid catabolic genes. In A. ory- be spatially much further apart so as not to be considered as part of zae (locus AO090003000205) and A. niger (An11g02230) the a gene cluster. Indeed a check for synteny (http://sybil.source- CYP51F2 is only 2 genes from the catabolic cluster. The Fernán- forge.net) of neighbouring CYPs in a number of fungi, including dez-Cañón lab has shown that CYP504A1 (AN8078.3) is a phenylac- A. nidulans, Neosartorya ﬁscheri, A. terreus, A. clavatus NRRL1, A. etate 2-hydroxylase and CYP504B1(AN1397.3) catalyzes 3- fumigatus AFU293, A. oryzae and A. niger CBS513.88, indicates that hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydrox- functional clusters of > 2 CYPs are limited and that the majority ylations (Mingot et al., 1999; Ferrer-Sevillano and Fernández- may function independently of other CYPs in distinct pathways. Cañón, 2007). These genes act upstream of the four genes men- An example of this can be seen for CYP51F2 (AN1901.3). In A. nidu- tioned above by hydroxylating the phenylacetate ring in prepara- lans it is one gene away from four genes involved in phenylacetate tion for dioxygenase ring cleavage. The CYP504 genes are not or tyrosine degradation (Fernández-Cañón and Peñalva, 1995). close to the catabolic cluster in A. nidulans, but CYP504B4 is only These include a 4-hydroxyphenylpyruvate dioxygenase one gene away from a homogentisate 1,2-dioxygenase, fumarylac- S56 D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61

Fig. 2. Potential P450 clusters (numbered as in text), defined as CYP genes with less than 7 genes between each other (see text), as located on chromosomes. Putative redox partners (bold); Pseudogenes (P in bold). Unique families (*). Known function (a, b, c, d, e, f, g, h, i, j, k,l. see Table 1); L-arm of the chromosome is at the bottom. Homologue in yeast (black underlined CYP name); Possible redox partners (cpr, nr1, msr, B5red in violet). CYP near possible redox partner (violet underlined); Functional cluster (in numbered box); Polyketide synthase (PKS in grey field); Nonribosomal peptide synthetase (NRPS in violet field); Data are from Cadre (http://www.cadre-genomes.org.uk/)(Mabey et al., 2004).

etoacetate hydroxylase pair in Nectria haematococca (chromosome Eukaryotes do not have bacterial style operons except in nema- 12) and CYP504A6 is 60 kb downstream (i.e. about 20–30 genes dis- todes (Qian and Zhang, 2008). The genes of pathways tend to be tant, based on approximate fungal gene size 2–3 Kb) of CYP504B4 scattered in most eukaryotes, though they may be regulated by in that genome. This suggests the pathway was contiguous in a common transcription factors. Fungi are an exception. The white common ancestor and it was broken up over time. The proximity rot fungal genome has large numbers of cytochrome P450 genes of CYP51F2 to this catabolic cluster suggests it may participate in and many appear in gene clusters with up to 11 CYPs (Doddapan- degradation of a substrate that is similar to phenylacetate in a eni et al., 2005; Yadav et al., 2006). These clusters are mainly built manner analogous to CYP504A1, or more probably reﬂects synteny by tandem duplication and the genes are not working in a single of unrelated genes. Despite a deﬁnitive function for CYP51F2, its pathway to make one product. Instead they are probably working presence next to a gene cluster dedicated to one function raises in catabolism of related substrates such as lignin degradation prod- the issue of secondary metabolite gene clusters in fungi, a very ucts. In contrast to these rather special degradative gene clusters, important consideration in the analysis of P450s in fungi. many secondary metabolites such as toxins and pigments are D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61 S57

Fig. 3. UPGMA tree of all 111 P450 protein sequences from A. nidulans. Pseudogene products are excluded due to large gaps in the sequences. