International Journal of Molecular Sciences

Article Blooming of Unusual Cytochrome P450s by Tandem Duplication in the Pathogenic coronatus

Mathula Lancelot Ngwenya 1, Wanping Chen 2, Albert Kotze Basson 1, Jabulani Siyabonga Shandu 1, Jae-Hyuk Yu 3 ID , David R. Nelson 4,* and Khajamohiddin Syed 1,* ID

1 Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, KwaDlangezwa 3886, South Africa; [email protected] (M.L.N.); [email protected] (A.K.B.); [email protected] (J.S.S.) 2 College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; [email protected] 3 Department of Bacteriology, University of Wisconsin-Madison, 3155 MSB, 1550 Linden Drive, Madison, WI 53706, USA; [email protected] 4 Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA * Correspondence: [email protected] (D.R.N.); [email protected] (K.S.)

 Received: 14 March 2018; Accepted: 14 May 2018; Published: 9 June 2018 

Abstract: While the Zygomycete fungus Conidiobolus coronatus primarily infects insects, it can be pathogenic to mammals as well, including humans. High variability in the treatment of this fungal infection with currently available drugs, including azole drugs is a very common phenomenon. Azoles bind to the cytochrome P450 monooxygenases (P450s/CYP) including CYP51, a sterol 14-α-demethylase, inhibiting the synthesis of cell membrane ergosterol and thus leading to the elimination of infecting fungi. Despite P450’s role as a drug target, to date, no information on C. coronatus P450s has been reported. Genome-wide data mining has revealed the presence of 142 P450s grouped into 12 families and 21 subfamilies in C. coronatus. Except for CYP51, the remaining 11 P450 families are new (CYP5854-CYP5864). Despite having a large number of P450s among entomopathogenic fungi, C. coronatus has the lowest number of P450 families, which suggests blooming P450s. Further analysis has revealed that 79% of the same family P450s is tandemly positioned, suggesting that P450 tandem duplication led to the blooming of P450s. The results of this study; i.e., unravelling the C. coronatus P450 content, will certainly help in designing experiments to understand P450s’ role in C. coronatus physiology, including a highly variable response to azole drugs with respect to P450s.

Keywords: cytochrome P450 monooxygenase; Conidiobolus coronatus; drug resistance; entomopathogens; P450 blooms; rare and neglected diseases; rhinoentomophthoramycosis; tandem duplications

1. Introduction Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins that are ubiquitously distributed in organisms belonging to different biological kingdoms [1]. P450s are well known for their stereo- and regio-specific enzymatic reactions [2] and thus play a key role in organisms’ physiology, both in primary and secondary metabolism [3]. Because of their important catalytic activities, P450s’ role in drug metabolism and drug discovery, in the generation of commercial products including antibiotics, in bioremediation, and in the production of biofuels has been explored [3].

Int. J. Mol. Sci. 2018, 19, 1711; doi:10.3390/ijms19061711 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 1711 2 of 14

Analysis of fungal species genomes has revealed the presence of a large number of P450s, with few exceptions. It is now well known that the number of P450s and diversity of P450s in a fungal species is largely affected by the fungal species’ lifestyle or their ecological niche [4–7]. This indicates that P450s play a key role in fungal species’ physiology in terms of their primary and secondary metabolism. Fungal P450s’ role in fungal species’ physiology and their biotechnological application has been thoroughly reviewed [8–11]. Among all fungal P450 applications, the use of P450s as drug targets against fungal pathogens is well explored [12,13] because of their binding affinity to azole drugs, which are widely used for treating fungal infections [14]. Most of the azole drugs bind and inhibit CYP51, a sterol 14α-demethylase, thus stopping the synthesis of cell membrane ergosterol, which is an indispensable component of fungal membranes [12]. Ergosterol depletion affects not only the structure of the membrane, but also several of its functions, including its hormone-like role that stimulates the growth and proliferation of fungal cells that eventually lead to the death of fungi [14–16]. Fungal pathogens were found to develop drug resistance [17] to anti-fungal drugs such as azole drugs and other drugs belonging to the classes of allylamine, echinocandin, nucleoside analog, and polyene [18]. Understanding the fungal pathogens’ physiology at the molecular level will help to unravel novel drug targets and develop novel drugs. Among fungal pathogens, Conidiobolus coronatus, an entomopathogen (pathogen of insects) developed the ability to infect animals (horses, sheep, and dogs), including humans [19]. C. coronatus lives in soil and decaying matter, especially dead leaves [20], in addition to the feces of amphibians, sheep, insects, and horses [19]. C. coronatus has a worldwide distribution, especially in the tropical rain forests of Africa. It has been found in the United Kingdom [21], on the eastern coast of the United States [22], in India [23,24], the western region of Africa, and recently in South Africa [25]. C. coronatus causes , an in humans, and is considered a rare and neglected disease [19]. The definitive route of infection has not been established, but it is believed that C. coronatus enters the human body via inhalation of spores or from minor trauma [26]. Conidiobolomycosis is a disease of the nasal submucosa and paranasal sinuses, which slowly spreads to the nasal skin, glabella, cheek, upper lip, and pharynx. Rarely, contiguous lymph nodes may be involved [24,27–30]. Infection of the nasal and paranasal mucocutaneous tissue is known as rhinoentomophthoramycosis. Recently, C. coronatus infection in the vagina has been reported [22], indicating that this fungus is capable of infecting other sites of the human body as well. C. coronatus is capable of producing mycotoxins that are known to be very toxic to insects [31,32]. This parasitic fungus will attack normal or stressed insects, gaining access by penetrating the host’s inner skin, and the host will eventually die because of tissue demolition, nutrient depreciation, and the production of toxins [33]. This entomopathogenic fungus has the potential of becoming a biological insect control, owing to its ability to infect and kill a varied range of insects [34]. In order to understand C. coronatus’s physiology at the molecular level, genome sequencing of this fungus has been carried out [35]. However, the genome sequencing study was focused on analyzing the evolution of cell wall digestion enzymes and no information on C. coronatus P450s has been presented [35]. Interestingly, conidiobolomycosis treatment employing amphotericin B, azoles and/or potassium iodide was found to be highly variable [19]. In a case study, a patient infected with C. coronatus showed no response when treated with amphotericin B; azoles (ketoconazole, fluconazole and itraconazole) and potassium iodide individually, and a combination of azole drug therapies were needed to treat the patient [36]. Sometimes treatment includes both the surgical removal of infected tissue and the use of different drugs [37]. High variability in susceptibility to azole drugs by C. coronatus was widely observed [19], including C. coronatus species that are resistant to azole drugs [38]. Since P450s were found to play a key role in organisms’ physiology, including serving as azole drug targets [12,14–17], it is necessary to unravel the P450 content of this fungus to be able to design experiments to understand P450s role in C. coronatus physiology and the variability in susceptibility of this fungus to azole drugs with respect to P450s. Int. J. Mol. Sci. 2018, 19, x 3 of 14

Int. J. Mol.experiments Sci. 2018, 19 to, 1711understand P450s role in C. coronatus physiology and the variability in susceptibility3 of 14 of this fungus to azole drugs with respect to P450s.

