Macrophomina phaseolina

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International Journal of Biological & Medical Research

Int J Biol Med Res. 2014; 5(3): 4246-4257

In silicoAnalysis of Chloroperoxidase and Lignin Peroxidase of Pathogenic Fungus

Macrophomina phaseolinaa b a b

Yeasmeen Ali , Mohd Omar Faruk Sikder , Lolo Wal Marzan , Farjana Sharmenb, Md Mursalin Khan ,a

Md Amzad Hosain , *aDepartment of Genetic Engineering and Biotechnology, University of Chittagong, Chittagong- 4331.

bBioinformatician, Basic and Applied Research on Jute Project, Bangladesh Jute Research Institute, Dhaka- 1207.

A R T I C L E I N F O A B S T R A C T

Keywords:

Motif

Botryosphaeriaceae

Physicochemical property

Active site

Secondary structure.

Original Article

Macrophomina phaseolina is a destructive fungus that affects more than 500 plant speciesthroughout the world. Lignin degradation of the host plant is crucial step for the pathogenesisof this fungus. Chloroperoxidase and lignin peroxidase are two important enzymes for lignindegradation. To develop an effective antagonist against this fungus, understanding theseproteins is necessary. In this study, we have reported physico-chemical characteristics,phylogenic relationship, secondary structure, 3-D structure, motifs of 7 chloroperoxidase and3 lignin peroxidase proteins. Moreover, pockets as well as conserved residues in the motifsequence were identified as the target for site-directed mutagenesis of chloroperoxidase and

lignin peroxidase. This mutagenesis or designing target ligands against these proteins maystop the ligninolytic activity of M. phaseolina, which would save economically important plants.

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1. Introduction

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Macrophomina phaseolina is a Botryosphaeriaceae fungus thatbelongs to phylum ascomycota. It is one of the most devastating soiland seed borne pathogens infecting more than 500 plant species[1]. The plant species includes jute [2], cotton [3], maize [4], pulses[5], sunflower [6] etc. This fungus under favorable environmentalconditions can cause diseases like charcoal rot, stem rot, dampingoff, seedling blight, collar rot, and root rot in various importantcrops [7].

Disease development is considered to be associated withseveral factors such as heat stress, soil water deficit, physiologicalstress or coarse soil texture [8]. Due to having sclerotia [thick-walled resistant hyphal mat] in soil and plant debris, it is difficult tocontrol M. phaseolina [9]. This pathogen is widely distributedthroughout the world especially in tropical and subtropicalcountries [10]. In Bangladesh, due to pathogenicity of this fungusonly jute fiber production rate is reduced by 30% [11].

In 2012, the genome sequence as well as gene prediction of M.phaseolina was accomplished. The genome size was about 49 Mband about 14,249 protein-coding genes were predicted [11]. Itpossesses ligninolytic activity, which is the result of combination ofdifferent enzymatic system such as slaccases, lignin peroxidases,

galactose oxidases, chloroperoxidases, haloperoxidases, and hemeperoxidases. Lignin peroxidase [Lip] is an extracellur enzyme [12],which with the help of a cofactor, Veratryl alcohol interacts withlignin polymer. Likewise, chloroperoxidase [CPO] is also anextraxellur enzyme that requires heme or vanadium as a cofactor[13]. This enzyme acts on lignin by oxidization of Cl tohypochlorous acid [HOCl] or a similarly reactive chlorineelectrophile[14]. However, the mechanism is not still wellunderstood.

Our study is to analyze the chloroperoxidase and ligninperoxidase of M. phaseolina, which will help to understand thepathogenesis of this devastating fungus. We have analyzed 7

chloroperoxidase and 3 lignin peroxidase enzymes and founddifferent important investigations such as physic-chemicalcharacteristics, functional motifs, 3D structure of these proteins.We also showed the active site pockets, different amino acids thatcould be structurally and functionally critical as well as

Copyright 2010 BioMedSciDirect Publications. All rights reserved.c

* Corresponding Author : Yeasmeen AliLecturer, Department of Genetic Engineering and Biotechnology,

University of Chittagong, Chittagong- 4331.

E-mail: [email protected]


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Yeasmeen Ali et.al Int J Biol Med Res. 2014; 5(3): 4246-4257

phylogenetic relationship among these peroxidases. All of thesefindings would decipher an effective way to block the activity ofchloroperoxidase and lignin peroxidase of M. phaseolina to protectdifferent economically important crop species.

