The CYPome (Cytochrome P450 complement) of Aspergillus nidulans

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
Article history:
Received 9 May 2008
Accepted 19 August 2008
Available online 12 September 2008
Index descriptor:
Cytochrome P450
CYPs
Haem-containing mono-oxygenases
Aspergillus nidulans
1. Introduction
abstract
Filamentous fungi produce a vast array of secondary metabolites
and an excellent review (Hoffmeister and Keller, 2007) has
identified the major structural classes of these compounds, many
of which may be harnessed as useful bioactive compounds, but
which have arisen so that the organism in question may occupy
a particular ecological niche. A consequence of this evolution is
that specific biosynthetic pathways may only exist in certain fungi
and not in others. Closer inspection of such pathways has revealed
that many of these compounds are tailored (modified) by the action
of a particular family of enzymes known as cytochromes
P450 (CYPs).
CYPs are found throughout all the biological kingdoms and are a
superfamily of haem-containing monooxygenases, interest in
which has resulted in extensive research papers and review articles,
(Ortiz de Montellano et al., 1995; Ortiz de Montellano et al.,
2005). Genome sequencing projects continue to reveal the ever
increasing number of these CYPs (presently > 8000, http://drnelson.utmem.edu/CytochromeP450.html,
numbering 1672 from
35 fungal genomes, (Intikhab et al., 2007), with 1384 already assigned
a CYP name http://drnelson.utmem.edu/CytochromeP450.html,
although the functions of the vast majority are
still unknown. The primary aim of this work was to identify the
complete CYPome of Aspergillus nidulans and match these with
the standardized nomenclature assigned by Nelson, D. R. originally
to version AN.2 of the genome sequence (http://drnel-
* Corresponding author.
E-mail address: D.Kelly@swansea.ac.uk (D.E. Kelly).
1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2008.08.010
Fungal Genetics and Biology 46 (2009) S53–S61
Contents lists available at ScienceDirect
Fungal Genetics and Biology
journal homepage: www.elsevier.com/locate/yfgbi
The cytochromes P450 (CYPs) are found in all biological kingdoms and genome sequencing projects continue
to reveal an ever increasing number. The principle aim of this paper is to identify the complete
CYPome of Aspergillus nidulans from the genome sequence version AN.3 deposited at the Broad institute,
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
89 CYP families, 21 of which are unique. We have identified 28 potential gene clusters associated with
one or more CYP genes and discussed those with putative PKS and NRPS associated function. The chromosomal
location of the genes, predicted cellular location of the proteins and possible function(s) are
discussed.
Ó 2008 Elsevier Inc. All rights reserved.
son.utmem.edu/CytochromeP450.html), but now to version AN.3
deposited at the Broad Institute (http://www.broad.mit.edu/annotations/fungi/aspergillus).
We describe the chromosomal location
of the genes, predicted cellular location of the proteins and focus
on defining function for as many of the CYPs found as possible,
while seeking clues to function for the remainder.
1.1. Sequence motifs as a tool to identify CYPs
The nomenclature for CYPs is based upon amino acid identity;
40% identity and above place a CYP in the same family, more than
55% identity places them in the same subfamily (Nebert et al.,
1987; Nelson et al., 1996). Families are designated a CYP number
based on those reserved for different taxonomic groups (http://
drnelson.utmem.edu/CytochromeP450.html), for fungi these are
CYP51-CYP69, CYP501-CYP699 and CYP5001-CYP6999. Detection
of CYP genes in a genome is greatly facilitated by the presence of
consensus amino acid sequences (Fig. 1): FXXGXXXCXG, the haem
binding domain containing the axial Cys ligand to the haem (highlighted).
EXXR motif found in the K – helix and the PER(W) domain.
Modifications in the haem binding region were found in CYP5120B1
(AN5335.3), this unusually has no conserved Cys downstream of
the normal EXXR and PER(W) motifs, in common with A. terreus,
CYP5121A1 Neosartorya fischeri, CYP5121A2 A. fumigatus and
CYP5121B1 from A. clavatus alignments, none of the five fungal sequences
had a Cys in this region. Instead the Cys is found at the
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
P450 structure. Modifications in the haem binding domain are
more usually found in CYPs with catalytic activity, often not

S54 D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61
Fig. 1. Consensus amino acid sequences in P450 from A. nidulans. Data were created
using WebLogo (http://weblogo.berkeley.edu/) (Crooks et al., 2004; Schneider and
Stephens, 1990). a - Oxygen binding and activation; b and c – ERR triad and d –
Heme binding.