2. Results2. Results and and Discussion Discussion

2.1. C.2.1. coronatus C. coronatus Has Has the Highestthe Highest Number Numbe ofr of P450s P450samong among Entomopathogenic Fungi Fungi GenomeGenome data data mining, mining identification,, identificati andon, annotationand annotation of P450s of P450s in C. in coronatus C. coronatusrevealed revealed the presencethe of 142presence P450s in of its 142 genome P450s (Figurein its genome1 and Table (Figure1). 1C. and coronatus Table 1).was C. foundcoronatus to havewas found the highest to have number the of highest number of P450s compared to other entomopathogenic and animal (including human) P450s compared to other entomopathogenic and animal (including human) pathogenic fungi (Table2). pathogenic fungi (Table 2).

Figure 1. Phylogenetic analysis of C. coronatus P450s. P450 families bloomed in C. coronatus are Figurehighlighted 1. Phylogenetic in different analysis colors. Bootstrap of C. coronatus replicationsP450s. used P450 to evaluate families each bloomed node of the in phylogeneticC. coronatus are highlightedtree are inpresented different in colors.Figure S1. Bootstrap replications used to evaluate each node of the phylogenetic tree are presented in Figure S1. Table 1. Annotation (family and subfamily assignment) of C. coronatus P450s. Table 1. C. coronatus Annotation (familyP450 and Subfamily subfamily assignment) of P450s. P450 Family No. of P450s in a Family Subfamily Name No. of P450s CYP51 F P450 Subfamily 1 1 P450 Family No. of P450s in a Family SubfamilyA Name No. of8 P450s B 4 CYP5854CYP51 C F 1 148 DA 1 8 EB 34 4 CYP5854 AC 5 1 48 CYP5855 * 36 BD 2 1 E 34

A 5 B 2 C 12 CYP5855 * 36 D 15 E 1 1 Int. J. Mol. Sci. 2018, 19, 1711 4 of 14

Table 1. Cont.

P450 Subfamily P450 Family No. of P450s in a Family Subfamily Name No. of P450s A 5 CYP5856 20 B 15 CYP5857 A 10 10 CYP5858 A 11 11 CYP5859 A 5 5 CYP5860 A 3 3 CYP5861 A 3 3 CYP5862 A 2 2 CYP5863 A 1 1 CYP5864 A 1 1 CYP-fragment # - 1 1 12 Families 21 subfamilies 142 142 * Belonging to the same family but not assigned to a subfamily because of short amino acid sequence; - not applicable; # not assigned to family because of short amino acid sequences (284 amino acids) and only showed 24% identity to named P450s.

Table 2. Comparative P450 analysis in entomopathogenic and animal (including human) pathogenic fungi.

No. of P450 Fungus Host P450 Count Reference Families Entomopathogenic fungus Arthropods (termites, thrips, Beauveria bassiana 83 49 [39] whiteflies, aphids, and beetles) Cordyceps militaris Butterflies and caterpillars 57 37 [40] Metarhizium acridum ≥200 insects 100 67 [41] Metarhizium anisopliae Locusts 123 87 [41] (formerly Metarhizium robertsii) Animal including human pathogen Humans and other animals (cats, dogs, rodents, squirrels, horses, 40 32 [7,42] and birds) Animals (including humans) and entomopathogenic fungus Insects, humans, and other animals Conidiobolus coronatus 142 12 This work (horses, sheep and dogs)

2.2. Large Number of New P450s Found in C. coronatus C. coronatus P450s can be grouped into 12 P450 families and 21 P450 subfamilies (Table1). Except for CYP51, the most conserved family in fungi [43], the remaining P450 families are new. It is quite interesting that C. coronatus, apart from CYP51, does not share a P450 family with other entomopathogenic fungi listed in Table2. Interestingly, C. coronatus lacks CYP61, the P450 involved in fungal membrane ergosterol synthesis [44], compared with pathogenic fungi listed in Table2. Among 142 P450s, one P450 (protein ID: 71373) is not assigned to a P450 family because of a short amino acid sequence and low sequence identity to other P450s. In addition, one P450 (protein ID: 2292) belonging to the CYP5854 family has not been assigned to a subfamily because of a short amino acid sequence and low identity to other subfamilies. P450s that are not full-length (<300 amino acids) were indicated with word “fragments” after their P450 subfamily (Figure1 and Tables S1 and S2). The new P450 families identified in C. coronatus include CYP5854-CYP5864 (Table1). Despite having a large number of P450s, C. coronatus has the lowest number of P450 families compared to entomopathogenic and animal (including human) pathogenic fungi (Table2), suggesting P450 blooms (a few families with many genes) in C. coronatus. Phylogenetic analysis revealed that CYP5859 P450s show a close relationship with CYP5855 P450s and seem to have evolved from CYP5855 P450s (Figure1). Int.Int. J. J. Mol. Mol. Sci. Sci.2018 2018, ,19 19,, 1711 x 55 of of 14 14

