# In Silico Characterization of Pectate Lyase Protein Sequences from Different Source Organisms

Authors: Amit Kumar Dubey; Sangeeta Yadav; Manish Kumar; Vinay Kumar Singh; Bijaya Ketan Sarangi; Dinesh Yadav
Journal: Enzyme Research
 (2010)

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## Abstract

A total of 121 protein sequences of pectate lyases were subjected to homology search, multiple sequence alignment, phylogenetic tree construction, and motif analysis. The phylogenetic tree constructed revealed different clusters based on different source organisms representing bacterial, fungal, plant, and nematode pectate lyases. The multiple accessions of bacterial, fungal, nematode, and plant pectate lyase protein sequences were placed closely revealing a sequence level similarity. The multiple sequence alignment of these pectate lyase protein sequences from different source organisms showed conserved regions at different stretches with maximum homology from amino acid residues 439–467, 715–816, and 829–910 which could be used for designing degenerate primers or probes specific for pectate lyases. The motif analysis revealed a conserved Pec_Lyase_C domain uniformly observed in all pectate lyases irrespective of variable sources suggesting its possible role in structural and enzymatic functions.


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## Body

## 1. Introduction

The enzymes hydrolyzing pectic substances ubiquitously present in the plant kingdom forming major components of middle lamella are referred as pectinases. The production, purification, biochemical characterization, and application of pectinases have been extensively reviewed [1–10]. The pectinases include polygalacturonases, pectic esterases, pectin lyases, and pectate lyases depending on their mode of action [1].Pectate lyase (PL, EC 4.2.2.2) cleaves theα-1,4 glycosidic bonds of polygalacturonic acid via a β-elimination reaction producing unsaturated ∆4, 5 bond at the nonreducing end of the polysaccharide and generates 4,5-unsaturated oligogalacturonates. Pectate lyase is widely distributed in diverse families of microorganisms and plants. The important members of bacterial family include Erwinia carotovora, Bacillus polymyxa, Klebsiella, Yersinia, Cytophaga, Pseudomonas, and Xanthomonas while in fungi Aspergillus, Fusarium, and Penicillium are the most predominant  source [9, 11–14].A number of pectate lyase genes have been cloned, sequenced, and expressed from different source organism, namely, bacteria [15–22], fungi [23–25], yeast [26], nematode [27] and plants [14, 28].The three-dimensional structures of various extracellular pectate lyase have been reported [29–36]. The pectate lyases, in general, have a parallel β-helix domain formed by parallel-strands folded into a large right-handed helix and a major loop region.Amino acid sequence homology-based classification of pectate lyases into distinct families suggesting the possible evolution from different lineages has been reported [20, 35, 37–44]. In silico analysis of pectin lyase protein sequences has been recently reported [45].This paper reportsin silicocharacterization of pectate lyase protein sequences from different source organisms for homology search, multiple sequence alignment, phylogenetic tree construction, and motif analysis using various bioinformatics tools.


