domain analysis of 3 keto acyl-coa synthase for structural variations in vitis vinifera and oryza...

Interdiscip Sci Comput Life Sci (2014) 6: 1–14

DOI: 10.1007/s12539-013-0017-8

Domain Analysis of 3 Keto Acyl-CoA Synthase for StructuralVariations in Vitis vinifera and Oryza brachyantha using

Comparative Modelling

Mamta Sagar∗, Neetesh Pandey, Naseha Qamar, Brijendra Singh, Akanksha Shukla(Department of Bioinformatics, University Institute of Engineering and Technology, Chhatrapati Shahuji Maharaj

University, Kanpur 208024, India)

Received 14 June 2013 / Revised 30 December 2013 / Accepted 21 January 2014

Abstract: The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS),found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase likeproteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein ofOryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor ; total 11 homologs werepredicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketidesynthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal andchalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were foundto have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acylsynthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitisvinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryzabrachyantha contains N terminal domain and C terminal domain and characterized using annotation of thesedomains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase(Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. Thissimilarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possiblerole in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha.Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein atdifferent positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites,were predicted in localized combination of conserved residues.Key words: Vitis vinifera, Oryza brachyantha, 3 keto acyl-CoA synthase, flavonoid, domain analysis, sequenceanalysis, KCS, active site, malonyl CoA binding site, structure alignment, binding site, Pyrone synthase.

List of abbreviations

ACP Acyl carrier proteinCHS Chalcone synthaseFA Fatty acidKCS 3 keto acyl CoA SynthaseGcs Germicidin synthasePS Pyrone synthasePKS polyketide synthaseJ3M9Z7 ORYBR & J3MFI6 ORYBR 3 keto acylCoA Synthase like protein in Oryza brachyanthaF6HH63 VITVI. 3 keto acyl CoA Synthase in VitisviniferaVLCFA Very long chain fatty acids

∗Corresponding author.E-mail: [email protected]

1 Introduction

Very-long-chain fatty acids (VLCFAs) are essentialprecursors of cuticular waxes and aliphatic suberins inroots. Condensation of C (2) units to an acyl CoAby 3-ketoacyl CoA synthase (KCS) is first committedstep in VLCFA biosynthesis. The very long chain fattyacids (VLCFA) incorporated into plant lipids are de-rived from the iterative addition of C2 units providedby malonyl-CoA to an acyl-CoA by the 3-ketoacyl-CoAsynthase (KCS) component of a fatty acid elongase(FAE) complex (Millar, A.A. et al. 1999; Lee SB etal. 2009). An Arabidopsis fatty acid elongase gene,KCS1 encodes a 3- ketoacyl-CoA synthase which is in-volved in very long chain fatty acid synthesis in vegeta-tive tissues, and wax biosynthesis. In Arabidopsis, waxbiosynthesis involves modification of most VLCFA by

2 Interdiscip Sci Comput Life Sci (2014) 6: 1–14

either the acyl reduction or decarbonylation pathway(Hannoufa et al. 1993; Jenks et al. 1995). A smoothlayer of epicuticular wax is found over the epidermalsurface in wild-type Arabidopsis leaf (Jenks, M.A. etal., 1995).

CHS is a key enzyme of plant flavonoid biosynthesis.Although it has various functions in plants but its rolein plant pathogen defence and insect resistance mecha-nism needs more precise definitions (Major, I. T. et al.2006; Robin D. Mellway et al. 2009). However, Palo RT(1984) described role of secondary metabolites as chem-ical defence against herbivores in Betula Spp, Salix Spp,and Populus Spp. CHS and its related genes are foundin several plants. One important related enzyme is 3keto acyl CoA synthase. Allthough 3 keto acyl CoAsynthase is well chracterised in Arabidopsis thaliana,Populus tricocarpa and other organism but this enzymeis not studied in Oryza brachyantha and no study re-ports KCS function in this organism except genomicannotation of whole genome sequence provided by Dr.Mingsheng Chen or Dr. Rod Wing (Jinfeng Chen et al.,2013). This enzyme has transferase activity, transfer-ring acyl groups other than amino-acyl groups. In thisstudy, homologs of 3 keto acyl CoA synthase sequenceof P . Trichocarpa x P.deltoides are predicted usinghomology search, these are similar 3-ketoacyl-synthasesynthase family proteins in Populus trichocarpa, Rici-nus communis and Gosypium hirsutum with similarityof more than 90% similarity. Uncharacterised proteinshomologs were also obtained, which are focus of ourstudy. Herein we have triesd to characterise these pro-teins. Conserved domain analysis, protein functionalanalysis followed by modelling and structural alignmentapproaches are used to predict role of unchracterisedproteins in Vitis vinifera and Oryza brachyantha. Pro-tein functional analysis was done using fold libraryand structure-based alignments for homologous proteinfamilies. This study provides functional annotation of3 keto acyl CoA synthase protein; A9PIU6, F6HH63,J3MFI6 and J3M9Z7 in T . Trichocarpa x T.deltoides,Vitis vinifera and Oryza brachyantha by Comparativedomain analysis based on sequence- sequence and se-quence structure comparison. Activity of these pro-teins has been characterized on the basis of structuralsimilarities and variations. Domains Germicidin syn-thase (streptomyces coelicolor) and Chalcone-stilbenesynthases (KAS) in 2-pyrone synthase (Gerbera hybrid)are analysed to predict role of protein in anthocyaninproduction and narigenin synthesis activity. Other do-mains, CHS 2 (Medicago sativa) B ketoacylsynthaseIII (Streptomyces sp.r1128 ). 3-oxoacyl-ACP synthaseIII (Mycobacterium tuberculosis), 3-oxoacyl-ACP syn-thase (Thermus thermophilus), β keto acyl-acyl carrierprotein synthase III (Escherichia coli) are also anal-ysed. Finally structural variations among dimer inter-face, product binding sites and malony CoA binding