2.3. P450 Signature Motifs EXXR and CXG Are Conserved in C. coronatus P450s 2.3. P450 Signature Motifs EXXR and CXG Are Conserved in C. coronatus P450s Among different motifs that are characteristic of P450s, two motifs, namely EXXR in the K-helix Among different motifs that are characteristic of P450s, two motifs, namely EXXR in the K-helix and FXXGXRXCXG (also known as CXG) in the heme-binding domain, are found to be conserved in and FXXGXRXCXG (also known as CXG) in the heme-binding domain, are found to be conserved P450s, with few exceptions [45–48]. These two motifs are largely explored in the identification of in P450s, with few exceptions [45–48]. These two motifs are largely explored in the identification P450s across biological kingdoms [48]. Analysis of EXXR and CXG motifs in C. coronatus P450s of P450s across biological kingdoms [48]. Analysis of EXXR and CXG motifs in C. coronatus P450s revealed that all P450s have both signature motifs (Table S3). Only a few P450s that are named as revealedfragments that (as all mentioned P450s have in bothSection signature 2.2) did motifs not ha (Tableve one S3). of Onlythese amotifs few P450s (Table that S3). are Thus, named these as fragmentsP450s named (as mentioned as fragments in Sectionrepresent 2.2 )pseudo-P450s. did not have oneAnalysis of these revealed motifs that (Table C. S3).coronatus Thus, P450sthese P450shave namedhighly asconserved fragments amino represent acids, pseudo-P450s. such as glutamic Analysis acid (E) revealed and cysteine that C. (C), coronatus in the P450smotifshave EXXR highly and conservedCXG, with amino the exception acids, such of CYP5857A4, as glutamic which acid (E) has and cysteine instead (C), inof theglutamic motifs acid EXXR in the and EXXR CXG, withmotif the (Table exception S3). ofNon-conservation CYP5857A4, which of glutamic has glycine acid insteadin EXXR of motifs glutamic is acidnot new in the and EXXR has motifbeen (Tablereported S3). for Non-conservation other P450s as of well glutamic [48]. acidAnalysis in EXXR of amino motifs acid is not patterns new and for has the been P450 reported families, for otherCYP5854-CYP5856, P450s as well [48revealed]. Analysis family-specific of amino acid amino patterns acid forpatterns the P450 at the families, EXXR CYP5854-CYP5856, and CXG motifs revealed(Figure 2). family-specific CYP5854 and aminoCYP5855 acid have patterns the same at E-S-M-R the EXXR amino and acid CXG pattern, motifs whereas (Figure2 CYP5856). CYP5854 has andan E-T/V-M-R CYP5855 have amino the acid same pattern E-S-M-R in the amino EXXR acid motif pattern, (Figure whereas 2). Comparativ CYP5856e analysis has an E-T/V-M-Rwith P450s aminoacross acid biological pattern kingdoms in the EXXR [48,49] motif revealed (Figure that2). Comparative the CYP5854 analysisand CYP5855 with P450sfamilies’ across EXXR biological amino kingdomsacid pattern [48, 49matched] revealed that that of thethe CYP5854 CYP94 family, and CYP5855 whereas families’ the CYP5856 EXXR amino family acid EXXR pattern amino matched acid thatpatterns of the partially CYP94 family, matched whereas those theof CYP5856the CYP53, family CYP92, EXXR and amino CYP176 acid P450 patterns families, partially indicating matched a thosepossible of the evolutionary CYP53, CYP92, relation and between CYP176 the P450 families. families, Contrary indicating to the a possibleEXXR motifs, evolutionary the amino relation acid betweenpatterns the at the families. CXG motifs Contrary of the to theCYP5854-CYP5856 EXXR motifs, the families amino did acid not patterns match at any the P450s CXG [48,49]. motifs ofThis the CYP5854-CYP5856strongly supports the families hypothesis did not that match each anyP450 P450s family [48 has,49 ].its This unique strongly signature supports amino the acid hypothesis patterns thatat the each EXXR P450 and family CXG has motifs its unique [48]. signature amino acid patterns at the EXXR and CXG motifs [48].

FigureFigure 2.2. Analysis of of amino acid acid patterns patterns at at th thee EXXR EXXR and and CXG CXG motifs motifs in in P450 P450 families families CYP5854-CYP5856.CYP5854-CYP5856. P450 protein sequencessequences used to deduce EXXR and CXG signature sequences sequences werewere presented presented in in Table Table S4. S4.

2.4.2.4. P450 P450 Blooming Blooming in in C.C. coronatuscoronatus ComparativeComparative analysisanalysis ofof P450sP450s revealedrevealed thatthat amongamong 11 P450 families, five five P450 P450 families families contributedcontributed 88%88% of P450s P450s to to the the total total number number of ofP450s P450s in C. in coronatusC. coronatus (Figure(Figure 2 and2 andTable Table 1). This1). Thisindicates indicates thatthat the thefive five P450 P450 families, families, namely namely CYP5854, CYP5854, CYP5855, CYP5855, CYP5856, CYP5856, CYP5858, CYP5858, and and CYP5857, CYP5857, areare highly highly bloomedbloomed in C. coronatus coronatus.. The The highest highest number number of ofmember member P450s P450s were were found found in CYP5854 in CYP5854 (48 (48P450s) P450s) followed followed by byCYP5855 CYP5855 (36 (36P450s), P450s), CYP5856 CYP5856 (20 (20P450s), P450s), CYP5858 CYP5858 (11 P450s), (11 P450s), and andCYP5857 CYP5857 (10 (10P450s) P450s) (Figure (Figure 3).3 ).Blooming Blooming of of certain certain P450 P450 families families in in an an organism organism indicates indicates that that these family

Int. J. Mol. Sci. 2018, 19, 1711 6 of 14 Int. J. Mol. Sci. 2018, 19, x 6 of 14 membersmembers playplay aa keykey rolerole inin anan organism’sorganism’s physiologyphysiology andand thusthus theythey areare expandedexpanded [[4,50].4,50]. InIn somesome Basidiomycetes,Basidiomycetes, P450P450 families families were were found found to beto expanded be expanded in order in toorder help to those help organisms those organisms to colonize to oncolonize wood on and wood wood-derived and wood-derived materials materials [4]. At present [4]. At the present reason the or needreason for or blooming need for ofblooming these five of P450these families five P450 in familiesC. coronatus in C.is coronatus not clear. is It not is noteworthy clear. It is noteworthy that none of that the P450none families of the P450 is expanded families inis otherexpanded entomopathogenic in other entomopathogen fungi listedic in fungi Table listed2. in Table 2.

FigureFigure 3.3. ComparativeComparative analysisanalysis ofof P450P450 familyfamily membersmembers inin C.C. coronatuscoronatus.. TheThe P450P450 familyfamily namename andand percentagepercentage inin thethe totaltotal numbernumber ofof P450sP450s areare shownshown inin thethe figure.figure. TheThe totaltotal numbernumber ofof P450sP450s inin aa P450P450 familyfamily isis presentedpresented inin TableTable1 .1.