requiring oxygen, such as prostacyclin synthases (CYP8A, Li et al.,
2008) and allene oxide synthases (CYP74, Song et al., 1993) and
may indicate a novel catalytic activity in this instance. Two further
CYPs, CYP62C1 (AN6414.3) and CYP66A1 (AN2607.3) possess the
invariant Cys, but residues upstream are replaced either by fewer
amino acids in the signature GXXCXG, or by an alternative residue
HXXXCXG respectively. It has been postulated that the haem binding
signature requires only the invariant Cys (Rupasinghe et al.,
2006).
1.2. Gene assignation
A complete list of all the A.nidulans CYPs is given in Table 1. The
total gene count is 119, including 8 pseudogenes (with in-frame
stop codons, frameshifts or deletions), representing 89 CYP families
(see Fig. 3 for their phylogenetic relationships), 21 of which are unique
to A. nidulans and representing approximately 1% of the
genome.
Closer inspection of protein sequences showed there is substantial
variation in sequence length across the CYPs, ranging from 286
to 750 residues. All sequences were further analysed to determine
incorrect exon calling, incorrect start or stop sequences and 41 sequences
were identified with different gene structure requiring
manual correction. Corrected proteins, after exclusion of pseudogenes
and a fusion protein contained between 417– 607 residues,
with an average of 511 amino acids.
During the course of this work, a comparative study for four filamentous
Ascomycetes by Deng et al., 2007 identified putative CYP
sequences for A. nidulans. We have since determined that previously
designated CYPs 539A4P (AN5478.3), 551A3P (AN5460.3),
630B3P (AN2191.3), 669A1P (AN0008.3) and 686A1P (AN8510.3)
were incorrect and are in fact pseudogenes. The completed analysis
revealed three new CYP sequences in the updated version AN.3
from Broad, these are AN10887.3, AN1703.3, AN8437.3 and named
CYP5095B1, CYP5128A1 and CYP5125A1 respectively. Table 1 illustrates
the linkage between all the A. nidulans CYPs, including pseudogenes,
their chromosomal location and AN.3 reference number
identified in this study.
1.3. Protein function
The natural substrates of fungal CYPs are largely unknown, but
will include precursors of membrane sterols in the ergosterol biosynthesis
pathway, involving CYP51 and CYP61 homologues (see
below) and it can be expected that many of the CYPs will be involved
in processes such as biosynthesis of pigments, antioxidants,
defence compounds, toxin inactivation, virulence factors as well as
in structural components of the cell (e.g. pyroverdine. dityrosine
biosynthesis, see below) and signalling compounds.
To date the functions of 13 CYPs from A. nidulans have been deduced
experimentally and these are given in Table 1. The relatively
slow progress of assigning function to specific CYPs poses a challenge,
but progress has been made recently through an interesting
approach by Bergmann et al., 2007. They have shown that by
inducing so-called ‘‘silent metabolic pathways” it was possible to
uncover the function of a number of genes including in this instance
two previously unknown CYPs, apdE (AN8411.3, CYP655A1)
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,
it is difficult and in most instances impossible, to predict the specific
functions of the A. nidulans CYPs simply from their sequence
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.,
2005). However, for some families there is sufficient knowledge
to be able to assign a putative function that may then be the subject
for further experiments.
The fungal sterol pathway is known to require two CYP activities.
The first CYP51 is the only P450 that has an orthologue (showing
22-23% sequence ID between kingdoms) found in all kingdoms
of life from bacteria, lower eukaryotes through fungi, plants and
animals (Yoshida et al., 2000). This is thought to be the most ancient
of CYPs (Aoyama et al., 1996; Nelson, 1999) and is a sterol
14a-demethylase involved in the biosynthesis of cholesterol in
animals, phytosterols in plants and ergosterol in fungi. CYP51 genes
have been cloned from a number of fungi including Saccharomyces
cerevisiae, Candida albicans and A. fumigatus (Bard et al., 1993; Mellado
et al., 2001) and some of the proteins have been characterised,
such that 41 conserved residues have been identified (Lepesheva
and Waterman, 2004). Unlike in S. cerevisiae where only one copy
of this essential gene (ERG11) is present, two CYP51 sequences are
present in A. nidulans CYP51F1 (AN8283.3, cyp51B) and CYP51F2
(AN1901.3, cyp51A).