sites are analysed on the basis of amino acids positionsin sequence and structure. Differences between C andN terminal domain are considered to analyse the func-tional variation. All proteins were found to be relatedto Chalcone and stilbene synthases; plant-specific PKSand related enzymes, also called type III PKS whichindicate the presence of cd00831domain.

2 Materials and Methods

2.1 Protein sequences of keto acyl CoA syn-thase were retrieved from NCBI ENTREZ database(www.ncbi.nlm.nih.gov). An isolated gene from Pop-ulus trichocarpa x Populus deltoides exposed to con-tinuous feeding by Malacosoma disstria Hubner (foresttent caterpillar) mid-instar larvae was characterised byits product 3 ketoacyl synthase. 3-ketoacyl-CoA syn-thase in Populus trichocarpa x P.deltoides (S Kawaiet al. 1996). This protein was taken as model pro-tein to perform local and global alignment. Proteinsequences and annotation of homologs of 3 keto-acylCoASynthase (EF148325) were retrieved from Uniprotdatabases (www.uniprot.org/help/uniprotkb).2.2 Uncharacterised proteins were obtained from re-sult of Homology search of 3 keto acyl CoA syn-thase using BLAST. Protein annotation of sequenceof Oryza brachyantha was obtained from Ensemble-plants database (www.plants.ensembl.org).Transcriptid OB09G23410.1, protein id OB09G23410.1 annota-tion is given here-Ave. residue weight: 109.221 g/mol,Charge: 19.5, Isoelectric point: 9.2732, location -Chromosome 9: 12, 008, 506-12, 010, 333 forwardstrand, Number of residues: 483 aa, (Jinfeng Chen,Mingsheng Chen et al., 2013). Four entries were re-lated to acyl-tansferase.2.3 Global alignment of Target sequence was doneto predict its homologs using FASTx program(www.ebi.ac.uk/Tools/sss/fasta/). Gap extensionpenalty and open gap penalty of −2 and −10 for pro-tein sequence are given respectively as negative score;total 50 related sequence of EF148325 (KCS from Pop-ulus hybrid) were predicted. For global sequence align-ment, matrix was based on Needleman wunsch algo-rithm (Needleman et al. 1970).2.4 Domain search analysis and Pair wise alignment(F. Smith et al. 1981) was performed using BLAST2.2.25 (Altschul et al. 1997) and Local alignment wasalso performed using WU BLAST to get homologs of 3-ketoacyl-CoA synthase of P . Trichocarpa x P.deltoides.CDD CDART: (www.ncbi.nlm.nih.gov/Structure/),conserved Domain Architecture Retrieval Tool Program(Marchler-Bauer A et al., 2013). The E-value corre-sponding to a given bit score is calculated