MATERIALS AND METHODS:

Retrieval of Macrophomina phaseolina chloroperoxidase andlignin peroxidase protein sequences:

Protein sequences of M. phaseolinawere retrieved from NCBIprotein database in FASTA format. 7 chloroperoxidase and 3 lignin

peroxidase protein sequences were selected for analysis.[http://www.ncbi.nlm.nih.gov/].

Physico-chemical characterization:

Different properties including number of amino acids,molecular weight, theoretical isoelectric point [pI], amino acidcomposition [%], number of positively [Arg + Lys] and negativelycharged [Asp + Glu] residues, extinction co-efficient, instabilityindex, aliphatic index and Grand Average of Hydropathicity[GRAVY] were calculated using ExPASy's ProtParam tool [15][http://expasy.org/tools/protparam.html]. The crystallizationtendency of the proteins was determined by using the CRYSTALP2

W E B S E R V E R [ 1 6 ] [ h t t p : / / b i o m i n e -ws.ece.ualberta.ca/CRYSTALP2.html] that accepts proteinsequences in the FASTA format. The CRYSTALP2 is a kernel-basedmethod that uses the composition and collocation of amino acids,hydrophobicity and isoelectric point of the given sequences toestimate the crystallization propensity of the proteins.

Characterization of secondary structure:

Secondary structure prediction was performed by SOPMA [17][Sel f -Optimized Predict ion Method with Al ignment ,http://npsapbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_sopma.html] tool The input protein sequences were given in

FASTA format. The number of conformational states was adjustedto four in order to predict Helix, Sheet, Coil and Turn while otherparameters were set as default. By using this software, Alpha helix[Hh], 310 helix, [Gg], Pi helix [Ii], Beta bridge [Bb], Extended strand[Ee], Beta turn [Tt], Bend region [Ss], Random coil [Cc], Ambigousstates, and other states were predicted.

Protein 3D structure prediction:

The 3D structures of chloroperoxidases and lignin peroxidaseswere predicted using the 3D-JIGSAW [version 3] comparativem o d e l i n g s e r v e r [ 1 8 ] [ h t t p : / /bmm.cancerresearchuk.org/~3djigsaw/], which acceptssequences in FASTA format. This program constructs 3D models forgiven protein sequences based on the known homologous proteinsstructure. It uses HMM for searching homologous templates insequence databases [PFAM+PDB+nr] and splits the query sequenceinto domains. The good templates with maximum coverage of thequeries are then used for modeling and the generated models arerepresented as PDB file format.

Detection of motif:

The motif scan tool [19, 20] [http://myhits.isb-sib.ch/cgi-bin/motif_scan] was used to identify the motifs and their locationsin the sequence of Lignin peroxidase and chloroperoxidase. Theseprotein sequences were given as input data [FASTA format] andscanned against 'PROSITE Patterns'. The selected motifs were thenvisualized by using Swiss- Pdb Viewer V4.1.0 [21]. Locations,nature, match score and match details of the motifs were analyzed.It was aimed to find out the conserved regions of these motifs. Atthis point of view, multiple sequence alignment [MSA] of thesemotifs was performed using EBI Clustal Omega [22].[https://www.ebi.ac.uk/Tools/msa/clustalo/]. As expected, anumber of conserved regions were found and their length,conserved amino acids were analyzed.

Active site prediction:

Active site analysis was performed using Computed Atlas ofSurface Topography of Protein [CASTp]. CASTp for automaticallylocating and measuring protein pockets and cavities, is based onprecise computational geometry methods, including alpha shapeand discrete flow theory. CASTp identifies and measures pocketsand pocket mouth openings, as well as cavities. The programspecifies the atoms lining pockets, pocket openings, and buried

cavities; the volume and area of pockets and cavities; and the areaand circumference of mouth openings. Most frequently, the largestpocket/cavity has been the active site, but there are a number ofinstructive exceptions [23]. Ligand volume and binding sitevolume are somewhat correlated, but the ligand seldom occupiesthe entire site. Auxiliary pockets near the active site have beensuggested as additional binding surface for designed ligands [24].Analysis of active sites is important for the modeled protein as aprecursor to further work on its docking studies, and to shape theprocess of making a grid before docking.

