Evolutionary Analysis and Structural Insights in Brain Type Cytochrome P450 /aromatase of Stinging Catfish, Heteropneustes Fossilis

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Evolutionary analysis and structural insights in Brain type Cytochrome P450 /Aromatase
of Stinging catfish, Heteropneustes fossilis
Article in Research journal of biotechnology · November 2015
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Research Journal of Biotechnology Vol. 10 (11) November (2015)
Res. J. Biotech Evolutionary analysis and structural insights in Brain type Cytochrome P450/Aromatase of Stinging catfish,
Heteropneustes fossilis Sundaray Jitendra Kumar, Rasal Kiran Dashrath*, Swain Pranati and Jayasankar Pallipuram Fish
Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Bhubaneswar 751 002, Odisha,
INDIA *kirancife@gmail.com; Kiran.Rasal@icar.gov.in
Abstract The brain type cytochrome p450 is the enzyme playing vital role in sexual
differentiation as well as secondary sexual character development. To date, most of the studies
on aromatase have focused on the expression pattern, promoter activity and function in sexual
differentiation. As the computational algorithms/tools are being developed, they have
revolutionized the way of study for in-depth analysis using existing nucleotide/protein sequence
data.
There is no evidence addressed for phylogenetic analysis for investigating conservation and 3D
structure of aromatase protein in the Stinging catfish. The stinging catfish, Heteropneustes
fossilis is one of the important species in aquaculture sector.
The evolutionary analysis was performed using Neighbor-joining and UPGMA methods using
CLC genomic 7.5.1 workbench tool followed by bootstrap test (100 replicates) performed to
validate the phylogenetic tree/cladogram. We have generated 3D structure of aromatase protein
of stinging catfish using homology modelling with help of template 4KQ8 (54 % identity) using
tool Modeller 9.1 which was further validated using SAVES server in Procheck and Verify3D.
The Ramachandran plot depicted that 89.4% residues lies in favored region followed by 9.7% in
additional as well as allowed region and 0.9 % residues found to be in outlier region.
In order to understand global network of aromatase protein, we have used STRING 9.1 tool and
speculated that this protein interacting with several other protein, strong association/interaction
with 2 proteins such as estradiol 17 beta dehydrogenase3, 3- beta steroid –dehydrogenase-1 with
0.988 and 0.979 confidence score respectively. In addition to this, we also investigated structural
and functional consequences of SNPs on structure of aromatase using algorithms like
PANTHER, PROVEAN and I- Mutant 2.0. We revealed impact of SNPs on the protein structure
and its function using sequence- based tools.
* Author for Correspondence
1
Keywords: Aromatase/Cytochrome P450, Homology Modelling, Non-synonymous Single Nucleotide
Polymorphism (nsSNP).
Introduction Despite the finding of Cytochrome P450/Aromatase about fifty years ago, there still remains
importance in the field of scientific research and increasing evidence for a role of aromatase in the
sexualization. The estrogen is important sex steroid playing major role in sex differentiation as well as
development of secondary sexual character and its biological synthesis is facilitated through enzyme
cytochrome P450 aromatase (CYP19) in almost all vertebrates
1-3
. This enzyme responsible for the conversion of androgens to estrogens is harnessed for
production of functional males in some teleost fishes
4
such as Chinook salmon, Oncorhynchus tshawytscha

5
; Nile tilapia, Oreochromis niloticus; rainbow trout,

Oncorhynchus mykiss
6
; Japanese flounder, Paralichthys olivaceus

7
and zebrafish, Danio rerio

8
. In case of mammals, it has been noted that this gene is having multiple promoters.
9-11
Interestingly, teleost fishes have two aromatase genes, one cyp19a1a (aromatase A) that is specific to the

gonads and cyp19a1b (aromatase B) due to genome duplication event which is strongly expressed in the
brain
12,13
. In fishes, two genes (Cyp19a1a and cyp19a1b) have been

identified such as Oncorhynchus mykiss
14
, Intalurus punctatus
15
, Oreochromis niloticus
16
, Carassius auratus
17
, Danio rerio
18-19
, Cyprinus carpio
20
, Trichogaster trichopterus
21
, Monoperus albus
22
, Melanotaenia fluviatilis
23
and Plecoglossus altivelis