E = mn2−5

where m and n are sequence length and s is bit score,

Interdiscip Sci Comput Life Sci (2014) 6: 1–14 3

It must attain the score x twice in a row for an HSPto get the score 2x, so E value decrease exponentiallywith score.2.5 Protein Functional Analysis (Quevillon E. etal. 2005) using the InterProScan program (http://www.ebi.ac.uk/Tools/services/web iprscan) was per-formed to provide structural view of domains on proteinsequence. InterProScan (E.M. Zdobnov et al. 2001) isa tool that combines different protein signature recog-nition methods from the InterPro.2.6 Sequences were searched against fold library usingenvironment-specific, substitution tables and structure-dependent gap penalties on web based FUGUE(v2.s.07) http://tardis.nibio.go.jp/result/fugue/21367/fugue.html.Fold library and substitution tables arebased on the HOMSTRAD database. (Shi J,, et al.2001). HOMSTRAD (HOMologous STRucture Align-ment Database) (http://tardis. nibio.go.jp/homstrad/)(Mizuguchi K et al. 1998) is a curated databaseof structure-based alignments for homologous proteinfamilies. Sequences of representative members of eachfamily were aligned on the basis of their 3D struc-tures using 3D structure of representative which useprograms MNYFIT, STAMP and COMPARER.2.7 Biological Annotation was retrieved from PDBedatabase http://www.ebi.ac.uk/pdbe-srv/view/entry/1mzj/summary.html via structural biology knowledge-base database. Protein databank (http://www.rcsb.org/pdb/explore/) was used to retrieve protein struc-tures in which domains were predicted.2.8 Geno3D (http://geno3d-pbil.ibcp.fr) was used formodelling uncharacterised protein. Modelling pro-tein was done using template Template pdb1cgzAand pdb3tsyA were used for modelling J3M9Z7 andF6HH63 protein (Combet C., 2002). Comparative(“homology”) modelling approximates the 3D structureof a target protein for which only the sequence is avail-able, provided an empirical 3D “template” structureis available with >30% sequence identity (i) Determi-nation of structurally conserved region (SCRs). (ii)Alignment of amino acid sequence of the unknown pro-tein with those of the reference protein(s) within theSCRs. (iii) Assignment of coordinates in the conservedregions. (iv) Prediction of conformations for the restof the protein chain, including loops. (v) Searchingfor the optimum side chain conformations for residuesthat differ from those in the reference protein. (vi)Energy minimization and molecular dynamics to re-fine the molecular structure so that steric introducedduring the (vii) Model building process can be relieved(Baker et al. 2001; Chothia et al. 1986 and Vitkup etal. 2001. Model of J3M9Z7 protein shows core region(66.4%), allowed region (24.1%), generously allowed re-gions (5.8%), Model energy was −15505.70 K/cal/moleand minimum disallowed region was 3.8%. disallowedregion increases (more than 4.1%-4.9%, if we further

decrease the energy. Therefore we have chosen modelwith −15505.70 kcal/mole energy and 3.8% disallowedregion, which was minimum among all conformations.

Model of F6HH63 shows core region (62%), allowedregion (29.12%), generously allowed regions (5.8%), ifdisallowed regions are kept minimum 2.3 %, and thenenergy of model −15594.60%. If we further decreaseenergy up to −15787.20 Kcal/mole, then disallowed re-gion slightly increases by 0.3% that is 2.6% which isminor difference and we do not prefer amino acids offunctional sites to fall in disallowed region, thereforewe have chosen this second model.

Protein J3MF16 and A9PIU6 9ROSI couldnot be modelled using Geno 3D (Fig. 6(a)),therefore modelling was also done by Swiss pdb(http://swissmodel.expasy.org) modelling tool (ArnoldK et al., 2006; Schwede T et al., 2003; Guex, N et al.,1997). It uses z score and qmean score to evaluatemodels, two models were obtained long and short.From the point of view of covering all functionalsites in structure, only long model was used forvisualisation. For J3MF16 protein, 1qlvA templatewas used with 17.98 identity and 2.10 A distance.For this model qmean z score was −4.73 and e valuewas 0.00e-1. Template 1tedA was used to modelA9PIU6 protein, which shows 17.05% identity and2.25 A distance. Qmean z score was −5.36 and evalue was 0.00e-1 for this protein. Although Qmeanz is very low score for both structures and does notshow good quality, but modelled structure of theseJ3MF16 and A9PIU6s protein have covered 106 to488 and 114 to 496 amino acid residues respectivelyand we have used this to show distant sites, shortrange models were not taken into consideration. TheQMEAN4 score is a composite score consisting ofa linear combination of 4 statistical potential terms(estimated model reliability between 0-1) (Fig. 6(b)). These four terms are C β interaction energy,All-atom pairwise energy, Solvation energy, Torsionangle energy (Benkert P, Biasini M, Schwede T. 2011).Pymol was used for visualisation of structure model.Pymol Visualisation tool (http://www.pymol.org) isan open-source molecular visualisation system createdby Warren Lyford DeLano2.9 Structure-structure alignment was done usingMatchalign programme of chimera. Molecular graphicsand analyses were performed with the UCSF Chimerapackage. Chimera is developed by the Resource for Bio-computing, Visualization, and Informatics at the Uni-versity of California, San Francisco (Pettersen et al.2004). Matchalign align amino acids in two structuresand calculate RMSD value. Structure of putative PKSand 2 pyrone synthase are used as refrence structureand aligned with modelled structure of 3 keto acyl syn-thase like protein in Vitis vinfera and Oryza brachyan-tha.


