(Cytochrome P450 Complement) of Aspergillus Nidulans
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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 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 1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2008.08.010 S54 D.E. Kelly et al. / Fungal Genetics and Biology 46 (2009) S53–S61 and apdB (AN8408.3, CYP685A1) involved in aspyridone biosynthesis. An alternative approach is to predict function based on se- quence 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.