Hu et al. (2007) have demonstrated that both CYP51F1 and F2
orthologues in A. fumigatus can act in a compensatory manner in
the ergosterol pathway, i.e. neither is essential individually, but a
double knockout is lethal. They postulate that CYP51F2 (CYP51A)
may encode the major 14a-demethylase activity required for
growth based on accumulation of multiple missense mutations
linked to azole resistance and CYP51F1 may serve a redundant role
with CYP51F2 or an alternative function under particular growth
conditions yet to be defined.
A BLASTP search (Altschul et al., 1997) against the ERG5 yeast
sequence (22-sterol desaturase) has revealed the second (single
copy) CYP in the ergosterol pathway in A. nidulans (AN4042.3,
CYP61A1).
A third and final CYP found in S. cerevisae is involved in the biosynthesis
of dityrosine, a major component of the spore wall surface
(Briza et al., 1996), DIT2 or CYP56. A homologue AN2706.3
(CYP56B1, 41% ID) is present in A. nidulans. Closer examination
shows that the adjacent upstream gene AN2705.3 has 35% ID with
DIT1 from S. cerevisae. Further homologues are also present in A.
fumigatus, A. orzyae and A. fisherianus and it therefore probable that
these genes code for the same function, i.e. that a dityrosine macromolecular
network is also present in the Aspergillus genus.
1.4. CYP clusters
Deng et al. (2007) defined a gene cluster as 4 or more CYPs present
within a 100 kb sliding window of genome sequence and this
revealed the presence of 3 gene clusters for A. nidulans. By sorting
CYPs into groups that have less than 7 genes between them (http://
drnelson.utmem.edu/CytochromeP450.html), an additional 10
gene clusters are observable, giving 13 in total. These are given in)-
Fig. 2 9 having only two CYPs as near neighbours, 1 having 3 CYPs,
2 having 4 CYPs in a cluster and 1 has potentially 5 CYPs grouped.
The function of only two of these groups is known, that for sterigmatocystin
biosynthesis, (Fig. 2, cluster 5) (Brown et al., 1996) and
aspyridone biosynthesis (Fig. 2 cluster 6) (BergmanN et al., 2007),
the remaining groups have no function assigned and as such may
be amenable to the same experimental approach. However, for

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 stcS e,f
CYP578C1 AN0459.3 VIIIR CYP649A1 *
AN3253.3 VIR CYP60A2 AN7818.3 IVR stcF e,f,g
CYP5076A1 AN0606.3 VIIIR CYP648A1 *
AN3256.3 VIR CYP62A1 AN7824.3 IVR stcB e,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 phacA k
CYP682E1 AN10573.3 IIIL ivoC a
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 ahbB b
CYP61A1 AN4042.3 IIR CYP647A1 *
AN8250.3 IIL
CYP59C1 AN10704.3 VR CYP660A1 AN4117.3 IIR CYP51F1 AN8283.3 IIL cyp51B i
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 apdB l
CYP53A3 AN10950.3 IVR bzuA c,d
CYP5080D1 AN5553.3 VR CYP655A1 AN8411.3 VL apdE l
CYP60B1 AN11013.3 IVR stcL e,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 phacB h
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 cyp51A i
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
Pseudogenes
VIR CYP573A3 AN7773.3 IVR CYP584E1 AN9384.3 VIIIL
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
these may well function individually, i.e. only one CYP in a pathway
(e.g. DIT2 above), or as in the case of the sterol pathway
(CYP51 and CYP61) even though present on the same chromosome
be spatially much further apart so as not to be considered as part of
a gene cluster. Indeed a check for synteny (http://sybil.sourceforge.net)
of neighbouring CYPs in a number of fungi, including
A. nidulans, Neosartorya fischeri, A. terreus, A. clavatus NRRL1, A.
fumigatus AFU293, A. oryzae and A. niger CBS513.88, indicates that
functional clusters of > 2 CYPs are limited and that the majority
may function independently of other CYPs in distinct pathways.
An example of this can be seen for CYP51F2 (AN1901.3). In A. nidulans
it is one gene away from four genes involved in phenylacetate
or tyrosine degradation (Fernández-Cañón and Peñalva, 1995).