Vitis viniferaF6HH63_VITVOryza brachyanthaJ3MF16_ORYBROryza brachyanthaJ3M9Z7_ORYBR

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Fig. 1 Graph is generated using bit score of domain sim-ilarity search for all protein domains present inthree proteins, shown on the right hand. ProteinJ3M9Z7 ORYBR shows lowest bit score range forall predicted domains.

3 Results and discussion

3.1 Domain analysis using domain profiling

Several domains very long 3 keto acyl CoA syn-thase, FAE1/Type III PKS-like protein (IPR013601),Thiolase-like, subgroup (IPR016038), Thiolase-like(IPR016039) and 3-Oxoacyl-(ACP) synthase III C-terminal (IPR013747) have been predicted in 3 ketoacyl CoA synthase of Populus trichocarpa x Populusdeltoides and its homologs which were obtained fromsimilarity search based on local alignment. Sequencesimilarity search revealed three uncharacterized 3 ketoacyl CoA synthase like protein, one protein F6HH63is from Vitis vinifera and other two proteins (J3MFI6,J3M9Z7) are from Oryza brachyantha. These three pro-teins do not show the presence of all domains. Very long3 keto acyl CoA synthase is found in all other fourtyseven (47) orthologs obtained from blast search. Secondsimilar domain is FAE1/Type III PKS-like proteins,this domain is found in 3-Ketoacyl-CoA synthases, typeIII PKS, fatty acid elongases and fatty acid condens-ing enzymes and are found in both prokaryotic and eu-karyotic species. This domain is mainly found in plantspecies.

Third similar domain is Thiolase-like, subgroup(IPR016038) and Thiolase-like (IPR016039) that aresubgroup of thiolase-like domains and usually occursin two similar copies that are arisen through duplica-tion. Such proteins are related to thiolase and chalconesynthase. The thiolase-like enzymes include thiolase,

B-ketoacyl-ACP synthases types I and Actinorhodinpolyketide β-keto acyl synthases 1 and 2. Thiolase-like (IPR016039) also include fatty oxidation com-plex β subunit (3-ketoacyl-CoA thiolase additionally.The CHS-like enzymes include CHS, Ketoacyl-ACPsynthase III (FabH); PKS, 3-hydroxy-3-methylglutarylCoA synthase, Dihydropinosylvin synthase. Third sim-ilar domain is 3-Oxoacyl-ACP synthase III C-terminal.This domain is found on 3-Oxoacyl-(ACP) synthase III,the enzyme responsible for initiating the chain of reac-tions of the fatty acid synthase in plants and bacteria.

3.2 Conserved domain prediction

Conserved domain prediction was performed usingCDD tool to analyse all conserved domains. Twentyfive (25) domains were predicted (Table 1), Chalconeand stilbene synthases; plant-specific PKS is also foundin 2-Pyrone Synthase complexed with acetoacetyl-Coain Gerbera hybrid cultivar and β keto acyl carrier pro-tein in E.coli. FAE1/Type III PKS-like protein is foundin Brassica napus (rape) as 3 keto acyl synthase. Simi-lar domain putative very-long-chain fatty acid condens-ing enzyme is found in CUT1 of Oryza sativa japonica;Ketoacyl-ACP synthase III (KASIII) initiates the elon-gation in type II “initiating” condensing enzymes are asubclass of decarboxylating condensing enzymes. Mem-bers of this family are described as 3-ketoacyl-CoA syn-thase and similar domain is found in B-Ketoacyl- ACPSynthase Iii (Fabh) of Escherichia coli. 3-Oxoacyl-(ACP) synthase III C terminal Chain B, Priming B-Keto acyl synthase from Polyketide Biosynthetic Path-way of Streptomyces sp. R1128. Chalcone and stilbenesynthases, C-terminal domain; this domain of CHS isreported to be structurally similar to domains in thi-olase and β-ketoacyl synthase. These are related tofatty acid synthesis pathway. Similar domain is foundin Pyrone Synthase (Pys) from Gerbera Hybrida.

Conserved domain prediction reveals domainsPLN02854, PLN02377 and PLN02192 with more sig-nificant negative e value of 0e-00 in Oryza brachyan-tha in comparison to positive e value of 0e+00 inVitis vinifera. Domains PLN00415, PLN02932 havebeen predicted with negative e value of 0e-00 inthese three proteins. These domains are 3 namedas 3 keto acyl CoA synthase. Negative e values de-fine significant alignment, in these protein, this in-dicate partial presence of domain 3 keto acyl CoAsynthase. Other domains have also been predicted,out of these PLN03169 are not characterised (Table1). Out of twenty five domains (25) domains 19 do-mains are common in KCS (J3M9Z7 and J3MFI6)of Vitis vinifera and Oryza brachyantha. (Table 1)Presence and absence of other six domains are ob-served in these organisms. Chalcone & stilbene syn-thase, C- terminal domain is absent in Vitis vinifera(F6HH63 VITVI) and present in Oryza brachayan-