24
.
It has been documented that teleost fish possesses highest level of brain aromatase (cyp19a1b) activity
compared to mammals
12-25
. These two isoforms possessed different tissue specificities, expression patterns, enzymatic
activities and regulation modes as evidenced in Zebrafish, Eupropean sebass, Nile tilapia, Common carp
and Yellowtail clownfish, and Catfish half-smooth tongue sole respectively.
4,20,26-30
Also, interestingly, teleost fishes are known for showing several forms of natural
hermaphroditism which was very rare in case of mammals.
31-33
Thus, the mechanisms underlying gonadal sex determination and differentiation obviously differ in fish as
compared to other vertebrates
34-36
. In our earlier investigation, we showed expression of brain type
aromatase in male and female Wrasse, Pseudolabrus

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sieboldin
37
. Although, the existence of two aromatase

catfish such as theoretical isoelectric point
(pI), molecular cyp19 genes was first evidenced in goldfish, Carassius
weight and total number of positive and
negative residues, auratus followed by in several other species. But some
extinction coefficient, half-life, instability
index, aliphatic interrogation is still remaining like sequence identity
index and grand average hydrophathy
(GRAVY) via among the diverse fish species, conserved region of
submitting protein sequence in FASTA
format. The expasy functional domains at the protein level, 3D structure of
tool, SMART was used for motif, domain
identification in protein and effect of mutation in the gene.
aromatase protein of catfish (http://smart.embl- heidelberg.de/). Currently with the wide availability of
sequence data in the form of nucleotide or protein level, we can gain previously
Alignment and Phylogenetic study: In
order to study the impossible insights into evolution of protein universe on
comparison among different protein
sequences, we have the domain, network and genome levels. Network level
used global multiple sequence alignment
program for studies allow us to understand the forces of evolution that
analysis of aromatase protein sequences
from different affect changes and conservation of protein networks. The
fishes. The multiple sequence alignment
(MSA) method is study of protein evolution had also become a key area of
widely used for assessing sequence
conservation and scientific research with discoveries such as the co-ordinated
conservation of protein domains in protein.
In this step, changes of key residues and substitution led to phenotype
CLC genomic workbench 7.5.1 was used
for multiple change
38
.
sequence alignment (MSA) analysis. Understanding phylogenetic relationship among different protein
Alongside, the improvement in the computational
sequences, we have seen the evolutionary
analysis by algorithms/tools made roadmap for studying the
depicting cladogram. The evolutionary
analysis of genome/proteome at wide scale. In this study, although,
aromatase protein among 31species was
conducted using there is availability of aromatase sequences and its encoded
CLC genomic workbench 7.5.1 using two
different protein sequences in the database for stinging catfish but
methods namely the UPMGA and
Neighbor joining lack of species-specific structural information. This led to
methods. The tree plots cladogram were
constructed using absence of knowledge of protein network and evolution in
CLC genomic workbench itself. fishes. In
this study, we have retrieved protein sequences of stinging catfish used for comparative as well as
Three dimensional structure generation
using homology evolutionary analysis. We have predicted the three
modeling and validation: In order to obtain
homologous dimensional structure and its protein-interaction network.
3D structure/template for Brain type
Cytochrome We have also analyzed the effect of non-synonymous SNPs
P450/Aromatase protein of Stinging
catfish, BLASTp on protein structure using sequence-based computational
(Basic local alignment) searches were
performed against tools.
protein databank. The 3D structure were retrieved from RCSB-PDB databank and used for homology
modelling Material and Methods We have used different bioinformatics algorithms/tools for studying the
aromatase protein of stinging catfish listed along with specified purpose.
using Modeller9.1. The 3D structure of aromatase was visualized by PyMOL which is open source
molecular visualization tool for interactive visualization and analysis of molecular structures
(http://www.pymol.org/). The predicted model evaluation along with stereo-chemical Collection of Brain
type cytochrome P450/Aromatase data: The UniProt is easily accessible database of protein sequence
(http://www.uniprot.org/). The brain type aromatase P450 protein sequence of stinging catfish was
retrieved from NCBI database with accession number (AID21724.1). Also, we have retrieved 31 species
of fish
quality assessment was carried out by using the SAVES (Structural Analysis and Verification Server)
which is integrated server (http://nihserver.mbi.ucla.edu/SAVES/). The ProSA (Protein Structure
Analysis) web server (https://prosa.services.came.sbg.ac.at/prosa) was used for refinement and validation
of protein structure. The ProSA aromatase protein sequences along with their accession number and their
source organisms were collected for multiple sequence alignment as well as for evolutionary
was used for checking model structural quality with potential errors and program shows plot of its residue
energies and Z-scores which determine overall quality of study (Table 1).
model
Physico-chemical Characterization, motif analysis of Aromatase P450: The various properties of proteins
were determined and characterized from the protein sequences. Protparam
(http://web.expasy.org/protoparam/) is expasy tool which is useful for computation of physical and
chemical parameters of given protein based on sequence. We have calculated physico-chemical properties
of Brain type Cytochrome P450/Aromatase sequence of stinging
2
39
.
Prediction of protein-protein interaction network of Brain type Cytochrome P450/Aromatase: We further
predicted the gene ontology of aromatase for biological, molecular functions which identified using
Uniprot (http://www.uniprot.org/). The STRING (Search Tool for the Retrieval of Interacting Proteins)
was used for studying the protein-protein interaction network of the Brain type Cytochrome
P450/Aromatase via submitting Stinging