2.5.2.5. ExtensiveExtensive TandemTandem DuplicationsDuplications LedLed toto P450P450 BloomingBlooming inin C.C. coronatuscoronatus P450P450 bloomingblooming isis onlyonly possible possible when when certain certain P450s P450s belong belong to to the the same same P450 P450 family family duplicated duplicated in largein large numbers numbers in an in organisman organism [50]. [50]. The The duplicated duplicated P450s P450s (paralogs) (paralogs) can be can identified be identified by observing by observing their locationtheir location in the genome,in the genome, intron-exon intron-exon organization, organizati and percentageon, and percentage identity at identity protein at level, proteinas described level, as elsewheredescribed [elsewhere4,6,13]. Tandem [4,6,13]. arrangement Tandem arrangement of P450s (P450s of P450s located (P450s one behindlocated theone other) behind belonging the other) to thebelonging same family to the is same a good family indication is a good of P450 indication duplications. of P450 Analysis duplications. of the C. Analysis coronatus ofgenome the C. coronatus revealed thatgenome 79% revealed of P450s (112that P450s)79% of belonging P450s (112 to P450s) the same belonging P450 family to the are same tandemly P450 locatedfamily (Figureare tandemly4 and Tablelocated S5). (Figure 4 and Table S5). AsAs shownshown inin FigureFigure4 4,, the the five five P450 P450 families families bloomed bloomed in in C.C. coronatuscoronatus werewere foundfound toto havehave thethe highesthighest numbernumber of of tandemly tandemly duplicated duplicated P450s. P450s. This This indicates indicates that that P450 P450 tandem tandem duplications duplications led toled the to bloomingthe blooming of these of these five P450five familiesP450 families in C. coronatusin C. coronatus. The CYP5855. The CYP5855 family family was found was to found have to the have highest the numberhighest number of tandemly of tandemly duplicated duplicated P450s (33 P450s P450s), (33 P450s), followed followed by the CYP5844by the CYP5844 family withfamily 32 with P450s, 32 theP450s, CYP5856 the CYP5856family with family 17 P450s,with 17the P450s, CYP5858 the familyCYP5858 with family 11 P450s, with and 11 theP450s, CYP5857 and the family CYP5857 with 10family P450s. with The 10 number P450s. of The tandemly number duplicated of tandemly P450s duplicated in the respective P450s familiesin the respective was shown families in Figure was4. Inshown addition in Figure to the 4. five In P450addition families to the bloomed five P450 in familiesC. coronatus bloomed, three in P450 C. coronatus families, were three found P450 families to have P450swere found that are to tandemlyhave P450s duplicated, that are tandemly namely CYP5861 duplicated, (2 P450s), namely CYP5859 CYP5861 (5 (2 P450s) P450s), and CYP5859 CYP5860 (5 (2P450s) P450s) and (Figure CYP58604 and (2 Table P450s) S5). (Figure It is interesting4 and Table to S5). note It thatis interesting P450s that to are note tandemly that P450s located that onare differenttandemly scaffolds located (portion on different of the genomescaffolds sequence (portion reconstructed of the genome from end-sequencedsequence reconstructed whole-genome from shotgunend-sequenced clones) whole-genome belong to the same shotgun subfamily clones) (Table belong S5). to This the stronglysame subfamily indicates that(Table these S5). P450s This indeedstrongly evolved indicates by that tandem these duplications. P450s indeed evolved by tandem duplications.

Int. J. Mol. Sci. 2018, 19, 1711 7 of 14 Int.Int. J. Mol. J. Mol. Sci. Sci. 2018 2018, 19, 19, x, x 7 of7 14 of 14

FigureFigure 4. Analysis 4. Analysis of tandem of tandem duplications duplications of of P450s P450s inin C. coronatus . The. The numbers numbers next next to bars to bars indicate indicate Figurethe number 4. Analysis of P450s of tandem located duplicationson the scaffold. of P450s in C. coronatus. The numbers next to bars indicate the number of P450s located on the scaffold. the number of P450s located on the scaffold. 2.6. C. coronatus P450s Have Same Gene Structure 2.6. C. coronatus P450s Have Same Gene Structure 2.6. C. coronatusAnother characteristicP450s Have Same of tandemly Gene Structure duplicated genes is that they have the same gene structure; Anotheri.e.,Another the same characteristic characteristic number and of sizeof tandemly tandemly of introns duplicated duplicated and exons. genesFungalgenes is P450s that that withthey they thehave have same the the genesame same structure gene gene structure; structure;have i.e.,i.e., thepreviously the same same number numberbeen reported and and sizesize and of of theintrons introns authors and and suggestedexons. exons. Fungal Fungalthat P450sthis P450sis with a typical withthe same thecharacteristic samegene structure gene of structuregene have havepreviouslyduplications previously been been[4,6,13]. reported reported In this and study, and the the geneauthors authors structure suggested suggested analysis that was that this carried this is isa aouttypical typical for P450characteristic characteristic families such of of geneas gene duplicationsduplicationsCYP5854, [4 CYP5857, ,[4,6,13].6,13]. In Inand this this CYP5860 study, study, gene (Figuresgene structure structure 5 and 6), analysisanalysis since most was of carried the members out out for for inP450 P450 these families families families such are such as as full-length P450s. CYP5854,CYP5854, CYP5857, CYP5857, and and CYP5860 CYP5860 (Figures (Figures5 5and and6 ),6), since since most most of of the the membersmembers in these families are are full-lengthfull-length P450s. P450s.

Figure 5. Gene-structure analysis of P450s in the CYP5854 family. Each P450 gene structure is presented with the P450 name, exons (red color bars), and introns (gap between bars). The size of the

exons (number at the top of the red bars) and introns (number in the gaps) is also shown in the FigureFigurefigure. 5. Gene-structure5. SomeGene-structure P450s’ analysisevolution analysis of was P450s of deduced P450s in the in from CYP5854 the otCYP5854her family. P450s family. Eachand their P450 Each origin gene P450 structureis geneindicated structure is presentedwith is withpresented thedotted P450 lines. with name, Blue the P450 exonsregions name, (red indicate exons color an (redbars), untranslated color and bars), introns DNA and region. (gap introns between The (gap location between bars). of the Thebars). respective size The of size theP450s of exons the (numberexonsis indicated (number at the topwith at of theirthe the top scaffold red of bars)the number. red and bars) introns and intr (numberons (number in the in gaps) the isgaps) also is shown also shown in the in figure. the figure. Some P450s’ evolution was deduced from other P450s and their origin is indicated with Some P450s’ evolution was deduced from other P450s and their origin is indicated with dotted lines. Bluedotted regions lines. indicate Blue regions an untranslated indicate an DNAuntranslated region. DNA Thelocation region. The of the location respective of the P450srespective is indicated P450s is indicated with their scaffold number. with their scaffold number.

Int. J. Mol. Sci. 2018, 19, 1711 8 of 14 Int. J. Mol. Sci. 2018, 19, x 8 of 14

Figure 6. Gene structure analysis of P450s in CYP CYP58575857 and CYP5860 family. Each P450 gene structure is presentedpresented withwith thethe P450P450 name,name, exons (red colorcolor bars)bars) andand intronsintrons (gap(gap betweenbetween bars).bars). The sizesize ofof exons (number at the top of the re redd bars) and introns (number in th thee gaps) is shown in in the figure. figure. Some P450s’ evolution was deduced from other P450s and their origin is indicated with dotted lines. lines. The location ofof thethe respectiverespective P450sP450s isis indicatedindicated withwith theirtheir scaffoldscaffold number.number.