Prediction of Evolutionary relationship:

Evolutionary tree among these 10 lignin degrading proteinsequences were aligned using multiple sequence alignment toolC l u s t a l o m e g a [ 2 5 ] [ h t t p s : / / w w w . e b i . a c . u k/Tools/services/web_clustalw2/toolform.ebi] , followed byconstruction of evolutionary tree using the protein sequences inFASTA format as input data The alignment parameters were tunedfinely to find out the best alignment and Neighbor Joining [NJ]method was selected to construct the tree.

RESULTS:

Physico-chemical characterization:

Isoelectric point [pI] is a pH in which net charge of protein iszero. pI of lignin peroxidase 2 and 3 were observed to lie in theacidic range, while the rest of the proteins occur in alkaline range.From the study of instability index, it was found that allperoxidases are stable [except Chloroperoxidase 7], as instabilityindex value less than 40 indicates stability of a protein


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sequences and ranked them according to the scores oframachandran plot. For each of the proteins, the top ranked 3Dmodel was selected from the given models [supplementary file, fig1]. The 3D models generated by 3D JIGSAW were then used furtherfor the motif detection and pocket findings.

Motif Identification:

Chloroperoxidase and lignin peroxidase are found in M.phaseolina as well as many other fungi, posing a major role in lignindegradation [11, 31]. Motifs for chloroperoxidase and ligninperoxidase were identified using Motif scan tool [Fig 1,

Supplementary file, table 4]. If we look into the conserved regions[Fig 2], a number of residues are revealed which are already knownto have important role in catalysis. These conserverd regions arepredominated by cysteine, proline, aspartate, asparagines,histidine, alanine and glycine.

Active site analysis:

The active sites of chloroperoxidase and lignin peroxidase werepredicted [fig 3]. Further, in this study, we have also reported thebest active site area of the experimental enzymes as well as thenumber of amino acids involved in it; showed the number ofpockets, with their area and volume [Supplementary file, fig 2].

In case of the lignin peroxidase, the volume of the pockets isquite similar and average is 2157 . There are some variations inthe area of pockets; for lignin peroxidase 2, it is 1100.9 2[Supplementary file, table 5]. On the other hand, for ligninperoxidase 3, it is highest 1857.8 2. For lignin peroxidase 1, thearea is between the two others-1542.7 2. But from thevisualization we can see a similar pocket structures.

In the analysis of the chloroperoxidases we find similar pocketsizes and structures too. For the chloroperoxidase 2,chloroperoxidase 3, chloroperoxidase 4, chloroperoxidase 5 andchloroperoxidase 6, the average pocket area is 2301.7 2 and

average pocket volume is 3428.6 3. Among these, the highest areashowed by chloroperoxidase 4 that is 3072.7 2 and volume is 50423. There are two chloroperoxidases show major variations; thoseare chloroperoxidase 1 and chloroperoxidase 7. Forchloroperoxidase 1, area is 697.6 2 and volume is 907.5 3. In caseof chloroperoxidase 7, we identified paramount variation; it istotally different from other chloroperoxidases regarding the area[208.4 2], volume [203.8 3] and pocket. From the visualization,we can see a similar pocket structure among the chloroperoxidasesother than the chloroperoxidase 7.

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[Supplementary file, Table 1]. Additionally, Aliphatic index [AI]refers to the relative volume of a protein occupied by its aliphaticside chains. The higher the Aliphatic index of proteins, the morethermally stable the proteins. Aliphatic index of chloroperoxidase1 [100.11] and 5 [90.84] classifies them as most thermostable,closely followed by other chloroperoxidases [chloroperoxidase 3,4, 6 and lignin peroxidase 9]. Grand average of hydropathicityindex [GRAVY] indicates the interaction of the proteins in water.Chloroperoxidase 1 [0.099] and lignin peroxidase 1 [0.022] arehydrophobic [due to positive GRAVY values]. GRAVY values ofother peroxidases were observed within a wide range of -0.055 to -0.54 [hydrophilic].

The amino acid composition of each peroxidase sequence wascalculated by using ExPASY's ProtParam tool [supplementary file,Table 2]. High percentage of alanine [above 9.9] and serine [above8.5] was found in all three lignin peroxidases compared to otheramino acid. In all chloroperoxidase [except chloroperoxidase 7],leucine content was found significant. Again, chloroperoxidase 2, 4and 5 have a glycine content above 8.9. Moreover, a goodpercentage of serine was found in chloroperoxidase 3 [9.6] and 7[9.2] and also chloroperoxidase 7 is proline rich [10.0%].