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catfish protein sequence (http://string-
44
.
db.org/newstring_cgi).
Here, we depicted, grand average of hydropathicity (GRAVY) was -0.078 for aromatase of stinging
catfish In silico mutagenesis study: To elucidate effect of SNPs
which indicated that protein is hydrophilic
in nature. on Aromatase protein structure, we have generated mutation using PyMoL tool. We have
analyzed the effect of
The Multiple Sequence Alignment (MSA)
can give insight SNPs on structure of protein using sequence based tools
into sequence conservation across several
species and can such as PANTHER, PROVEAN and I-Mutant 2.0. The
allow identification of those sections of the
sequence, most functional impact of nsSNPs predicted was analyzed by
critical to protein function PANTHER
(Protein Analysis Througth Evolutionary Relationships; www.pantherdb.org/tools/csnp). This tool
estimates the likelihood of a particular non-synonymous (amino-acid changing) coding SNP to cause a
functional impact on the protein using Hidden Markov Models (HMM) based modeling and evolutionary
relationship. It calculates the subPSEC (substitution position-specific evolutionary conservation) score
based on an alignment of evolutionarily related proteins.
PROVEAN (Protein Variation Effect Analyzer) is a tool which predicts impact of an amino-acid
substitution or indel on the biological function of a protein (http://provean.jcvi.org/index.php). A query
sequence was provided in FASTA format and score prediction based on default threshold -2.5. Finally,
I-Mutant2.0 (http://folding.biofold.org/cgi-bin/i-mutant2.0) is a Support Vector Machine -based server for
the automatic prediction of protein stability changes upon single-site mutations. The RI value (Reliability
Index) is computed only when the sign of the stability change is predicted and is evaluated from the
output of the support vector machine. The input FASTA sequence of protein along with the residues
change was provided for analysis of DDG value (Kcal/mol).
Results and Discussion The current improvement in the field of bioinformatics has been facilitated to
understand the global network of genes and their encoded protein. In this study, in order to get the
evolutionary in-sights of brain type Cytochrome P450/aromatase of commercially important stinging
catfish, H. fosilis, we have used several bioinformatics tools for analysis. After, retrieval of protein
sequences, we first analyzed the physico-chemical parameter of aromatase protein of stinging catfish. The
various characteristics were determined using ProtParam tool of the ExPASY proteomic server
3
and contrary reveals hydrophobicity of the protein
45
.We have analyzed the MSA among 31 species of fishes using retrieved
aromatase protein sequences using CLC genomic workbench 7.5.1 (Figure 1). Subsequently, the
phylogenetic software has been developed such as PhyML, RaxML, MEGA and Bayesian inference such
as MrBayes for profiling proteins with respect to evolutionary study
46-52
. In our study, phylogenetic tree was constructed using CLC