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 3.1 (Kumar et al., 2004). synthesized by gene clusters in fungi. Recently the gene LaeA has nonribosomal peptide synthase (NRPS) genes. These might only in- been identified as a global regulator of many of these gene clusters volve a single P450 so they would be missed in the search above. In (Perrin et al., 2007). This particular observation offers the possibil- fact, 11 single P450s were found eight genes or less from a PKS, ity of detecting the clusters by coordinate expression of adjacent Supplementary Table S1, or NRPS gene Supplementary Table S2. genes on microarrays when LaeA function is perturbed. Analysis When looking at the neighbourhood of the 13 CYP gene clusters of known clusters has led to some common features that make pre- found earlier, nine of these also were near PKS or NRPS genes. This diction of clusters possible based on gene associations. Further- method identified 20 potential secondary metabolite gene clusters more, the presence of some genes in the cluster may have where P450s might serve to decorate the products of PKS or NRPS predictive value in trying to understand what the cluster is making. genes. In all, 32 of 111 functional P450s in A. nidulans (29%) were We have applied this method to look for clusters of genes in A. near PKS or NRPS genes. nidulans associated with CYPs to use as a method for uncovering One illustration given is of a probable toxin gene cluster CYP function. (AN3269.3 - AN3281.3) with four CYP genes (Fig. 2 cluster 9), a PKS, a major facilitator superfamily (MFS) transporter to export 2. Cytochrome P450s in proximity to PKS and NRPS genes the product and two fungal transcription factors of the Zn(II)2Csy6 type (Fig. 4 and supplementary table S3 for sequence identifiers). The 13 CYP gene clusters discussed above (Fig. 2) were found by This gene cluster also contains a terpene cyclase gene scanning for P450 genes separated by seven or fewer genes. The (AN3277.3) 63% identical to aristolochene synthase ari1 of Penicil- unusual property of fungi keeping secondary metabolite synthesis lium roqueforti. Aristolochene synthase genes are farnesylpyro- genes in clusters prompted the search for P450s that were near(de- phosphate (FPP) cyclases that form sesquiterpene rings. This fined here as <8 genes distant) polyketide synthase (PKS) genes or cluster is probably joining a sesquiterpene ring to a PKS product S58 D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61

Fig. 4. The terpene cyclase region from A. nidulans is aligned with six other genome’s homologous regions keeping the terpene cyclase as the alignment point. CYPs are indicated with filled arrows. The terpene cyclase proteins are similar to Penicillium roqueforti aristolochene synthase as indicated: A. nidulans 63%, A. clavatus 62%, A. oryzae 55%, A. flavus 55%, A. terreus 63%, Penicillium citrinum 61%, Podospora anserina 54%. Sequence ID’s are indicated in S3. and decorating the result with four P450 oxidations. The figure in- gene clusters seem to be regulated separately. It is relevant to note cludes the region around six other fungal terpene cyclase genes. that A. terreus has a toxin cluster orthologous to the compactin There is remarkable similarity in the P450 content near these cluster that makes lovastatin. It is not known, but it may be possi- genes. The A. clavatus cluster has all four of the CYPs in the same ble that the lovastatin gene cluster is just downstream of the ter- spatial arrangement indicating orthologous functions between pene cyclase gene as seen in the orthologous cluster in P. the two clusters. The PKS gene is larger in A. clavatus suggesting citrinum. Note that CYP567D, CYP567E and CYP667A genes are all additional modules so the PKS product is probably larger. A. clava- present as seen in the A. nidulans and A. clavatus clusters. This sug- tus has the best BLAST matches to the ZnCys MFS pair on the acces- gests that these other clusters may be making a similar compound sion NW_001517093.1. ZnCys = XM_001267927.1, as the P. citrinum cluster, but with a PKS addition. This view is MFS = XM_001267928.1. It appears this end of the gene cluster strengthened by the 65% identity of orf14 to AN3279.3 and A. clav- has moved in A. clavatus. The A. nidulans and A. clavatus clusters atus 3848. Orf14 is also 54% identical to A. oryzae 0102 and the A. are orthologous from the CYP667A genes to the CYP567D genes. flavus ortholog. Orf11 is the best BLAST hit among patent se- AN3276.3, a short chain dehydrogenase (DH), is 93% identical to quences to the A. clavatus 3845 sequence, though the identity is the A. clavatus 3845 sequence. The gene clusters in A. oryzae and only 31%. The synteny between these clusters suggests that orf11 A. flavus are nearly identical and they contain two of the three is a potential ortholog of AN3278.3 and A. clavatus 3847. P450 families found in A. nidulans and A. clavatus. There is a new Podospora anserina has the terpene cyclase and CYP567E as seen CYP in these two clusters and a PKS gene is not nearby. A. oryzae earlier, but it has an NRPS gene instead of a PKS. It may be related