Table 1 Information obtained from Uniprot databse

Uiprot id/

accession numberName of protein Organism

A9PIU6 9ROSI 3 keto acyl CoA synthase P . Trichocarpa X P.deltoides

F6HH63 VITVI Putative uncharacterized protein OS=Vitis vinifera (Orderedlocus name-VIT 11s0016g04700)

Vitis vinifera

J3MFI6 ORYBR Recommended name-3 keto acyl CoA synthase encoded byOB06G27800 Uncharacterized protein

Oryza brachyantha

J3M9Z7 ORYBR encoded by OB05G34210 uncharacterized protein Oryza brachyantha

tha (J3M9Z7 and J3MFI6). 3 oxoacyl synthase IIIis absent in Oryza brachyantha (J3MFI6 ORYBR)but present in protein (J3M9Z7 ORYBR) of Oryzabrachyantha and Vitis vinifera. Chalcone and stilbenesynthase and N-terminal domain is absent in Oryzabrachyantha (J3M9Z7 ORYBR) and Vitis vinifera(F6HH63 VITVI) but present in Oryza brachyantha(J3MFI6 ORYBR). Chalcone and stillbene synthase ispresent in Vitis vinifera and absent in Oryza brachyan-tha. These are the critical differences of this proteinamong these plant organisms. Presence of N and Cterminal domain is related to biochemical activity inpathway leads to the synthesis of Chalcone stilbene syn-thase. C terminal domain is present in three proteins.It is clear from this study that protein J3MFI6 ORYBRcontain both N and C terminal domain of chalconeand stilbene synthase, thus it is confirmed that thisprotein has functionality of Chalcone and stilbene syn-thase. The C-terminal domain of Chalcone synthase isreported to be structurally similar to domains in thio-lase and β-ketoacyl synthase. All three proteins containC terminal domain which share similar structure withdomain of thiolase and β keto acyl synthase. Differencesin N-terminal domain is responsible for distinguish ac-tivity. N terminal domain is absent in J3M9Z7 proteinof Oryza brachyantha and F6HH63 of Vitis vinifera.Activity of these proteins can be characterised on thebasis of these feature. The protein J3MF16 of Oryzabrachyantha contains N terminal domain and C termi-nal domain and thus can be characterised using anno-tation of these domains but modelling of this proteinwas difficult because of low sequence identity (17.98)with template protein. Several uncharacterised domainPRK09352, PLN03172 and PLN02854 are present inboth varieties of Oryza brachyantha and Vitis vinifera,allthough PRK05963 uncharacterised domain is ab-sent in Oryza brachyantha (J3MFI6 ORYBR) and Vi-tis vinifera but present in J3M9Z7 of Oryza brachyan-tha. PRK05963 uncharacterised domain absent inJ3MFI6 of Oryza brachyanta and Vitis vinifera. Ensm-bleplants database entry shows molecular function ofOryza brrachyantha is transferase, catalytic and trans-ferring acyl groups and annotation source is UniPro-

tKB/TrEMBL: A6MCZ1 9ORYZ.PLN02854, PLN02377, PLN02932, PLN00415 do-

mains present in 3-ketoacyl-CoA synthase enzymewhich are found in bryophytes and pteridophytesplants, other pln domains are related to chalcone syn-thase like protein (Fig. 2). Other predicted domainsare from Populus hybrids which are related to flavonoidpathway.

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J3MFI6_ORYBR (Oryza brachyantha)

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F6HH63_VITVI (Vitis vinifera)

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Fig. 2 Local alignment using NCBI-BLAST: Similarityscore of homologs in various organisms are depicted.

3.2.1 Sequence homology analysis of 3 ketoacyl CoA synthase in Oryza brachyantha(J3M9Z7), Oryza brachyanta (J3MFI6)and Vitis vinifera (F6HH63)

J3MFI6 protein of Oryza brachyanta shows thatit is more similar to hypothetical protein OsI 23590(EAZ01557.1) Oryza sativa Indica Group and Oryzasativa Japonica Group) hypothetical protein SOR-BIDRAFT 10g023290 (Sorghum bicolor) share 94%(100% query coverage) and 96% (96% query coverage)similarity. Twenty protein entries were named as 3 ketoacyl CoA synthase. Out of these, eleven (11) 3 ketoacyl CoA synthase proteins are from Sorghum bicolor.Nine entries were searched from Glycine max (Fig. 2).


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J3MFI6_ORYBR (Oryza brachyantha)J3MFI6_ORYBR (Oryza brachyantha)

Fig. 3 All homologs of 3 keto acyl CoA synthase are shown in various organisms.