These include a 4-hydroxyphenylpyruvate dioxygenase
D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61 S55
(AN1899.3), homogentisate 1,2-dioxygenase (AN1897.3), fumarylacetoacetate
hydrolase (AN1896.3), and maleylacetoacetate isomerase
(AN1895.3). In A. terreus, CYP51F2 (ATEG_05917) is four
genes from the four aromatic amino acid catabolic genes. In A. oryzae
(locus AO090003000205) and A. niger (An11g02230) the
CYP51F2 is only 2 genes from the catabolic cluster. The Fernández-Cañón
lab has shown that CYP504A1 (AN8078.3) is a phenylacetate
2-hydroxylase and CYP504B1 (AN1397.3) catalyzes 3hydroxyphenylacetate
and 3,4-dihydroxyphenylacetate 6-hydroxylations
(Mingot et al., 1999; Ferrer-Sevillano and Fernández-
Cañón, 2007). These genes act upstream of the four genes mentioned
above by hydroxylating the phenylacetate ring in preparation
for dioxygenase ring cleavage. The CYP504 genes are not
close to the catabolic cluster in A. nidulans, but CYP504B4 is only
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
12) and CYP504A6 is 60 kb downstream (i.e. about 20–30 genes distant,
based on approximate fungal gene size 2–3 Kb) of CYP504B4
in that genome. This suggests the pathway was contiguous in a
common ancestor and it was broken up over time. The proximity
of CYP51F2 to this catabolic cluster suggests it may participate in
degradation of a substrate that is similar to phenylacetate in a
manner analogous to CYP504A1, or more probably reflects synteny
of unrelated genes. Despite a definitive function for CYP51F2, its
presence next to a gene cluster dedicated to one function raises
the issue of secondary metabolite gene clusters in fungi, a very
important consideration in the analysis of P450s in fungi.
Eukaryotes do not have bacterial style operons except in nematodes
(Qian and Zhang, 2008). The genes of pathways tend to be
scattered in most eukaryotes, though they may be regulated by
common transcription factors. Fungi are an exception. The white
rot fungal genome has large numbers of cytochrome P450 genes
and many appear in gene clusters with up to 11 CYPs (Doddapaneni
et al., 2005; Yadav et al., 2006). These clusters are mainly built
by tandem duplication and the genes are not working in a single
pathway to make one product. Instead they are probably working
in catabolism of related substrates such as lignin degradation products.
In contrast to these rather special degradative gene clusters,
many secondary metabolites such as toxins and pigments are

synthesized by gene clusters in fungi. Recently the gene LaeA has
been identified as a global regulator of many of these gene clusters
(Perrin et al., 2007). This particular observation offers the possibility
of detecting the clusters by coordinate expression of adjacent
genes on microarrays when LaeA function is perturbed. Analysis
of known clusters has led to some common features that make prediction
of clusters possible based on gene associations. Furthermore,
the presence of some genes in the cluster may have
predictive value in trying to understand what the cluster is making.
We have applied this method to look for clusters of genes in A.
nidulans associated with CYPs to use as a method for uncovering
CYP function.
2. Cytochrome P450s in proximity to PKS and NRPS genes
The 13 CYP gene clusters discussed above (Fig. 2) were found by
scanning for P450 genes separated by seven or fewer genes. The
unusual property of fungi keeping secondary metabolite synthesis
genes in clusters prompted the search for P450s that were near(defined
here as

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 includes
the region around six other fungal terpene cyclase genes.
There is remarkable similarity in the P450 content near these
genes. The A. clavatus cluster has all four of the CYPs in the same
spatial arrangement indicating orthologous functions between
the two clusters. The PKS gene is larger in A. clavatus suggesting
additional modules so the PKS product is probably larger. A. clavatus
has the best BLAST matches to the ZnCys MFS pair on the accession
NW_001517093.1. ZnCys = XM_001267927.1,
MFS = XM_001267928.1. It appears this end of the gene cluster
has moved in A. clavatus. The A. nidulans and A. clavatus clusters
are orthologous from the CYP667A genes to the CYP567D genes.
AN3276.3, a short chain dehydrogenase (DH), is 93% identical to
the A. clavatus 3845 sequence. The gene clusters in A. oryzae and
A. flavus are nearly identical and they contain two of the three
P450 families found in A. nidulans and A. clavatus. There is a new
CYP in these two clusters and a PKS gene is not nearby. A. oryzae
and A. flavus do not have a CYP667A gene in their genomes. The
A. oryzae ZnCys 0113 sequence and its A. flavus ortholog are 47%
identical to A. clavatus 3839. They are not related to AN3269.3 or
AN3280.3.