As shownshown in in Figure Figure5, the5, sizethe ofsize the of exons the inexons CYP5854 in CYP5854 P450s is conserved.P450s is conserved. The number The of intronsnumber and of exonsintrons in and some exons P450s in is some also highly P450s conserved. is also highly P450s cons thaterved. are short P450s contain that theare sameshort size contain exons the as full-lengthsame size P450s,exons as indicating full-length their P450s, origin indicati from full-lengthng their origin P450s. from This wasfull-length clearly observedP450s. This for was some clearly CYP5854 observed P450s suchfor some as CYP5854-fragment2, CYP5854 P450s which such originated as CYP5854-fragment2, from CYP5854A2, CYP5854A2-fragment4,which originated from which CYP5854A2, originated fromCYP5854A2-fragment4, CYP5854-fragment3 andwhich CYP5854E1, originated which from originated CYP5854-fragment3 from CYP5854E2 and (Figure CYP5854E1,5). which originatedThe phenomenon from CYP5854E2 of the (Figure same 5). gene structure was also observed in CYP5857 and CYP5860 P450sThe (Figure phenomenon6). The originof the same of P450s gene from structure other was P450s also in observed the CYP5857 in CYP5857 family and was CYP5860 observed P450s as well,(Figurewhere 6). The CYP5857A5 origin of P450sP450s seemfrom toother have P450s originated in the fromCYP5857 CYP5857A1 family was (Figure observed6). The as two well, members where ofCYP5857A5 the CYP5860 P450s family seem have to thehave same originated gene structure from CYP5857A1 (Figure6). (Figure 6). The two members of the CYP5860 family have the same gene structure (Figure 6). 2.7. C. coronatus Has the Lowest P450 Diversity among Entomopathogenic Fungi 2.7. C. coronatus Has the Lowest P450 Diversity among Entomopathogenic Fungi Another characteristic of tandem duplication of P450s is that organisms have the lowest P450 diversityAnother percentage characteristic [7,49,51 of]. tandem The P450 duplication diversity of percentage P450s is that is a organisms good indication have the of P450lowest family P450 blooming.diversity percentage The lowest [7,49,51]. P450 diversity The P450 means diversity that certain percentage P450 familiesis a good are indication bloomed inof anP450 organism. family Analysisblooming. of The the lowest P450 diversityP450 diversity percentage means revealedthat certain that P450C. coronatusfamilies arehas bloomed only 8% in P450 an organism. diversity, despiteAnalysis having of the a P450 large diversity number of percentage P450s in its revealed genome that (Figure C. 7coronatus). has only 8% P450 diversity, despiteComparative having a large analysis number of theof P450s P450 in diversity its genome percentage (Figure 7). with other entomopathogenic fungi revealedComparative that C. coronatus analysishas of the the lowest P450 diversity diversity (Figure percentage7). This with further other indicates entomopathogenic that extensive fungi P450 duplicationrevealed that led C. to coronatus blooming has of certainthe lowest P450 diversity families and(Figure thus 7). the This lowest further P450 indicates diversity that percentage. extensive P450 duplication led to blooming of certain P450 families and thus the lowest P450 diversity percentage.

Int. J. Mol. Sci. 2018, 19, 1711 9 of 14 Int. J. Mol. Sci. 2018, 19, x 9 of 14

Figure 7. ComparativeComparative analysis analysis of ofP450 P450 diversity diversity perc percentageentage among among entomopathogenic entomopathogenic fungi. fungi. The Thecorresponding corresponding values values are presented are presented in Table in Table S6. S6.

2.8. Functional Prediction of C. coronatus P450s Except forfor CYP51CYP51 P450, P450, all all P450s P450s in C.in coronatusC. coronatusare new,are new, implying implying that functionalthat functional analysis analysis based onbased characterized on characterized homologs homologs is not possible. is not However,possible. However, as indicated as earlier, indicated CYP51 earlier, of C. coronatusCYP51 ofcan C. becoronatus predicted can to be be predicted involved into ergosterolbe involved biosynthesis in ergosterol based biosynthesis on the characterized based on homolog the characterized P450 [43]. Ithomolog is interesting P450 [43]. to see It is the interesting role of bloomed to see the P450s role in ofC. bloomed coronatus P450s, as these in C. familiescoronatus are, as uniquethese families to this fungusare unique compared to this tofungus other entomopathogeniccompared to other fungi.entomo Itpathogenic is interesting fungi. to note It is that interesting another P450, to note CYP61, that involvedanother P450, in fungal CYP61, membrane involved ergosterol in fungal synthesis membrane [44] is ergosterol not present synthesis in C. coronatus [44] is. not present in C. coronatus. 3. Materials and Methods 3. Materials and Methods 3.1. Genome Data Mining for P450s 3.1. Genome Data Mining for P450s The genome sequence of C. coronatus NRRL28638 v1.0 has been published [35] and is available at the JGIThe MycoCosm genome sequence database of (Available C. coronatus online: NRRL28638 https://genome.jgi.doe.gov/Conco1/Conco1.home. v1.0 has been published [35] and is available htmlat the), which JGI wasMycoCosm data-mined database for P450s. (Available The data miningonline: of P450shttps://genome.jgi.doe.gov/Conco1/ was carried out as described elsewhereConco1.home.html), [4,6,52], with which slight was modifications. data-mined for Briefly, P450s. the TheC. data coronatus mininggenome of P450s was was mined carried for out P450s as usingdescribed InterPro elsewhere code “IPR001128”.[4,6,52], with Theslight hit modifications. protein sequences Briefly, were the downloaded C. coronatus andgenome subjected was mined to the NCBIfor P450sBatch usingWeb CD-SearchInterPro code Tool [53“IPR001128”.]. Proteins that Th groupede hit protein under sequences the P450 superfamily were downloaded were selected and forsubjected further to analysis. the NCBI Batch Web CD-Search Tool [53]. Proteins that grouped under the P450 superfamily were selected for further analysis. 3.2. Annotation of P450s 3.2. AnnotationThe above of selected P450s P450 proteins were subjected to BLAST analysis against all named fungal P450sThe at theabove Cytochrome selected P450 P450 proteins Homepage were [54 ]subjecte to identifyd to aBLAST homolog analysis P450. Theagainst name all of named the homolog fungal P450P450s and at the percentage Cytochrome of identity P450 Homepage to the homolog [54] P450to identify were recorded a homolog (Table P450. S1). The Based name on of the the percentage homolog identityP450 and to percentage homologous of P450s, identity a family to the and homolog subfamily P450 were were assigned recorded to C. (Table coronatus S1).P450s. Based This on wasthe donepercentage by following identity the to rules homologous set by the P450s, International a family P450 and Nomenclature subfamily were Committee assigned [55 to–57 C.]; i.e.,coronatus >40% identityP450s. This for was a family done and by >55%following identity the rules for a set subfamily. by the InternationalC. coronatus P450sP450 Nomenclature that had <40% Committee and <55% identity[55–57]; i.e., to the >40% named identity fungal for P450s a family were and assigned >55% identity to new for families a subfamily. and new C. subfamilies,coronatus P450s respectively that had (Table<40% and S1). Percentage<55% identity identity to the among namedC. coronatusfungal P450sP450s were was alsoassigned used toto assignnew families P450 families and new and subfamilies.subfamilies, respectively Information (Table on homolog S1). Percentage P450s that identity are used among in the C. naming coronatus of P450sC. coronatus was alsoP450s used was to listedassign in P450 Table families S1 along and with subfamilies. the C. coronatus InformationP450s identifiedon homolog and P450s annotated that are in thisused study. in theC. naming coronatus of P450C. coronatus protein P450s sequences was listed along in with Table their S1 amino along acidwith length the C. arecoronatus presented P450s in identified Table S2. and annotated in this study. C. coronatus P450 protein sequences along with their amino acid length are presented in Table S2.