and A. flavus do not have a CYP667A gene in their genomes. The by descent, but it has changed significantly. A. oryzae ZnCys 0113 sequence and its A. flavus ortholog are 47% In a second example the gene neighbourhood of CYP genes may identical to A. clavatus 3839. They are not related to AN3269.3 or predict the function of a secondary metabolite cluster, such as a 20 AN3280.3. gene cluster on chromosome VI from AN9210.3 to AN11201.3. This The terpene cyclase of A. terreus (sequence from AF198360.) is region contains five CYP genes (cluster 8 in Fig. 2). The genes in the the known aristolochene synthase with crystal structures deter- cluster are characteristic for a metabolite cluster, i.e. a Zn(II)2Cys6 mined, (Shishova et al., 2008). This gene is the best match in the binuclear cluster transcription factor (AN9221.3), a PKS A. terreus genome to the other terpene cyclases in the figure and (AN11198.3), an NRPS (AN9226.3), an acyl-CoA synthetase-like probably has orthologous function. The region around this gene gene often seen in conjunction with PKS pathways, an efflux pum- is not in Genbank so the neighbouring genes are not known. How- p(AN9219.3) and an FAD binding monooxygenase (AN9224.3). The ever, two adjacent genes in A. terreus XM_001212369 and gene that gives a clue to the function of this cluster is AN11201.3 a XM_001212370 on NT_165927 are possible orthologs of dimethylallyltryptophan synthase-like gene. These enzymes cata- AN3276.3 and AN3280.3, but the surrounding genes are not re- lyze the first step in ergot alkaloid biosynthesis (Coyle and Panac- lated to the genes in Fig. 4. cione, 2005) and this suggests this cluster may synthesize an P. citrinum makes the compound ML-236B (compactin) an alkaloid product. inhibitor of HMG-CoA reductase and a precursor of pravastatin A final example here is taken from siderophore biosynthesis. A. (Baba et al., 2006). The compactin gene cluster is just downstream nidulans makes two siderophores, an external siderophore tria- of the cluster shown, following orf11 (Fig. 4). The two adjacent cetylfusarinine and an internal siderophore called ferricrocin D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61 S59

(Eisendle et al., 2003). Both pathways begin with the gene sidA (L- fusion. It is similar to CYP102 bacterial fusions and probably repre- ornithine-N5-monooxygenase). This gene is not a P450. Both inter- sents a lateral gene transfer with later diversification in eukary- nal and external siderophores are constructed by two different otes. The eukaryotic CYP505 clan has two families CYP505 and NRPS genes sidC and sidD respectively. The gene AN0607.3 is an CYP541. CYP541 has no fusion to a reductase partner, so it may NRPS gene orthologous to sidC (Eisendle et al., 2003). Adjacent to be an example of a fusion protein being broken. The next closest this gene is CYP5076A1 (AN0606.3). It is known that the internal family to CYP505 and CYP541 is CYP540. Three of the CYP540 siderophore ferricrocin is hydroxylated in A. fumigatus. The P450 genes were mentioned above as having reductase partners adja- gene CYP5076A1 is a prime candidate to perform that hydroxyl- cent to them. It seems probable that all three of these CYP families ation in A. nidulans and thus based on its location, it is predicted descended from a common ancestor that had a fusion structure. to be a ferricrocin hydroxylase. The ferricrocin hydroxylase in A. CYP505 has four subfamilies. In Phanerochaete chrysosporium fumigatus has not been identified and there is no P450 next to there are five CYP505D sequences in a 60 Kb window on scaffold the sidC gene in A. fumigatus. The related P450 CYP5076C2 in A. 3 from 150,000 to 210,000. This tandem arrangement of very sim- fumigatus is adjacent to a different NRPS gene called ftmA ilar genes argues against a pathway and in favour of a catabolic (Afu8g00170) that is the brevianamide synthetase gene (Maiya function with slightly different or overlapping substrate prefer- et al., 2006). This gene cluster has a dimethylallyltryptophan syn- ences for each. Two other CYP505D genes are elsewhere in the thase-like gene as mentioned earlier, and it is involved in making white rot genome. Since white rot is known for its metabolic deg- brevianamide F, a precursor to several known alkaloids. A. fumiga- radation abilities as a consumer, these seven CYP505D genes prob- tus has another CYP that could be a candidate for the ferricrocin ably contribute to that mechanism. In other species the CYP505 hydroxylase function: CYP5076D1 (AFUA_4G09470). genes can be biosynthetic rather than degradative. CYP505B1 is We have examined some of the 20 secondary metabolite clus- FUM6 in the fumonisin biosynthetic pathway of Fusarium verticil- ters predicted for A. nidulans that include at least one P450 gene. lioides (Seo et al., 2001). FUM2 (CYP65AH1) is the fumonisin C10- There are additional clusters that do not involve P450s. Perrin hydroxylase in the same pathway (Proctor et al., 2006). There are et al., 2007 have systematically searched for these clusters in A. 14 named CYP505A genes in filamentous fungi. Some of them fumigatus and found 22, though only nine contained P450 genes. may help utilize fatty acids as a carbon source since CYP505A1,or If each filamentous fungal genome contains 20 or more secondary P450foxy, is a known fatty acid hydroxylase like CYP102A1 in B. metabolite clusters, there will soon be hundreds of these gene clus- megaterium (Kitazume et al., 2008). Others may function in gene ters to compare. The CYPs seem to be a highly variable part of these clusters to make a toxin like fumonisin as seen in some Fusarium clusters. With some comparative data as gleaned from Fig. 4 it may species. Neighbourhoods near the gene should be examined for be possible to create new metabolites by either deleting P450s or evidence of gene cluster characteristics analogous to the polyke- by adding P450s from homologous clusters. tide synthases approach used here and for fungal specific transcription factor(s). 2.1. Redox partners

For functional activity most CYPs require the input of two suc- 3. Subcellular localisation of A. nidulans CYPs cessive electrons from NADPH via a NADPH cytochrome P450 reductase and/or cytochrome b5 reducatase and cytochrome b5 Eukaryotic cytochromes P450 are usually found bound to mem- (Lamb et al., 1999; de Vetten et al., 1999). Mining the genome se- branes, in most instances anchored on the cytoplasmic surface of quence of A. nidulans further we have identified 8 putative NADPH the endoplasmic reticulum (ER) through a short N-terminal hydro- cytochrome P450 reductase sequences (Fig. 2). AN0595.3 on chro- phobic sequence (Nelson and Strobel, 1988), while some mamma- mosome VIII has 91% sequence ID to cprA from A. niger and 88% ID lian CYPs are known to be located on the matrix side of the to cprA from A. fumigatus and represents the most probable candi- mitochondrial inner membrane. The latter include those involved date reductase for the majority of the CYPs. However, a second in sterol, steroid and bile acid biosynthesis. We have undertaken reductase with 51% sequence ID to cprA (A.fumigatus) AN5838.3 a bioinformatics analysis of the amino acid sequences of all CYPs is present on chromosome I and interestingly adjacent to CYP630B2 from A. nidulans in order to define possible ER signal peptides, (AN5837.3). We postulate that this may be specific for this CYP and the presence, abundance and relationships between trans- whose function is yet to be elucidated. AN8920.3 present on chro- membrane regions and re-entrant loops. mosome VII is immediately downstream of CYP540A2, but has the Using TargetP (Emanuelsson et al., 2000), cross-referenced with additional feature of a siderophore-interacting FAD binding do- the two different modes (the neural network and HMM versions) main, that may provide a clue to function. In the same way, of SignalP (Bendtsen et al., 2004), 75 of the proteins were predicted AN3862.3 is immediately downstream of CYP540B4 (chromosome to possess N-terminal signal peptides. Typical microsomal CYPs II) and might also be specific for this CYP. Lah et al., 2008 have fur- possess a stretch of about 20 hydrophobic residues serving as a ther identified members of this protein family with similar domain membrane anchor in the endoplasmic reticulum and current mod- architecture, which include a novel reductase 1 (NR1), AN10283.3 els still represent the triangular fold common to the CYPs lying flat and a methionine synthase reductase (MSR), AN2042.3, one gene on the membrane surface with the haem parallel or inclined to the away fron CYP552A2. AN11198.3 is a putative cytochrome b5 membrane (Nelson and Strobel, 1988; Ohta et al., 1992). In two reductase and neighbour of CYP540D1 AN9218.3 in the middle of mammalian CYPs (CYP2C5 and CYP2B4, Williams et al., 2000) the cluster 8 (chromosome VI) with 5 CYPs and which appears to be presence of F’ and/or G’ helices form a hydrophobic surface near specific for A. nidulans. Finally, AN6835.3 is annotated as CYP505A8 the N-terminus of the protein and it is thought that this portion on chromosome I and is a member of clan CYP505, a group of CYPs of the CYP may lie in close proximity to the membrane and possi- that are fused to their reductase partner and found previously in bly be partially inserted (De-Lemos-Chiarandini et al., 1987). Fusarium oxysporum (CYP505A1, P450foxy)(Nakayama et al.,1996; Transmembrane region topology prediction, using the TMHMM Kitazume et al., 2000) and Bacillus megaterium (CYP102, BM3, Narhi program (Sonnhammer et al., 1998; Krogh et al., 2001), and subse- and Fulco. 