genomic workbench 7.5.1 using UPGMA method which assumes constant rate of evolutionand Neighbor
Joining method, well suited for trees with varying rates of evolution. We have used Kimura protein
algorithm which assumes equal amino acid frequency and equal substitution rates while analysis.
The earlier study demonstrated that, bootstrapping method generates reliable trees using re-sampling the
given dataset several times
52-54
. We revealed that aromatase of stinging catfish was more related with Ictarulus
punctatus,Silurus meridionalis and Tachysurus fulvidraco and Clarias spp. while distantly related with
carps (Figure 2a). This could be due to duplicated gene events in the fishes over the period of time. The
conserved domains of the Aromatase were the I-helix region, the Ozol’s peptide region, the aromatase-
specific region and the heme-binding region. Overall, this analysis strongly indicates that aromatase of
stinging catfish is part of the aromatase evolutionary clade (Figure 2b).
Further, in order to understand structure level information of aromatase protein of stinging catfish, we
have searched template PDB structure for homology modelling using BLASTp. We have retrieved
homologous structure 4KQ8 with 54% identity and performed homology modelling using Modeller9.13
(Figure 3). The Modeller9.13 tool is highly versatile homology modeling tool developed by sailab and
used in protein structure prediction
55,56
.
40
. We speculated that molecular weight 56657.2; Theoretical pI 7.98; total number of negatively
charged residues (Asp + Glu) was 53; total number of positively
The DOPE Score was -57782.65234 for generated model of aromatase. The generated 3D model of
aromatase of charged residues (Arg + Lys) was 55.
stinging catfish was validated using SAVES server and Ramachandran plot showed good quality of model
with If the instability index of a given protein is < 40, it is predicted as stable while > 40 indicated as
unstable protein 41-42
. In this work, value of an instability index of aromataseprotein was 44.55 which indicated unstable
protein. The GRAVY index for given proteins is calculated as the sum of the hydropathy values of all the
aa divided by the number of residues in the protein sequence
43
. A GRAVY index > 0 indicates a protein is

hydrophobic
most favored region 89.9 % with other statistics described in tabular form (Table 2; Figure 4). The model
possessed 85.02% of the residues had an averaged 3D-1D score >= 0.2 using Verify3D server in the
SAVES (Figure 5).The Verify3D determines the compatibility of 3D model with its own amino acid
sequence by allocating a structural-class based on its position and environment (alpha, beta, loop, polar,
nonpolar etc.) and also comparing the results to the

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good 3D structures. We have depicted our predicted structure having more than 80% residues in the
averaged 3D-1D score which indicates good quality model. The ProSA analysis showed Z score value
-1.995 which indicates good quality model. Protein-protein interaction
4
investigation is a wide-ranging approach to know the organization of desire proteome. The functional
network analysis of protein will be helpful for drug discovery, to understand metabolic pathways and to
predict or develop genotype-phenotype associations
57-58
.