Int. J. Mol. Sci. 2018, 19, 1711 10 of 14

3.3. Phylogenetic Analysis of P450s The phylogenetic tree of C. coronatus P450s was constructed as described previously [51], with slight modifications. Briefly, the protein sequences were aligned by MUSCLE embedded in MEGA 7 [58]. Then, the best-fit substitution model for alignment was determined by the IQ-TREE web server (Available online: http://iqtree.cibiv.univie.ac.at/)[59]. Finally, the tree was constructed in MEGA7 by the maximum likelihood method, along with the best-fit substitution model and 100 bootstrap replications [60]. iTOL was used to view and highlight the tree [61].

3.4. Analysis of EXXR and CXG Motifs An analysis of EXXR and CXG motifs in C. coronatus P450s was carried out as described elsewhere [48]. Briefly, a multiple sequence alignment of P450s was carried out using Clustal Omega [62] and then a manual search was performed for the presence of EXXR and CXG motifs (Table S3). The WebLogo for EXXR and CXG motifs for the P450 families, CYP5854-CYP5856, was deduced as described elsewhere [48] following the WebLogo program [63]. WebLogos were deduced using the default parameter with the amino acid sequence type. The criteria for the selection of P450 families for EXXR and CXG amino acid pattern analysis are based on the number of P450s in a P450 family [49]. Three P450 families, CYP5854-CYP5856, have more than 15 P450s for analysis (Table S4) compared to other new P450 families, thus they qualify for EXXR and CXG motif analysis.

3.5. Identification of Tandemly Duplicated P450s Tandem P450 gene duplications were assessed as described elsewhere [4,6]. Briefly, the physical location of P450 genes, such as scaffold number, start and end point of the P450 gene on the DNA strand, was recorded by scanning the C. coronatus genome using the P450 protein ID (Table S5). P450s that are tandemly arranged and belong to the same family are regarded as duplicated P450s. The percentage P450 duplications are calculated as the percentage of P450s duplicated in the total number of P450s.

3.6. Gene Structure Analysis Gene structure analysis of P450s was carried out as described elsewhere [4,6,7,13]. Briefly, the C. coronatus genome was mined using P450 protein ID. P450 gene structure graphics downloaded from the C. coronatus genome database were aligned in such a way that exons with the same size would align together. The length of exons was noted as an indication of possible gene duplication, if P450s showed conservation in the size of exons. P450s whose gene structures contained the same sizes of exons or introns were presented in the figures.

3.7. P450 Diversity Percentage Analysis P450 diversity percentage analysis was carried out as described elsewhere [7,49,51]. Briefly, the P450 diversity percentage in C. coronatus was measured as a percentage contribution of the number of P450 families in the total number of P450s.

3.8. Comparative Analysis of P450s Entomopathogenic and animal (including human) pathogenic fungi P450s were retrieved from published articles [7,39–42] and used for comparative analysis with C. coronatus P450s.

4. Conclusions This study is the first of its kind on an in silico analysis of P450s in C. coronatus. Except for CYP51 P450, a conserved P450 in fungi and a primary target of azole drugs, all P450s were found to be new in C. coronatus. Unprecedented blooming of novel P450s in C. coronatus compared to other fungal species was observed. It would be interesting to observe the role of bloomed P450s in C. coronatus physiology, Int. J. Mol. Sci. 2018, 19, 1711 11 of 14 considering this fungus has a broad host range and is capable of infecting humans and other animals, in addition to insects.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/6/1711/ s1. Author Contributions: K.S. conceived and designed the experiments; M.L.N., W.C., A.K.B., J.S.S., J-H.Y., D.R.N., and K.S. performed the experiments, analysed the data and contributed analysis tools. M.L.N., W.C., A.K.B., J.S.S., J-H.Y., D.R.N., and K.S. were involved in writing the manuscript. All authors reviewed and approved the manuscript. Acknowledgments: Mathula Lancelot Ngwenya, Albert Kotze Basson, Jabulani Siyabonga Shandu and Khajamohiddin Syed express sincere gratitude to the University of Zululand Research Committee for funding. Khajamohiddin Syed is grateful to the National Research Foundation, South Africa for a research grant (Grant No: 114159). The authors also want to extend their thanks to Barbara Bradley, Pretoria, South Africa for English language editing. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; the collection, analysis, or interpretation of data, the writing of the manuscript or the decision to publish the results.

References

1. Nelson, D.R. Cytochrome P450 diversity in tree of life. Biochim. Biophys. Acta 2018, 1866, 141–154. [CrossRef] [PubMed] 2. Bernhardt, R. Cytochromes P450 as versatile biocatalysts. J. Biotechnol. 2006, 124, 128–145. [CrossRef] [PubMed] 3. Montellano, P.R.O. Cytochrome P450: Structure, Mechanism, and Biochemistry, 4th ed.; Springer: Basel, Switzerland, 2015; pp. 1–912. ISBN 978-3-319-12108-6. 4. Syed, K.; Shale, K.; Pagadala, N.S.; Tuszynski, J. Systematic identification and evolutionary analysis of catalytically versatile cytochrome P450 monooxygenase families enriched in model basidiomycete fungi. PLoS ONE 2014, 9, e86683. [CrossRef][PubMed] 5. Kgosiemang, I.K.R.; Mashele, S.S.; Syed, K. Comparative genomics and evolutionary analysis of cytochrome P450 monooxygenases in fungal subphylum Saccharomycotina. J. Pure Appl. Microbiol. 2014, 8, 291–302. 6. Qhanya, L.B.; Matowane, G.; Chen, W.; Sun, Y.; Letsimo, E.M.; Parvez, M.; Yu, J.H.; Mashele, S.S.; Syed, K. Genome-wide annotation and comparative analysis of cytochrome P450 monooxygenases in Basidiomycete biotrophic plant pathogens. PLoS ONE 2015, 10, e0142100. [CrossRef][PubMed] 7. Matowane, R.G.; Wieteska, L.; Bamal, H.D.; Kgosiemang, I.K.R.; Van Wyk, M.; Manume, N.A.; Abdalla, S.M.H.; Mashele, S.S.; Gront, D.; Syed, K. In silico analysis of cytochrome P450 monooxygenases in chronic granulomatous infectious fungus Sporothrix schenckii: Special focus on CYP51. Biochim. Biophys. Acta 2018, 1866, 166–177. [CrossRef] [PubMed] 8. Sakaki, T. Practical application of cytochrome P450. Biol. Pharm. Bull. 2012, 35, 844–849. [CrossRef][PubMed] 9. Girhard, M.; Bakkes, P.J.; Mahmoud, O.; Urlacher, V.B. P450 Biotechnology. In Cytochrome P450: Structure, Mechanism, and Biochemistry, 4th ed.; Montellano, P.R.O., Ed.; Springer: Basel, Switzerland, 2015; pp. 451–520. ISBN 978-3-319-12108-6. 10. Durairaj, P.; Hur, J.S.; Yun, H. Versatile biocatalysis of fungal cytochrome P450 monooxygenases. Microb. Cell Fact. 2016, 15, 125. [CrossRef][PubMed] 11. Shin, J.; Kim, J.E.; Lee, Y.W.; Son, H. Fungal cytochrome P450s and the P450 complement (CYPome) of graminearum. Toxins 2018, 10, 112. [CrossRef][PubMed] 12. Kelly, S.L.; Kelly, D.E. Microbial cytochromes P450: Biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us? Phil. Trans. R. Soc. B 2013, 368.[CrossRef] [PubMed] 13. Jawallapersand, P.; Mashele, S.S.; Kovaˇciˇc,L.; Stojan, J.; Komel, R.; Pakala, S.B.; Kraševec, N.; Syed, K. Cytochrome P450 monooxygenase CYP53 family in fungi: Comparative structural and evolutionary analysis and its role as a common alternative anti-fungal drug target. PLoS ONE 2014, 9, e107209. [CrossRef] [PubMed] 14. Sheehan, D.J.; Hitchcock, C.A.; Sibley, C.M. Current and emerging azole antifungal agents. Clin. Microbiol. Rev. 1999, 12, 40–79. [PubMed] Int. J. Mol. Sci. 2018, 19, 1711 12 of 14