This shows that J3MF16 protein of Oryza brachyanthais highly similar to 3 keto acyl synthase of Sorghum bi-color. All though similarity to KCS of Oryza sativa In-dica, Oryza sativa japonica is not considered significantbecause Oryza brachyantha is originated from Oryza.

Total 32 homologs were predicted in 3 keto acyl CoAsynthase (J3M9Z7) of Oryza brachyantha in differentorganism (see Fig. 2, 3 for more description); Vitisvinifera (72.6%) similarity, Glycine max (79.0%), Cu-cumis sativus (76%).

Total thirty one (31) 3 keto acyl CoA synthase pro-tein of Putative uncharacterized protein Vitis viniferawere predicted in various organisms with differentsimilarity. These are highly similar; Glycine max(77.7%), Vitis vinifera (86.4%) and Cocumis sativus(79.6%). Brachypodium distachyon (81%), Medicagotruncatula, Camellia oleifera (85%). Maximum ho-mologs of 3 keto acyl CoA synthase of Vitis viniferaand Oryza brachyantha were predicted in Glycine max,Vitis vinifera and Cucumus sativus. More homologsof Oryza brachyantha in Brachypodium distachyon arepredicted than other organism (Fig. 2). Similarityis higher in protein of Medicago truncatula, Camelliaoleifera (85%) and Vitis vinifera (86.4).

3.3 Domain prediction using sequence struc-ture comparison

Various domains were predicted in P . Trichocarpa xP.deltoides, Vitis viinfera and Oryza brachyantha bysequence structure comparison. Sequence structureswere aligned for each domain. All four protein showssimilarity to Gcs (hs3v7ia) and a type III PKS pu-tative PKS from Streptomyces coelicolor with 14.23,14.08, 13.15, 13.23 score. Chalcone and stilbene syn-

thases (KAS) was predicted in 3 keto acyl CoA syn-thase like protein of P . Trichocarpa x P.deltoides,Vitis viinfera, Oryza brachyanta with 13.08, 13.93,17.74 16.02 score respectively. These hits are 2-pyrone synthase (Gerbera hybrid) chalcone synthase2 (Medicago sativa), B-ketoacylsynthase III (Strepto-myces sp.r1128 ), 3-oxoacyl-[ACP] synthase III (My-cobacterium tuberculosis) 3-oxoacyl-[acyl-carrier pro-tein] synthase (Thermus thermophilus), B-ketoacyl-ACP synthase III (Escherichia coli).

Other domain hs2f82a profile hits from 3-hydroxy-3-methylglutaryl CoA synthases of Brassica juncea Bras-sica juncea were also predicted in Vitis vinifera, popu-lus hybrids and Oryza brachyantha with 4.01, 4.25, 4.25score. This enzyme is involved in isoprenoid biosyn-thetic process. Amino acids tyrosine, arginine, lysine,serine, asparagines, cystiene and aspartate are involvedin hydrogen bond formation.

3.4 Functional characterisation

Structure and functional characterisation of proteinswas done using their structure alignment with similarcharacterised proteins, obtained from sequence struc-ture alignment using fold library. All protein domainsrepresent transferase activity which is predicted in 3keto acyl CoA synthase like protein of Vitis viniferaand Oryza brachyantha (Table 2). Gcs from Strepto-myces coelicolor is a type III PKS with broad substrateflexibility. Chemler, J.A. and Buchholz, T.J., 2012 pro-vide unanticipated information about the interactionsbetween type III PKSs and ACPs that will facilitate en-gineering of novel polyketides. The Gcs (3v7i) structurecontains an unusual helical bundle of unknown functionthat appears to extend the dimerization interface. A


Table 2 Conserved domain prediction: All conserved domains present in three uncharacterised protein inOryza brachyantha and Vitis vinifera

Domain Name Domain DescriptionOryza brachyantha

J3M9Z7 ORYBR

Oryza brachyantha

J3MFI6 ORYBR

Vitis vinifera

F6HH63 VITV

CHS like Chalcone and stilbene synthases; plant-specificPKS

2.97e-124 5.51e-129 4.14e-128

FAE1 CUT1 RppA FAE1/Type III PKS-like protein; The mem-bers of this family are described as 3-ketoacyl-CoA synthases

0e-00 0e-00 0e+00

ACP syn III C 3-Oxoacyl-ACP synthase III C terminal; Thisdomain is found on 3-Oxoacyl-ACP

4.66e-11 3.27e-08 4.51e-09

KAS III Ketoacyl-ACP synthase III (KASIII) initiatesthe elongation in type II

4.54e-15 5.29e-17 2.02e-17

Init cond enzymes “initiating” condensing enzymes are a subclassof decarboxylating condensing enzymes