## 2. Materials and Methods

A total of 121 protein sequences of pectate lyases of different source organism available in GenBank were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/). The accession numbers of pectate lyases protein sequences along with the source organism are listed in Table 1.Table 1
List of pectate lyase protein sequences with respective accession number from different source organisms.
GroupTotal  numberAccession number (Source organism name)Nematode06AAQ09004.1[Meloidogyne incognita],  AAL66022.1|AF455757 1[Meloidogyne javanica],  AAF80747.1[Globodera rostochiensis],  BAE48375.1[Bursaphelenchus mucronatus],  BAE48371.1[Bursaphelenchus xylophilus],  BAI44500.1[Aphelenchus avenae]Plant17CAA47630.1[Nicotiana tabacum],  NP 001150723.1[Zea mays], AAA33398.1[Lilium longiflorum], CAA70735.1[Zinnia elegans], AAQ84042.1[Malus x domestica], BAE48664.1| Prunus mume], AAY85180.1[Gossypium hirsutum], AAF63756.1|AF243475 1[Vitis vinifera], BAB59066.1[Salix gilgiana], gi|1256509|emb|CAA63496.1[Musa acuminata], AAK66161.1[Fragaria x ananassa], BAF43573.1[Prunus persica], ACF40835.1[Manilkara zapota], ABG66729.2[Carica papaya], AAM63307.1[ Arabidopsis thaliana], ABR26682.1[Fragaria chiloensis],  ABD47739.1[Eucalyptus globulus subsp. GlobulesFungi10AAA80568.1[Emericella nidulans], ABM60783.1[Penicillium citrinum],AAA75471.1[Glomerella cingulata], AAC64368.1[Fusarium oxysporum f. sp. lycopersici],  ACS78057.1[ Fusarium oxysporum f. cubense],  EEY55044.1[Phytophthora infestans T30-4],  XP 749217.1[Aspergillus fumigatus Af293],  AAA80568.1[Emericella nidulans],  ABM60783.1[Penicillium citrinum],  EEY23761.1[Verticillium albo-atrum VaMs.102]Bacteria87AAB46398.1[Pseudomonas viridiflava], AAC60448.1[Pseudomonas marginalis], AAL56657.1[Pseudomonas syringae pv. glycinea], AAC41522.1[Xanthomonas campestris],AAM38405.1[Xanthomonas axonopodis pv citri str 306], YP 001915548.1|[Xanthomonas oryzae PXO99A], AAY50632.1|[Xanthomonas campestris pv campestris str 8004], YP 001314702.1[Sinorhizobium medicae WSM419], ZP06003184.1[Selenomonas noxia ATCC 43541], ZP 04658159.1[Selenomonas flueggei ATCC 43531], ACH58409.1|[Bacillus subtilis], BAA05383.1[Bacillus sp. YA-14], ZP 05725973.1[Dickeya dadantii Ech586], NP 866630.1[Rhodopirellula baltica SH1], NP 228243.1[Thermotoga maritima MSB8],ACR10769.1[Teredinibacter turneraeT7901], ZP 04713986.1[Alteromonas macleodiiATCC 27126], ZP 04334947.1[Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111], AAZ56201.1[Thermobifida fusca YX],AAW84086.1[Bacillus halodurans], ACD11362.1[Bacillus pumilus], AAC38059.1[Pseudonocardia sp.], ZP 04608172.1[Micromonospora sp ATCC 39149], ZP 05021985.1[Streptomyces sviceus ATCC 33406], YP 677777.1[Cytophaga hutchinsonii ATCC 33406], CAA43401.1[Pectobacterium carotovorum],CAA47821.1[Erwinia chrysanthemi],ABF59812.1[Erwinia sp. BTC105], NP 866628.1[Rhodopirellula baltica SH 1],ZP 01721007.1[Algoriphagus sp PR1], ZP 01851785.1[Planctomyces maris DSM8797], ACL96719.1[Caulobacter crescentus NA1000], AAO79220.1[Bacteroides thetaiotaomicron VPI-5482], ABC76269.1[Syntrophus aciditrophicus SB],YP 618077.1[Sphingopyxis