15. Shapiro, R.S.; Robbins, N.; Cowen, L.E. Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol. Mol. Biol. Rev. 2011, 75, 213–267. [CrossRef][PubMed] 16. Hargrove, T.Y.; Wawrzak, Z.; Lamb, D.C.; Guengerich, F.P.; Lepesheva, G.I. Structure-fuctional characterization of cytochrome P450 sterol 14α-demethylase (CYP51B) from fumigatus and molecular basis for the development of antifungal drugs. J. Biol. Chem. 2015, 290, 23916–23934. [CrossRef] [PubMed] 17. Sanglard, D. Emerging threats in antifungal-resistant fungal pathogens. Front. Med. 2016, 3, 11. [CrossRef] [PubMed] 18. Ghannoum, M.A.; Rice, L.B. Antifungal agents: Mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin. Microbiol. Rev. 1999, 12, 501–517. [PubMed] 19. Shaikh, N.; Hussain, K.A.; Petraitiene, R.; Schuetz, A.N.; Walsh, T.J. Entomophthoramycosis: A neglected tropical . Clin. Microbiol. Infect. 2016, 22, 688–694. [CrossRef][PubMed] 20. Page, R.M.; Humber, R.A. Phototropism in Conidiobolus coronatus. Mycologia 1973, 65, 335–354. [CrossRef] [PubMed] 21. Smith, M.F.; Callaghan, A.A. Quantitative survey of Conidiobolus and Basidiobolus in soils and litter. Trans. Br. Mycol. Soc. 1987, 89, 179–185. [CrossRef] 22. Subramanian, C.; Sobel, J.D. A case of Conidiobolus coronatus in the vagina. Med. Mycol. 2011, 49, 427–429. [CrossRef][PubMed] 23. Chowdhary, A.; Randhawa, H.S.; Khan, Z.U.; Ahmad, S.; Khanna, G.; Gupta, R.; Chakravarti, A.; Roy, P. Rhinoentomophthoromycosis due to Conidiobolus coronatus. A case report and an overview of the disease in India. Med. Mycol. 2010, 48, 870–879. [CrossRef][PubMed] 24. Dutta, S.; Sarkar, S.; Linka, U.; Dora, S. Conidiobolomycosis: A case report of rare fungal infection from the eastern India. Indian Dermatol. Online J. 2015, 6, 393–395. [CrossRef][PubMed] 25. Hoogendijk, C.F.; Pretorius, E.; Marx, J.; van Heerden, W.E.P.; Imhof, A.; Schneemann, M. Detection of villous conidia of Conidiobolus coronatus in a blood sample by scanning electron microscopy investigation. Ultrastruct. Pathol. 2006, 30, 53–58. [CrossRef][PubMed] 26. Gugnani, H.C. A review of due to . Eur. J. Epidemiol. 1999, 15, 923–929. [CrossRef][PubMed] 27. Rippon, J.W. Medical Mycology: The Pathogenic Fungi and the Pathogenic Actinomycetes, 3rd ed.; W.B. Saunders Co.: Philadelphia, PA, USA, 1988; pp. 506–531. ISBN 0-7216-2444-8. 28. Kwon-Chung, K.J.; Bennett, J.E. Medical Mycology; Lea and Febiger: Philadelphia, PA, USA, 1992; p. 866, ISBN 0-8121-1463-9. 29. Ribes, J.A.; Vanover-Sams, C.L.; Baker, D.J. Zygomycetes in human disease. Clin. Microbiol. Rev. 2000, 13, 236–301. [CrossRef][PubMed] 30. Ellis, D.H. Systemic zygomycosis. In Topley and Wilson’ Microbiology and Microbial Infections. Medical Mycology, 10th ed.; Merz, W.G., Hay, R.J., Eds.; Edward Arnold: London, UK, 2005; pp. 659–686. 31. Paszkiewicz, M.; Tyma, M.; Lig˛eza-Zuber,˙ M.; Włóka, E.; Bogu´s,M.I.; Stepnowski, P. Mycotoxin production by entomopathogenic fungus Conidiobolus coronatus. Int. J. Environ. Agric. Res. 2017, 3, 33–40. 32. Paszkiewicz, M.; Tyma, M.; Lig˛eza-Zuber,˙ M.; Włóka, E.; Bogu´s,M.I.; Stepnowski, P. Trichothecenes production by entomopathogenic fungus Conidiobolus Coronatus. Adv. Toxicol. Toxic Eff. 2016, 1, 7–14. [CrossRef] 33. Bogus, M.I.; Maria, S. Histopathology of Conidiobolus coronatus () infection in Galleria mellonella (lepidoptera) larvae. Acta Parasitol. 2000, 45, 48–54. 34. Malinowski, H. Entomopathogenic fungi as insecticides in protecting the forest. Prog. Plant Prot. 2009, 49, 865–873. 35. Chang, Y.; Wang, S.; Sekimoto, S.; Aerts, A.L.; Choi, C.; Clum, A.; LaButti, K.M.; Lindquist, E.A.; Yee Ngan, C.; Ohm, R.A.; et al. Phylogenomic analyses indicate that early fungi evolved digesting cell walls of algal ancestors of land plants. Genome Biol. Evol. 2015, 7, 1590–1601. [CrossRef][PubMed] 36. Valle, A.C.; Wanke, B.; Lazéra, M.S.; Monteiro, P.C.; Viegas, M.L. Entomophthoramycosis by Conidiobolus coronatus. Report of a case successfully treated with the combination of itraconazole and fluconazole. Rev. Inst. Med. Trop. Sao Paulo 2001, 43, 233–236. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 1711 13 of 14