The terpene cyclase of A. terreus (sequence from AF198360.) is
the known aristolochene synthase with crystal structures determined,
(Shishova et al., 2008). This gene is the best match in the
A. terreus genome to the other terpene cyclases in the figure and
probably has orthologous function. The region around this gene
is not in Genbank so the neighbouring genes are not known. However,
two adjacent genes in A. terreus XM_001212369 and
XM_001212370 on NT_165927 are possible orthologs of
AN3276.3 and AN3280.3, but the surrounding genes are not related
to the genes in Fig. 4.
P. citrinum makes the compound ML-236B (compactin) an
inhibitor of HMG-CoA reductase and a precursor of pravastatin
(Baba et al., 2006). The compactin gene cluster is just downstream
of the cluster shown, following orf11 (Fig. 4). The two adjacent
gene clusters seem to be regulated separately. It is relevant to note
that A. terreus has a toxin cluster orthologous to the compactin
cluster that makes lovastatin. It is not known, but it may be possible
that the lovastatin gene cluster is just downstream of the terpene
cyclase gene as seen in the orthologous cluster in P.
citrinum. Note that CYP567D, CYP567E and CYP667A genes are all
present as seen in the A. nidulans and A. clavatus clusters. This suggests
that these other clusters may be making a similar compound
as the P. citrinum cluster, but with a PKS addition. This view is
strengthened by the 65% identity of orf14 to AN3279.3 and A. clavatus
3848. Orf14 is also 54% identical to A. oryzae 0102 and the A.
flavus ortholog. Orf11 is the best BLAST hit among patent sequences
to the A. clavatus 3845 sequence, though the identity is
only 31%. The synteny between these clusters suggests that orf11
is a potential ortholog of AN3278.3 and A. clavatus 3847.
Podospora anserina has the terpene cyclase and CYP567E as seen
earlier, but it has an NRPS gene instead of a PKS. It may be related
by descent, but it has changed significantly.
In a second example the gene neighbourhood of CYP genes may
predict the function of a secondary metabolite cluster, such as a 20
gene cluster on chromosome VI from AN9210.3 to AN11201.3. This
region contains five CYP genes (cluster 8 in Fig. 2). The genes in the
cluster are characteristic for a metabolite cluster, i.e. a Zn(II)2Cys6
binuclear cluster transcription factor (AN9221.3), a PKS
(AN11198.3), an NRPS (AN9226.3), an acyl-CoA synthetase-like
gene often seen in conjunction with PKS pathways, an efflux pump(AN9219.3)
and an FAD binding monooxygenase (AN9224.3). The
gene that gives a clue to the function of this cluster is AN11201.3 a
dimethylallyltryptophan synthase-like gene. These enzymes catalyze
the first step in ergot alkaloid biosynthesis (Coyle and Panaccione,
2005) and this suggests this cluster may synthesize an
alkaloid product.
A final example here is taken from siderophore biosynthesis. A.
nidulans makes two siderophores, an external siderophore triacetylfusarinine
and an internal siderophore called ferricrocin

(Eisendle et al., 2003). Both pathways begin with the gene sidA (Lornithine-N5-monooxygenase).
This gene is not a P450. Both internal
and external siderophores are constructed by two different
NRPS genes sidC and sidD respectively. The gene AN0607.3 is an
NRPS gene orthologous to sidC (Eisendle et al., 2003). Adjacent to
this gene is CYP5076A1 (AN0606.3). It is known that the internal
siderophore ferricrocin is hydroxylated in A. fumigatus. The P450
gene CYP5076A1 is a prime candidate to perform that hydroxylation
in A. nidulans and thus based on its location, it is predicted
to be a ferricrocin hydroxylase. The ferricrocin hydroxylase in A.
fumigatus has not been identified and there is no P450 next to
the sidC gene in A. fumigatus. The related P450 CYP5076C2 in A.
fumigatus is adjacent to a different NRPS gene called ftmA
(Afu8g00170) that is the brevianamide synthetase gene (Maiya
et al., 2006). This gene cluster has a dimethylallyltryptophan synthase-like
gene as mentioned earlier, and it is involved in making
brevianamide F, a precursor to several known alkaloids. A. fumigatus
has another CYP that could be a candidate for the ferricrocin
hydroxylase function: CYP5076D1 (AFUA_4G09470).
We have examined some of the 20 secondary metabolite clusters
predicted for A. nidulans that include at least one P450 gene.