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Figure 1: Multiple sequence alignment of H. fossilis along with 30 fish species showing conserved
amino acids with specified colors. The conserved Cystiene (C) residues are indicated by yellow color
5

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Figure 2: Phylogenetic tree and cladogram of brain type cytochrome P450 aromatase of H. fossilis
along with 30 fish species constructed by neighbor-joining method with bootstrap value 100 using
CLC genomic workbench 7.5.1.
6

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Figure 3: The 3D structure of Aromatase protein of H. fossilis predicted using homology modelling
started with Methionine and ended with glutamine (Showing helix, beta sheet and coils)
Figure 4: Ramachandran Plot of Aromatase protein showing residues in most favored (red),
additionally allowed (yellow), generously allowed (pale yellow) and disallowed regions (white)
generated by PROCHECK.
7

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Figure 5: The plot which indicates 85.02% of the residues had an averaged 3D-1D score >= 0.2
using Verify 3D server.
Figure 6: Interaction networks between Aromatase of H. fossilis and other proteins as predicted by
STRING
8

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Table 1 Retrieved amino acid sequences of brain type cytochrome P450 aromatase of fish species
S.N. Name of Organisms Accession number
1. Heteropneustes fossilis AID21724.1 2. Clarias fuscus AFB77217.1 3. Clarias gariepinus ADH29766.1
4. Ictalurus punctatus AAL14612.1 5. Tachysurus fulvidraco AAU25806.2 6. Danio rerio AAV41033.1 7.
Carassius auratus BAA23757.1 8. Gobiocypris rarus ADB44882.1 9. Cyprinus carpio ACB13198.1 10.
Rutilus rutilus BAD91038.1 11. Pimephales prome CAC38767.1 12. Oncorhynchus mykiss CAG32835.1
13. Amphiprion ocellaris BAP59135.1 14. Melanotaenia fluviatilis AED99847.1 15. Rhabdosargus sarba
ABC70868.1 16. Scatophagus argus AFV47578.2 17. Micropogonias undulates AEL31294.1 18.
Odontesthes bonariensis AAQ88434.2 19. Oreochromis mossambicus AAD31030.1 20. Oryzias
melastigma AGN04323.1 21. Fundulus heteroclitus AAR97269.1 22. Kryptolebias marmoratus
BAF03499.1 23 Sebastes schlegelii AGG53952.1 24. Larimichthys crocea ACO35042.1 25. Oreochromis
niloticus AAO62626.1 26. Epinephelus coioides AAR97602.1 27. Kryptolebias marmoratus ABC68613.1
28. Epinephelus akaara AAS58447.1 29. Poecilia vivipara AIN75487.1 30. Fundulus heteroclitus
AAS76199.1 31. Oreochromis niloticus AAG49480.2
Table 2 Ramachandran Plot statistics for predicted model of aromatase of H. fossilis
No. of Residues Percentage
Most favoured regions [A,B,L] 396 89.4%
Additional allowed regions [a,b,l,p] 38 8.6%
Generously allowed regions [~a,~b,~l,~p] 5 1.1%
Disallowed regions [XX] 4 0.9%
Non-glycine and non-proline residues 443 100.0%
End-residues (excl. Gly and Pro) 2
Glycine residues 29
Proline residues 21
Total number of residues 495
9

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Table 3 Protein-protein interacting networks of aromatase of H. fossilis as determined by STRING
tool
cyp19a1b Cytochrome P450, family 19,
subfamily A, polypeptide 1b (513 aa)
Predicted Functional Partners:
hsd17b3 Estradiol 17 beta-dehydrogenase
3 (306 aa)
10
• • 0.988
hsd3b1 3 beta-hydroxysteroid
dehydrogenase 1 (374 aa)
• • 0.979
ugt1ab UDP glucuronosyltransferase 1
family a, b (526 aa)
• • • 0.975
hsd17b1 Hydroxysteroid (17-beta) dehydrogenase 1 (293 aa)
• 0.972
LOC553532 LOC553532 protein Fragment
(402 aa)
• 0.965
nr0b1 Nuclear receptor subfamily 0,
group B, member 1 (264 aa)
• 0.962
zgc:172315 Hypothetical protein
LOC792506 (527 aa)
• • 0.953
ENSDARG00000058845 Annotation not available (242 aa) • • 0.953 zgc:112490 Hypothetical protein
LOC550352 (543 aa)
• • 0.953
zgc:112208 Hypothetical protein
LOC550398 (252 aa)
• • • 0.918
Table 4 Impact analysis of SNPs in aromatase of H. fossilisusing PANTHER-cSNP, PROVEAN and
I-Mutant2.0
Amino acids change
Protein position
subPSEC score
P
deleterious
Prediction by PANTHER- cSNP