alaskensisRB2256], EER49733.1[Asticcacaulis excentricusCB 48], YP 527779.1[Saccharophagus degradans2-40], YP 356933.1[Pelobacter carbinolicus DSM 2380], CAB40884.1[Paenibacillus barcinonensis BP23], YP 001615581.1[Sorangium cellulosum 'So ce 56'], YP 002573715.1[Anaerocellum thermophilum DSM6725], ACU58213.1[Chitinophaga pinensis DSM 2588], ZP 00943832.1[Ralstonia solanacearum UW 551], CAG74004.1[Pectobacterium atrosepticum SCRI1043], YP 432149.1[Hahella chejuensis KCTC 2396], ZP 05805856.1[Streptomyces flavogriseus ATCC 33331], ZP 05542020.1[Streptomyces griseoflavus Tu4000], ZP 04688982.1[Streptomyces ghanaensis ATCC 14672], ZP 05530610.1[Streptomyces viridochromogenes DSM 40736], NP 827559.1[Streptomyces avermitilis MA-4680], BAH85845.1| [Streptomyces thermocarboxydus], ZP 05514668.1[Streptomyces hygroscopicus ATCC 53653], YP 001506642.1[Frankia sp EAN1pec], ZP 05011753.1[Streptomyces pristinaespiralis ATCC 25486], ACV08000.1[Jonesia denitrificans DSM 20603], ZP 04367351.1[Cellulomonas flavigena DSM 20109]YP 001106556.1[Saccharopolyspora erythraea NRRL 2338], ACU38032.1[Actinosynnema mirum DSM 43827], AAL51034.1|AF454849 1[Klebsiella oxytoca],NP 149220.1[Clostridium acetobutylicum ATCC 824], YP 003248772.1[Fibrobacter_succinogenes ssp succinogens], ZP 04805707.1[Clostridium_cellulovorans_743B], YP002505581.1[Clostridium_cellulolyticum_H10], ZP 05495790.1[Clostridium_papyrosolvens_DSM_2782], AAG24437.1|AF303225 1[Bacillus alcalophilus],BAD63679.1[Bacillus_clausii_KSM-K16], YP 003091439.1[Pedobacter_heparinus_DSM_2366], EER72050.1[Sphingobacterium_spiritivorum_ATCC_33861], ZP 06173220.1[Brevundimonas subvibrioides ATCC15264], ABJ82092.1[Candidatus solibacter  usitatus Ellin6076], EEX43407.1[Bacteroides finegoldii DSM 17565], ZP 06083351.1[Bacteroides sp. 2 1 22], YP 001196575.1[Flavobacterium johnsoniae UW101], ACU08313.1[Flavobacteriaceae bacterium 3519-10], YP 001817270.1[Opitutus terrae PB90-1], YP 003003775.1[Dickeya zeae Ech1591], AAD25394.1[Azospirillum irakense], EER49094.1[Asticcacaulis excentricus CB 48], YP 003061197.1[Hirschia baltica ATCC 49814], AAG29353.1|AF279264 1[Cellvibrio japonicus], ZP 02927550.1[Verrucomicrobium spinosumDSM 4136], ZP 03725417.1[Opitutaceae bacterium TAV2], YP 003259879.1[Pectobacterium wasabiae WPP163], ACT13209.1[Pectobacterium carotovorum subsp. carotovorum PC1], AAM87379.1|AE013987 2[Yersinia pestis KIM 10], YP 001175221.1[Enterobactersp.638], ABR71284.1[Marinomonas sp.MWYL1], YP 003243108.1[Geobacillus sp. Y412MC10], YP 001018637.1[prochlorococcus marinus str MIT 9303], YP 382660.1[Synechococcus sp.CC9605]The program ClustalW [46] was used for multiple sequence alignment. Mega 4 was used for dendrogram construction by Neighbor-Joining (NJ) method [47]. For domain search, the Pfam site (http://www.sanger.ac.uk/software/pfam/search.html) was used. Domain analysis was done using MEME (http://meme.sdsc.edu/meme/meme.html) [48]. The conserved protein motifs deduced by MEME were characterized for biological function analysis using protein BLAST, and domains were studied with Interproscan providing the best possible match based on highest similarity score.