There are some properties of the proteins such as isoelectricpoint, hydrophobicity, and the frequency of certain collocated diand tripeptides that are pivotal indicators of crystallization [16].The CRYSTALP2 accounts all such characteristics of givensequences for estimating the confidence of crystallization.Thehigher the confidence, the more probable that protein iscrystallizable and vice versa. Among the 7 chloroperoxidases and 3lignin peroxidases, chloroperoxidase 5 showed the highestconfidence of crystalizaiton that was 0.61 which was followed bychloroperoxidase 2 [0.565] and chloroperoxidase 6 [0.523],respectively [supplementary file, table 1]. In contrast, ligninperoxidase 3 represented the least confidence of crystallization[0.221]. Each of the the remaining chloroperoxidases exhibitedhigher confidence of crystallization than that of remaining ligninperoxidases. From the CRYSTALP2 result, it can be assumed that

chloroperoxidases are more likely to be crystallized than ligninperoxidases. Among all chloroperoxidase, chloroperoxidase 5represents maximum propensity for crystallization.

Characterization of secondary structure:

SOPMA analysis of secondary structure of chloroperoxidaseand lignin peroxidase protein sequences results the predominanceof random coil which is followed by alpha helix, extended strand,and beta sheet, respectively [supplementary file, table 3]. In case ofChloroperoxidase 1, Lignin Peroxidase 1, it was found that alphahelix exceed the random coil.

Protein 3D structure:

The 3D structure of protein is very crucial for comprehending

the protein functions, their sub-cellular localization as well asprotein-protein interactions. Based on homology modeling, 3D-JIGSAW results showed five 3D models for each of the given



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Fig 1: Motifs in Chloroperoxidase and Lignin peroxidase. Insets show most common amino acids in motifs.



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Fig 2: Conserved regions in chloroperoxidase [top] and lignin peroxidases [bottom] motifs using boxshade.

Active site analysis:

The active sites of chloroperoxidase and lignin peroxidasewere predicted [fig 3]. Further, in this study, we have also reportedthe best active site area of the experimental enzymes as well as thenumber of amino acids involved in it; showed the number of

pockets, with their area and volume [Supplementary file, fig 2].In case of the lignin peroxidase, the volume of the pockets is

quite similar and average is 2157 . There are some variations inthe area of pockets; for lignin peroxidase 2, it is 1100.9 2[Supplementary file, table 5]. On the other hand, for ligninperoxidase 3, it is highest 1857.8 2. For lignin peroxidase 1, thearea is between the two others-1542.7 2. But from thevisualization we can see a similar pocket structures.

In the analysis of the chloroperoxidases we find similar pocketsizes and structures too. For the chloroperoxidase 2,chloroperoxidase 3, chloroperoxidase 4, chloroperoxidase 5 andchloroperoxidase 6, the average pocket area is 2301.7 2 and

average pocket volume is 3428.6 3. Among these, the highest areashowed by chloroperoxidase 4 that is 3072.7 2 and volume is

5042 3. There are two chloroperoxidases show majorvariations; those are chloroperoxidase 1 and chloroperoxidase 7.For chloroperoxidase 1, area is 697.6 2 and volume is 907.5 3. Incase of chloroperoxidase 7, we identified paramount variation; it istotally different from other chloroperoxidases regarding the area

[208.4 2], volume [203.8 3] and pocket. From the visualization,we can see a similar pocket structure among thechloroperoxidases other than the chloroperoxidase 7.

Fig 3: Pockets [active site] of the enzymes



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Evolutionary relationship:

Phylogenetic tree shows evolutionary relationship among all the ten proteins. There are two major clades on the tree. Allchloroperoxidases [except chloroperoxidase 1] reside in a common clade whereas, three lignin peroxidases reside in another tree.Chloroperoxidase 1 seems to be the outgroup of the tree.

Table 1: Physico-chemical parameters of chloroperoxidase and lignin peroxidase:

Fig 4: Phylogenetic relationship among chloroperoxidases and lignin peroxidases



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Table 2: Composition of amino acid of chloroperoxidase and lignin peroxidase (in %):

Table 3: Secondary structure prediction of chloroeroxidase and lignin peroxidase (in %):



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Table 4: Identification of motifs:

Table 5: Active site Prediction



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Fig 2: Pockets (active site) in motif of chloroperoxidase and lognin peroxidase.