There are additional clusters that do not involve P450s. Perrin
et al., 2007 have systematically searched for these clusters in A.
fumigatus and found 22, though only nine contained P450 genes.
If each filamentous fungal genome contains 20 or more secondary
metabolite clusters, there will soon be hundreds of these gene clusters
to compare. The CYPs seem to be a highly variable part of these
clusters. With some comparative data as gleaned from Fig. 4 it may
be possible to create new metabolites by either deleting P450s or
by adding P450s from homologous clusters.
2.1. Redox partners
For functional activity most CYPs require the input of two successive
electrons from NADPH via a NADPH cytochrome P450
reductase and/or cytochrome b5 reducatase and cytochrome b5
(Lamb et al., 1999; de Vetten et al., 1999). Mining the genome sequence
of A. nidulans further we have identified 8 putative NADPH
cytochrome P450 reductase sequences (Fig. 2). AN0595.3 on chromosome
VIII has 91% sequence ID to cprA from A. niger and 88% ID
to cprA from A. fumigatus and represents the most probable candidate
reductase for the majority of the CYPs. However, a second
reductase with 51% sequence ID to cprA (A.fumigatus) AN5838.3
is present on chromosome I and interestingly adjacent to CYP630B2
(AN5837.3). We postulate that this may be specific for this CYP
whose function is yet to be elucidated. AN8920.3 present on chromosome
VII is immediately downstream of CYP540A2, but has the
additional feature of a siderophore-interacting FAD binding domain,
that may provide a clue to function. In the same way,
AN3862.3 is immediately downstream of CYP540B4 (chromosome
II) and might also be specific for this CYP. Lah et al., 2008 have further
identified members of this protein family with similar domain
architecture, which include a novel reductase 1 (NR1), AN10283.3
and a methionine synthase reductase (MSR), AN2042.3, one gene
away fron CYP552A2. AN11198.3 is a putative cytochrome b5
reductase and neighbour of CYP540D1 AN9218.3 in the middle of
cluster 8 (chromosome VI) with 5 CYPs and which appears to be
specific for A. nidulans. Finally, AN6835.3 is annotated as CYP505A8
on chromosome I and is a member of clan CYP505, a group of CYPs
that are fused to their reductase partner and found previously in
Fusarium oxysporum (CYP505A1, P450foxy) (Nakayama et al.,1996;
Kitazume et al., 2000) and Bacillus megaterium (CYP102, BM3, Narhi
and Fulco. 1986) are responsible for fatty acid hydroxylation.
Fusions between CYP genes and a reductase partner are rare,
with most of the known examples being bacterial. CYP505 is the
only eukaryotic family with an NADPH cytochrome P450 reductase
D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61 S59
fusion. It is similar to CYP102 bacterial fusions and probably represents
a lateral gene transfer with later diversification in eukaryotes.
The eukaryotic CYP505 clan has two families CYP505 and
CYP541. CYP541 has no fusion to a reductase partner, so it may
be an example of a fusion protein being broken. The next closest
family to CYP505 and CYP541 is CYP540. Three of the CYP540
genes were mentioned above as having reductase partners adjacent
to them. It seems probable that all three of these CYP families
descended from a common ancestor that had a fusion structure.
CYP505 has four subfamilies. In Phanerochaete chrysosporium
there are five CYP505D sequences in a 60 Kb window on scaffold
3 from 150,000 to 210,000. This tandem arrangement of very similar
genes argues against a pathway and in favour of a catabolic
function with slightly different or overlapping substrate preferences
for each. Two other CYP505D genes are elsewhere in the
white rot genome. Since white rot is known for its metabolic degradation
abilities as a consumer, these seven CYP505D genes probably
contribute to that mechanism. In other species the CYP505
genes can be biosynthetic rather than degradative. CYP505B1 is
FUM6 in the fumonisin biosynthetic pathway of Fusarium verticillioides
(Seo et al., 2001). FUM2 (CYP65AH1) is the fumonisin C10hydroxylase
in the same pathway (Proctor et al., 2006). There are
14 named CYP505A genes in filamentous fungi. Some of them
may help utilize fatty acids as a carbon source since CYP505A1, or
P450foxy, is a known fatty acid hydroxylase like CYP102A1 in B.
megaterium (Kitazume et al., 2008). Others may function in gene
clusters to make a toxin like fumonisin as seen in some Fusarium
species. Neighbourhoods near the gene should be examined for
evidence of gene cluster characteristics analogous to the polyketide
synthases approach used here and for fungal specific transcription
factor(s).