1986) are responsible for fatty acid hydroxylation. quently corrected for coincident signal peptides predicted using Fusions between CYP genes and a reductase partner are rare, Signal P (Bendtsen et al., 2004) and TargetP (Emanuelsson et al., with most of the known examples being bacterial. CYP505 is the 2000), suggests that 83 of the CYPs do not possess any transmem- only eukaryotic family with an NADPH cytochrome P450 reductase brane regions, however using TMLoop (Lasso et al., 2006), 2 of S60 D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61 these sequences (CYP620E1 and CYP654A1) were predicted to pos- Bergmann, S., Schumann, J., Scherlach, K., Lange, C., Brakhage, A.A., Hertweck, C., sess re-entrant loops, suggesting at least some anchoring to the 2007. Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans. Nat. Chem. Biol. 3 (4), 213–217. membrane in regions other than the N-terminus signal peptide. Fi- Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of nally, 34 of the proteins were not predicted to possess N-terminal signal peptides, SignalP 3.0. J. Mol. Biol 340, 783–795. signal peptides. Of these, one protein (CYP61A1) possesses a C-ter- Briza, P., Kalchhauser, H., Pittenaver, E., Allmaier, G., Breitenbach, M., 1996. N, N’- Bisformyl dityrosine is an in vivo precursor of the yeast ascopspore wall. Eur. J. minal XDEL motif and 4 proteins were found to contain the C-ter- Biochem. 239, 124–131. minal ER retention KKXX motifs (searched for with a cut-off of the Brown, D.W., Yu, J.H., Kelkar, H.S., Fernades, M., Nesbitt, T.C., Keller, N.P., Adams, last 20 residues), CYP504B1, CYP631B1, CYP5080D1 and CYP552E1. T.H., Leonard, T.J., 1996. Twenty-five co-regulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans. Proc. Natl. Acad. Sci. Of the four, only CYP504B1 does not also possess an N-terminal sig- U.S.A. 93, 1418–1422. nal peptide, giving a total of 72 proteins targeted to the ER. Coyle, C.M., Panaccione, D.G., 2005. An ergot alkaloid biosynthesis gene and clustered hypothetical genes from Aspergillus fumigatus. Applied and Environ. Micro. 71, 3112–3118. 4. Conclusion Crooks, G.E., Hon, G., Chandonia, J.M., Brenner, S.E., 2004. WebLogo; A sequence logo generator. Genome Res. 14, 1188–1190. Filamentous fungi have 41 (Neurospora) to 152 (Aspergillus ory- De-Lemos-Chiarandini, C., Frey, A.B., Sabatini, D.D., Kreibich, G., 1987. Determination of the membrane topology of the Phenobarbital-inducible rat zae) P450s. Only two have established roles in housekeeping func- liver cytochrome P450 isozyme PB-4 using site-specific antibodies. J. Cell Biol. tions in sterol biosynthesis CYP51 and CYP61. The others are 104, 209–219. enzymes specific to the purpose(s) essential or useful to the fun- Deng, J., Carbone, I., Dean, R.A., 2007. The evolutionary history of Cytochrome P450 genes in four filamentous Ascomycetes. BMC Evolutionary Biology. 7, 30. gus. Often this involves pathogenesis and the production of toxins De Vetten, N., Ter Horst, J., Van Schaik, H.-P., De Boer, A., Mol, J., Koes, R., 1999. A and virulence factors, or the utilization of specific carbon sources. cytochrome b5 is required for full activity of flavonoid 3’, 5’-hydroxylase, a Many studies have been done on deciphering the gene clusters that cytochrome P450 involved in the formation of blue flower colours. Proc. Natl. Acad. Sci.USA 96, 778–783. make these toxins such as sterigmatocystin, aflatoxin and fumon- Doddapaneni, H., Chakraborty, R., Yadav, J.S., 2005. Genome-wide structural and isin. Frequently these clusters contain CYP genes. Here we have evolutionary analysis of the P450 monooxygenase genes (P450ome) in the presented a detailed account of the A. nidulans CYPs and putative white rot fungus Phanerochaete chrysosporium, evidence for gene duplications redox partners and we have identified those that are in probable and extensive gene clustering. BMC Genomics. 14 (6), 92. 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