37. Choon, S.E.; Kang, J.; Neafie, R.C.; Ragsdale, B.; Klassen-Fischer, M.; Carlson, J.A. Conidiobolomycosis in a young Malaysian woman showing chronic localized fibrosing leukocytoclastic vasculitis: A case report and meta-analysis focusing on clinicopathologic and therapeutic correlations with outcome. Am. J. Dermatopathol. 2012, 34, 511–522. [CrossRef][PubMed] 38. Guarro, J.; Aguilar, C.; Pujol, I. In-vitro antifungal susceptibilities of Basidiobolus and Conidiobolus spp. strains. J. Antimicrob. Chemother. 1999, 44, 557–560. [CrossRef][PubMed] 39. Xiao, G.; Ying, S.H.; Zheng, P.; Wang, Z.L.; Zhang, S.; Xie, X.Q.; Shang, Y.; Leger, R.J.S.; Zhao, G.P.; Wang, C.; et al. Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana. Sci. Rep. 2012, 2, 483. [CrossRef][PubMed] 40. Zheng, P.; Xia, Y.; Xiao, G.; Xiong, C.; Hu, X.; Zhang, S.; Zheng, H.; Huang, Y.; Zhou, Y.; Wang, S.; et al. Genome sequence of the insect Cordyceps militaris, a valued traditional Chinese medicine. Genome Biol. 2011, 12, R116. [CrossRef][PubMed] 41. Gao, Q.; Jin, K.; Ying, S.H.; Zhang, Y.; Xiao, G.; Shang, Y.; Duan, Z.; Hu, X.; Xie, X.Q.; Zhou, G.; et al. Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet. 2011, 7, e1001264. [CrossRef][PubMed] 42. Cuomo, C.A.; Rodriguez-Del Valle, N.; Perez-Sanchez, L.; Abouelleil, A.; Goldberg, J.; Young, S.; Zeng, Q.; Birren, B.W. Genome sequence of the pathogenic fungus Sporothrix schenckii (ATCC 58251). Genome Announc. 2014, 2.[CrossRef][PubMed] 43. Lepesheva, G.I.; Waterman, M.R. Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms. Biochim. Biophys. Acta 2007, 1770, 467–477. [CrossRef][PubMed] 44. Kelly, S.L.; Lamb, D.C.; Baldwin, B.C.; Corran, A.J.; Kelly, D.E. Characterization of Saccharomyces cerevisiae CYP61, Sterol ∆22-desaturase, and inhibition by azole antifungal agents. J. Biol. Chem. 1997, 272, 9986–9988. [CrossRef][PubMed] 45. Gotoh, O. Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J. Biol. Chem. 1992, 267, 83–90. [PubMed] 46. Sirim, D.; Widmann, M.; Wagner, F.; Pleiss, J. Prediction and analysis of the modular structure of cytochrome P450 monooxygenases. BMC Struct. Biol. 2010, 10, 34. [CrossRef][PubMed] 47. Gricman, Ł.; Vogel, C.; Pleiss, J. Conservation analysis of class-specific positions in cytochrome P450 monooxygenases: Functional and structural relevance. Proteins 2014, 82, 491–504. [CrossRef][PubMed] 48. Syed, K.; Mashele, S.S. Comparative analysis of P450 signature motifs EXXR and CXG in the large and diverse kingdom of fungi: Identification of evolutionarily conserved amino acid patterns characteristic of P450 family. PLoS ONE 2014, 9, e95616. [CrossRef][PubMed] 49. Sello, M.M.; Jafta, N.; Nelson, D.R.; Chen, W.; Yu, J.H.; Parvez, M.; Kgosiemang, I.K.R.; Monyaki, R.; Raselemane, S.C.; Qhanya, L.B.; et al. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes. Sci. Rep. 2015, 5, 11572. [CrossRef][PubMed] 50. Feyereisen, R. Arthropod CYPomes illustrate the tempo and mode in P450 evolution. Biochim. Biophys. Acta 2011, 1814, 19–28. [CrossRef][PubMed] 51. Parvez, M.; Qhanya, L.B.; Mthakathi, N.T.; Kgosiemang, I.K.R.; Bamal, H.D.; Pagadala, N.S.; Xie, T.; Yang, H.; Chen, H.; Theron, C.W.; et al. Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s. Sci. Rep. 2016, 6, 33099. [CrossRef][PubMed] 52. Syed, K.; Shale, K.; Nazir, K.N.H.; Krasevec, N.; Mashele, S.S.; Pagadala, N.S. Genome-wide identification, annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila. Genes Genom. 2014, 36, 321–333. [CrossRef] 53. Marchler-Bauer, A.; Bo, Y.; Han, L.; He, J.; Lanczycki, C.J.; Lu, S.; Chitsaz, F.; Derbyshire, M.K.; Geer, R.C.; Gonzales, N.R.; et al. CDD/SPARCLE: Functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2016, 45, D200–D203. [CrossRef][PubMed] 54. Nelson, D.R. The cytochrome p450 homepage. Hum. Genom. 2009, 4, 59–65. 55. Nelson, D.R.; Kamataki, T.; Waxman, D.J.; Guengerich, F.P.; Estabrook, R.W.; Feyereisen, R.; Gonzalez, F.J.; Coon, M.J.; Gunsalus, I.C.; Gotoh, O.; et al. The P450 superfamily: Update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol. 1993, 12, 1–51. [CrossRef][PubMed] 56. Nelson, D.R. Cytochrome P450 nomenclature. Methods Mol. Biol. 1998, 107, 15–24. [PubMed] Int. J. Mol. Sci. 2018, 19, 1711 14 of 14

57. Nelson, D.R. Cytochrome P450 Nomenclature, 2004. Methods Mol. Biol. 2006, 320, 1–10. [CrossRef][PubMed] 58. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [CrossRef] [PubMed] 59. Trifinopoulos, J.; Nguyen, L.T.; von Haeseler, A.; Minh, B.Q. W-IQ-TREE: A fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 2016, 44, W232–W235. [CrossRef][PubMed] 60. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [CrossRef][PubMed] 61. Letunic, I.; Bork, P. Interactive tree of life (iTOL) v3: An online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016, 44, W242–W245. [CrossRef][PubMed] 62. Sievers, F.; Higgins, D.G. Clustal Omega, accurate alignment of very large numbers of sequences. Methods Mol. Biol. 2014, 1079, 105–116. [CrossRef][PubMed] 63. Crooks, G.E.; Hon, G.; Chandonia, J.M.; Brenner, S.E. WebLogo: A sequence logo generator. Genome Res. 2004, 14, 1188–1190. [CrossRef][PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).