3. Subcellular localisation of A. nidulans CYPs
Eukaryotic cytochromes P450 are usually found bound to membranes,
in most instances anchored on the cytoplasmic surface of
the endoplasmic reticulum (ER) through a short N-terminal hydrophobic
sequence (Nelson and Strobel, 1988), while some mammalian
CYPs are known to be located on the matrix side of the
mitochondrial inner membrane. The latter include those involved
in sterol, steroid and bile acid biosynthesis. We have undertaken
a bioinformatics analysis of the amino acid sequences of all CYPs
from A. nidulans in order to define possible ER signal peptides,
and the presence, abundance and relationships between transmembrane
regions and re-entrant loops.
Using TargetP (Emanuelsson et al., 2000), cross-referenced with
the two different modes (the neural network and HMM versions)
of SignalP (Bendtsen et al., 2004), 75 of the proteins were predicted
to possess N-terminal signal peptides. Typical microsomal CYPs
possess a stretch of about 20 hydrophobic residues serving as a
membrane anchor in the endoplasmic reticulum and current models
still represent the triangular fold common to the CYPs lying flat
on the membrane surface with the haem parallel or inclined to the
membrane (Nelson and Strobel, 1988; Ohta et al., 1992). In two
mammalian CYPs (CYP2C5 and CYP2B4, Williams et al., 2000) the
presence of F’ and/or G’ helices form a hydrophobic surface near
the N-terminus of the protein and it is thought that this portion
of the CYP may lie in close proximity to the membrane and possibly
be partially inserted (De-Lemos-Chiarandini et al., 1987).
Transmembrane region topology prediction, using the TMHMM
program (Sonnhammer et al., 1998; Krogh et al., 2001), and subsequently
corrected for coincident signal peptides predicted using
Signal P (Bendtsen et al., 2004) and TargetP (Emanuelsson et al.,
2000), suggests that 83 of the CYPs do not possess any transmembrane
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 possess
re-entrant loops, suggesting at least some anchoring to the
membrane in regions other than the N-terminus signal peptide. Finally,
34 of the proteins were not predicted to possess N-terminal
signal peptides. Of these, one protein (CYP61A1) possesses a C-terminal
XDEL motif and 4 proteins were found to contain the C-terminal
ER retention KKXX motifs (searched for with a cut-off of the
last 20 residues), CYP504B1, CYP631B1, CYP5080D1 and CYP552E1.
Of the four, only CYP504B1 does not also possess an N-terminal signal
peptide, giving a total of 72 proteins targeted to the ER.
4. Conclusion
Filamentous fungi have 41 (Neurospora) to 152 (Aspergillus oryzae)
P450s. Only two have established roles in housekeeping functions
in sterol biosynthesis CYP51 and CYP61. The others are
enzymes specific to the purpose(s) essential or useful to the fungus.
Often this involves pathogenesis and the production of toxins
and virulence factors, or the utilization of specific carbon sources.
Many studies have been done on deciphering the gene clusters that
make these toxins such as sterigmatocystin, aflatoxin and fumonisin.
Frequently these clusters contain CYP genes. Here we have
presented a detailed account of the A. nidulans CYPs and putative
redox partners and we have identified those that are in probable
gene clusters based on the nature of the neighbouring genes. Thirteen
CYP gene clusters with between 2–5 CYPs are documented in
Fig. 1. Fifteen more CYP clusters were found with one CYP eight
genes or less from a PKS or NRPS gene (Tables S1 and S2). Eleven
more clusters without any CYP genes were also found bringing
the total to 39 potential gene clusters. Comparative genomic analysis
for one of these clusters, that of terpene cyclase (Fig. 2) was
done and illustrates linkage of genotype to function at the protein
level. The toxins that given species make are often known and a
bioinformatics comparison of the toxin lists to the clusters could
provide a candidate or at least a short list of clusters to test for production
of a given toxin. The occurrence of these gene clusters
greatly simplifies the task of linking genotype to phenotype since
the numbers are reduced from hundreds of genes to dozens of clusters,
many of which are already characterized. The remaining
uncharacterized clusters represent obvious targets for future work.
Acknowledgments
We thank Cornell, M. at E-fungi data base (http://www.efungi.org.uk/index.html)
for useful discussions and the European Union
(Eurofung) is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.fgb.2008.08.010.
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