1.19e-11 2.63e-15 1.40e-14

Chal sti synt C Chalcone and stilbene synthases, C-terminaldomain;

3.14e-07 2.56e-05 1.19e-06

Ketoacyl-synt su-perfamily

Ketoacyl-synthase superfamily 4.54e-15 5.29e-17 2.02e-17

Cond enzymessuperfamily

Condensing enzymes 1.19e-11 2.63e-15 1.40e-14

PLN02192,PLN02854,PLN02377

3-ketoacyl-CoA synthase 0e-00 0e-00 0e+00

PLN02932,PLN00415

3-ketoacyl-CoA synthase 0e-00 0e-00 0e-00

BcsA Predicted naringenin-CHS [Secondarymetabolites biosynthesis, transport]

7.90e-26 3.00e-26 8.39e-26

FabH 3-oxoacyl-[acyl-carrier-protein] 6.38e-16 3.57e-18 2.35e-11

PRK12879 3-oxoacyl-(acyl carrier protein) synthase III;Reviewed

2.16e-03 1.12e-04 4.10e-07

PLNO3169 Not characterized 7.09e-08 3.77e-11 3.77e-11

PRK09352 3-oxoacyl-(acyl carrier protein) synthase III;Reviewed

1.99e-06 1.14e-04 8.39e-05

PLN03172 CHS family protein; Provisional 1.29e-05 1.52e-07 4.10e-07

PLN03168 chalcone synthase; Provisional 1.49e-05 1.71e-05 1.06e-04

PLN02326 3-oxoacyl-[acyl-carrier-protein] synthase III 3.14e-03 Absent 2.91e-04

PRK05963 3-oxoacyl-(acyl carrier protein) synthase II;Reviewed

7.24e-03 Absent Absent

Chal sti synt N Chalcone and stilbene synthases, N-terminaldomain; This domain of chalcone

Absent 2.56e-05 Absent

PLNO3171 CHS-like protein; Provisional Absent 1.44e-06 3.90e-07

Chal sti synt Chalcone and stilbene synthases Absent Absent 1.19e-06

pair of arginine residues adjacent to the active site af-fect catalytic activity but not ACP binding. (Chemler,J.A., Buchholz, T.J., 2012).

PKSs generate molecular diversity by utilizing dif-ferent starter molecules for the biosynthesis of anti-microbial phytoalexins, anthocyanin floral pigments,and inducers of Rhizobium nodulation genes. Similar-ity of 3 keto acyl CoA synthase like protein of Vitisvinifera and Oryza brachyantha to Pyrone synthase and

CHS share a common three-dimensional fold, a set ofconserved catalytic residues, and similar CoA bindingsites, but four positions are varied in CHS (Jez, J.M.,Austin, M.B., 2001). KAS 2-pyrone synthase (1EE0)from gerbera hybrid provides information about its pos-sibility to involve in proanthocyanidine production. Bi-ological annotation from databases reveals anthocyaninbiosynthetic process which may be related to proantho-cyanidine production because sequence is obtained from


Populus hybrid exposed to forest tent caterpillar.CHS (1i88) synthesizes a tetraketide by sequential

condensation of three acetyl anions which is derivedfrom malonyl-CoA decarboxylation to a p-coumaroylmoiety attached (Jez, J.M., Bowman, M.E., 2001) Simi-larity to KAS chalcone synthase 2 from Medicago sativareveal role of these 3 keto acyl CoA synthase like proteinof Vitis vinifera and Oryza brachyantha in Naringenineand CHS like activity.

Similarity to KAS-B-ketoacylsynthase III from Strep-tomyces sp.r1128 and KAS oxoacyl-ACP synthase fromThermus thermophilus reveal, role of these 3 keto acylCoA synthase like protein of Vitis vinifera and Oryzabrachyantha in B-ketoacyl-ACP synthase III activityand 3-oxoacyl-ACP synthase activity. B-keto acyl syn-thase III from Streptomyces sp.r1128 reveals an exten-sive loop region at the dimer interface that appears toaffect the selectivity for the primer unit. (Pan, H., Tsai,S., 2002)

Additionally, Similarity to KAS, 3-oxoacyl-ACP syn-thase III Mycobacterium tuberculosis give hypotheticalview of activities; fatty-acyl-coa binding, Catalytic ac-tivity, 3-oxoacyl-ACP synthase activity, Protein bind-ing and Transferase activity. This also shows theirinvolvement in protein homodimerisation. Protein ofβ-ketoacyl-ACP synthase III (mtFabH) of Mycobac-terium tuberculosis catalyze a Claisen-type condensa-tion (Scarsdale, J.N., Kazanina, G, 2001) B-ketoacyl-ACP synthase III (1HNJ) of E. coli also share KASdomain with these protein.