## 3. Results and Discussion

A total of 121 pectate lyases sequences from different source organisms subjected to phylogenetic tree construction revealed major clusters of bacterial, fungal, plant, and nematode pectate lyases. The pectate lyase from bacterial source was the predominant comprising of 87 accession numbers. The different accession of bacterial pectate lyase formed three major clusters as shown in Figure1. The plant, fungal, and nematode pectate lyases formed separate clusters signifying the sequence-based similarity with reference to different source organisms. The multiple accessions of bacterial, fungal, plant, and nematode pectate lyases were placed closely in the clusters signifying the greater degree of sequence level similarity. Similar phylogenetic tree revealing clustering of pectin lyases protein sequences based on different source organism has been reported [45].Figure 1
Phylogenetic tree of pectate lyase protein sequences from different source organism constructed by NJ method.The multiple sequence alignment of these protein sequences revealed conserved regions at different stretches, namely, from 439–467, 715–816, and 829–918 amino acid residues (Figures2(a), 2(b), and 2(c)). This region could be used for designing degenerate primers or probes for PCR-based amplification or hybridization-based detection of pectate lyase sequences from different source organisms.(a) Multiple sequence alignment of pectate lyase protein sequences showing maximum homology from amino acid residues 439–467. (b) Multiple sequence alignment of pectate lyase protein sequences showing maximum homology from amino acid residues 715–816. (c) Multiple sequence alignment of pectate lyase protein sequences showing maximum homology from amino acid residues 829–910.
(a)(b)(c)A total of five motifs labelled as 1, 2, 3, 4, and 5 were observed in only 91 sequences when subjected to MEME. The distribution of these motifs among 92 pectate lyase accession number is shown in Table2.Table 2
Distribution of motifs among 91 pectate lyase proteins sequences from different source organisms.
S. no.Accession no.Motif 1Motif 2Motif 3Motif 4Motif 51AAB46398++2CAB40884+3CAA47630++++4CAA70735++++5CAA43401+6CAA47821+7NP866630+++8YP003003775+9ZP05725973++10NP866628++++11ZP05805856+12YP002573715+13YP001506642+14NP001150723++++15BAE48375+16BAE48371+17AAD25394+18ACV08000+19ACU58213++20AAC60448+++21BAI44500+22YP001314702++23AAQ09004+24AAL66022.1|AF455757_1+25AAF80747+26AAC64368+27AAA33398++++28ACU38032+29ACR10769+++30ACU08313+31BAH85845+32ACS78057+33ZP04367351+34AAW84086++35AAL56657++++36AAK66161++++37AAQ84042++++38AAC41522++39AAA80568+++40ZP06003184++41ACH58409+++42AAF63756.1|AF243475_1++++43ZP04713986+44ZP04334947++45ABG66729++++46ABR26682++++47ACD11362++48XP749217++49BAA05383++50BAB59066++++51ZP01851785+++52ABM60783+++53BAF43573++++54ABF59812+55AAY85180++++56EEY55044+57EEY23761++58YP001615581++59ZP05542020+60ZP05530610+61ZP05514668+62ZP05011753+63ZP04688982+64YP001106556+65NP827559+66YP432149+67ABD47739+++68ZP00943832+69AAM63307++++70AAC38059+++71AAA75471++72ACL96719++73AAZ56201++74AAY50632+++75AAM38405++76YP618077++77ZP04608172++78ZP05021985++79ZP04658159++80YP001915548++81NP228243+++82YP356933+83YP677777++84ACF40835++++85CAA63496++++86AAO79220+++87ABC76269++88ZP01721007++89BAE48664++++90EER49733++91AAL51034.1|AF454849_1+92YP527779.1|+The motifs with width and best possible match amino acid sequences are shown in Table3. All these motifs showed similarity with pec_lyase_C domain which is quite prevalent in pectin lyase sequences as reported earlier [45]. As the mechanism of pectin lyase and pectate lyase is quite similar, it is expected to show similar motifs. The motif 1 of 29 amino acid residues with sequence IAFNHFGEGLVQRMPRCRHGYFHVVNNDY and motif 4 of 50 amino acid residues with a sequence HNSLSNCHDGLIDAIHGSTAITISNNYMTHHDKVMLLGHSDSYTQDKNMQ were observed in 47 and 39 pectate lyase protein sequences (Table 3) signifying their possible role in the structural and catalytic attributes of pectate lyases.Table 3
Different motifs commonly observed in pectate lyases protein sequences with best possible match amino acid sequences.
Motif numberWidthSequenceOccurrence in pectate lyase protein sequences129IAFNHFGEGLVQRMPRCRHGYFHVVNNDY47250NPRPGTLRHAVIQDEPLWIVFKRDMVIQLKQELIMNSFKTIDGRGVNVHI16350CITIQFVTNIIIHGIHIHDCKPTGNAMVRSSPSHYGWRTMADGDGISIFG16450HNSLSNCHDGLIDAIHGSTAITISNNYMTHHDKVMLLGHSDSYTQDKNMQ39549SSSQTMTVDGGGARYAHDKVFQHNGPGTFVIKNFQVQDFGKLYRSCGNC27Further when the motif best possible match amino acid sequence was subjected to BLAST  to reveal its identity, it was observed that the  motifs 1, 2, 3, and 4 represents  Pec_Lyase_C  superfamily while motif 5 represents  pectate lyase superfamily. The exact function of these motifs in influencing the catalytic activity of the pectate lyase needs to be investigated.Thein silico characterization of pectate lyases protein sequences from different source organisms has revealed sequence level similarity specific for different groups which    could be utilized for  designing strategy for cloning the putative genes based on PCR amplification using degenerate primers.



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*Source: 950230-2010-09-19.xml*