PROVEAN score
PROVEAN Prediction (Cutoff =- 2.5)
I-mutant (DDG value)
Stability Prediction
Q/H 128 -2.6119 0.40418 Tolerated -1.299 Neutral -0.36 Decrease
T/A 388 -3.04387 0.51096 Deleterious -3.772 Deleterious -0.50 Decrease
R/G 371 -4.28368 0.78308 Deleterious -5.406 Deleterious -1.75 Decrease
The STRING maps revealed aromatase of stinging catfish interacting with several proteins (Table 3) but
strong association/interaction was with 2 proteins/enzymes such as estradiol 17 beta dehydrogenase 3,
3-beta steroid – dehydrogenase-1, having 0.988 and 0.979 confidence score respectively (Figure 6).
Moreover, to investigate structural and functional impact of SNPs present in coding region of aromatase
of stinging catfish, we have performed varied computational analysis.
We have used sequence-based algorithms such as PANTHER, PROVEAN, I-Mutant2.0 etc. In previous
studies also, these computational tools could detect non- synonymous mutations and proved to be useful
techniques in predicting the effect of nsSNPs on other proteins
59-63
.
In earlier studies, mutation in the aromatase gene resulted into genetic disorder, also on bone
mineralization and their effect on glucose as well as lipid metabolism
64-69
. In case of humans, MutCYP, bioinformatics online tools

is available

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to investigate or classify the effect of the missense mutation in the aromatase gene
11
network. This study has provided the clue to undertake 70
. The experimental evidences also
future in vivo experimental investigation
for understanding reported site directed mutagenesis in aromatase gene at the
the effect of mutation on biological as well
as molecular p.Q123E, p.Q123H, p.T310S, p.T310C, p.R365K,
level changes in aromatase activity due to
mutation. p.R365A
71
.In case of fishes, polymorphism in a portion of the gene regulatory region of aromatase gene
was reported in the three strain of Nile tilapia
72
. Thus, in case of fishes, there is no strong evidence regarding whether
mutation will affect sex change, behavior in fish and/or other genetic disorder.
Acknowledgement The work was supported by the grant from Indian Council of Agricultural Research
(ICAR), Ministry of Agriculture, and Government of India.
In this study, we have created mutation in the three positions at the p.Q128H, p.T388A and p.R371G
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(Received 20
th
March 2015, accepted 10

th
May 2015)
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such as Chinook salmon, Oncorhynchus tshawytscha

; Nile tilapia, Oreochromis niloticus; rainbow trout,

; Japanese flounder, Paralichthys olivaceus

and zebrafish, Danio rerio

Interestingly, teleost fishes have two aromatase genes, one cyp19a1a (aromatase A) that is specific to the

. In fishes, two genes (Cyp19a1a and cyp19a1b) have been

and Plecoglossus altivelis

. Although, the existence of two aromatase

. In our study, phylogenetic tree was constructed using CLC

. A GRAVY index > 0 indicates a protein is

Prediction by PANTHER- cSNP

. In case of humans, MutCYP, bioinformatics online tools

. In case of fishes, it is plausible that mutation in the aromatase led to

problems like late puberty and regulations of gonadal sex

Molecular reproduction and development,

were in aromatase protein of stinging catfish network.

The PANTHER, PROVEAN and I-Mutant2.0 algorithms

March 2015, accepted 10

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