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DISCUSSION:

Instability index and aliphatic index indicate the relativethermostability of the proteins, whereas GRAVY values showhydrophilic nature of most of them. lignin peroxidases have highpercentage of alanine and serine. On the other hand,chloroperoxidases are predominated by leucine, glycine, prolineand serine. These amino acids have important role in proteinstructure. Serine is small in size and hence reasonably common tooccur within the tight turns on the protein surface or within theinterior of a protein. Serine side-chain hydroxyl oxygen can form ahydrogen bond with the protein backbone [26]. Leucine is ahydrophobic amino acid as it has a branched hydrocarbon sidechains usually buried in folded proteins. The hydrophobic effectaccounts for stabilization of water-soluble proteins [24]. Highpercentage of glycine may be responsible for the stability of triplehelical structure, since incorporation of large amino acids can causesteric hindrance [27]. Increased proportion of proline residues issignificant not only to act as structural disruptor of the secondarystructural elements but also to point outward and stabilize the helix[28].

From the CRYSTALP2 result, it is clear that chloroperoxidasesare more likely to be crystallized than lignin peroxidases. Among allchloroperoxidase, chloroperoxidase 5 represents maximumpropensity for crystallization. The knowledge of crystallizationtendency of protein has a great importance in structural biologythat provides valuable insight into the structure-functionrelationship of the proteins [16]. Moreover, crystallographicknowledge of proteins has immense roles in pharmaceutical,biotechnological and chemical industries for rational drugdesigning, protein engineering and other applications. [29].Secondary structure analysis showed predominance of random coilover other structures. Large number of random coil gives theprotein more flexibility and self-assembly [30]. Protein 3D modelswere created using homology modeling that were validated byRamachandran plot.

Motif scan tool was used as motif finder in these proteins.Conserved regions of these motifs are important zone to mitigatetheir activities. It is possible to inactivate these proteins bydesigning ligands against these conserved sites in 3D proteinstructure. Moreover, any type of site directed mutagenesis can beperformed in these regions to inactivate these proteins. To do so, wehave to acquire a deep knowledge on it. cysteine, proline, aspartate,asparagines, histidine, alanine and glycine are major amino acidthat not only have structural role but also important in catalyticfunction. Cysteine is important for disulfide linkage as well as formetal binding [32]. Another important residue is proline. It isunable to adopt a normal helical conformation because it

introduces kinks into alpha helices. Aspartate and asparagine arefrequently involved in protein active sites. Because of their negativecharge, they can interact with positively charged non-proteinatoms. Since Aspartate has a shorter side-chain, it is slightly morerigid within protein structures. Thus, it has a slightly strongerpreference to be involved in protein active sites [26]. Histidine is the

most common amino acid in protein active sites. Theyefficiently bind metal ions, often acting together with cysteines.Alanine plays important role in substrate recognition or specificity,particularly in interactions with non-reactive atoms such ascarbon. The uniqueness of Glycine [H as R group] also means that itcan play a distinct functional role, such as using its sidechain-lessbackbone to bind to phosphates [26].

Predicted active sites were of similar structure in same proteinfamily. Volume, area and charge density of active sites weresatisfactory. All of the chloroperoxidase proteins [exceptchloroperoxidase 1] share a common ancestral point [marked byarrow, Fig 4] after being diverged from root of the tree. Fungallignin peroxidase 1 and 3 are more closely related than ligninperoxidase 2. Chloroperoxidase 1 seems to be the outgroup of thetree. Probably this protein has acquired a lot of change in itssequence over time without losing its catalytic activity.

CONCLUSION:

A number of plants for example crop, fiber or othercommercially valuable species are infected by Macrophominaphaseolina fungus and causes a great lose throughout the world.For the pathogenesis of this fungus, different genes are involved.Among these genes, chloroperoxidase and lignin peroxidase are

two important genes that are required for a crucial step that islignin degradation. In present study, we have analyzed physico-chemical characteristics, secondary and tertiary structure as wellas motifs in 7 chloroperoxidase and 3 lignin peroxidase. Duringthis analysis, we found amino acids like cysteine, aspertate,asparagine, proline, histidine and glycine, which play vital roleboth structurally and functionally. Moreover, we have searched theactive sites in these 10 proteins and found highest pocket area forchloroperoxidase 4 and lignin peroxidase 3. Study of evolutionaryrelationship reveals that all of the proteins shared a commonancestor. All of these findings help us to understand the charactersof these proteins. This will facilitate to design a potent targetagainst chloroperoxidase and lignin peroxidase and protect thefungal susceptible crop species.

CONFLICT OF INTERESTS

The authors declare that there is no conflict of interestsregarding the publication of this paper.

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