Protein J3MF16 of Oryza brachyantha has 17.74

score in sequence structure alignment which is highest.Presumably, this protein has activities which are dis-cussed above. Its similarity to KAS 2-pyrone synthase(1EE0) from Gerbera hybrid gives important clue aboutits possibility to involve in proanthocyanidine produc-tion. Allthough other three protein of P . Trichocarpax P.deltoides, Vitis viinfera, Oryza brachyantha alsocontain Chalcone and stilbene synthases (KAS) with13.08, 13.93, 16.02 score respectively. All similar struc-tures has transferase activity, transferring acyl groups,transferring acyl groups other than amino-acyl groups(Fig. 4, 5).3.4.1 Structural similarities and variations

among active site, dimer interface, prod-uct binding sites and malonyl CoA bind-ing site in 3 keto acyl CoA synthase ofVitis vinifera and Oryza brachyantha

Structure obtained from homology modelling of theseproteins were used for predictions of these sites andthier actual positions in the structure. Models wereevaluated on the basis of minimum energy and mini-mum disallowed regions in ramachandran plot (Fig. 6).Amino acids involved in fatty acid synthesis are il-lustrated, starter molecule is loaded onto the activesite cysteine, a carboxylative condensation reaction ex-tends the polyketide chain which incorporated intoplant lipids are derived from the iterative addition ofC2 units. Malonyl-CoA provide C2 unit to an acyl-CoAby the 3-ketoacyl-CoA synthase (KCS). Several malonylCoA binding site have been predicted, these are I-229,DQ-232-233, F-392, V-451, GRA-515-517, these are not

Fig. 4 β-ketoacyl-ACP synthase, type III and PKS: B-Ketoacyl-ACP Synthase III (Fabh) from Escherichia Coli (1EBL A),2-Pyrone Synthase(1 EE0 A) Complexed With Acetoacetyl-Coa from Gerbera hybrid cultivar and other relatedprotein sequences are aligned with active site in yellow colour (number denotes gap between amino acids residues).


Fig. 5 Chalcone and stilbene synthases domain: Conserved and aligned amino acids are shown in various color according tosecondary structure (helix-red; sheet-blue; 310 helix-maroon; solvent accessible lower case; solvent inaccessible-uppercase, H- bond-main chain –amide-bold; H- bond-main chain carbonyl-underline; disulfide bond-cedilla, positive φtorsion angle-positive) depicted in 2-pyrone synthase (1ee0a; Gerbera hybrid) CHS 2 (1i88a; Medicago sativa), β-ketoacyl synthase III(1mzj; Streptomyces sp. r1128, 3-oxoacyl-ACP synthase III (1hzp; Mycobacterium tuberculosis)3-oxoacyl-[ACP] synthase (1ub7; Thermus thermophilus) β -ketoacyl ACP synthase III (1hnj; Escherichia coli).

16000

14000

12000

10000

8000

6000

4000

2000

0

Energy(kcal/mole)F6HH63_VITVI

Energy(kcal/mole)J3M9Z7_ORYBR

1 2 3 4 5 6 7 8 9 10Model

(a)

1.5

1.0

0.5

0

0.5

1.0

1.5

Nor

mal

ised

QM

EA

N4

scor

e

0 100

Comparison withnon-redundant set of PDB structures

200 300

(b) (c)

400

|Z-score|<1

1<|Z-score|<2

|Z-score|>2

Query model

500 600Protein size

Z-score QMEAN: 4.73Z-score Cbeta: 1.84Z-score all_atom: 3.52Z-score solvation: 1.08Z-score torsion: 3.82

1.5

1.0

0.5

0

0.5

1.0

1.5

Nor

mal

ised

QM

EA

N4

scor

e

0 100

Comparison withnon-redundant set of PDB structures

200 300 400

|Z-score|<1

1<|Z-score|<2

|Z-score|>2

Query model

500 600Protein size

Z-score QMEAN: 4.73Z-score Cbeta: 1.84Z-score all_atom: 3.52Z-score solvation: 1.08Z-score torsion: 3.82

|Z-score|<1

1<|Z-score|<2

|Z-score|>2

Query model

score QMEAN: 4.73score Cbeta: 1.84score all_atom: 3.52score solvation: 1.08score torsion: 3.82

|Z-score|<1

1<|Z-score|<2

|Z-score|>2

Query model

score QMEAN: 4.73score Cbeta: 1.84score all_atom: 3.52score solvation: 1.08score torsion: 3.82

Fig. 6 (a) Histogram depicting energy (-Kcal/mole) used for evaluation of modelled structures of Z3M9Z7 ORYBR andF6HH63 VITVI. (b) Z score was calculated using C β interaction energy, all atom pairwise energy, salvation energyand torsion angle energy for J3MF16 ORYBR model. (c) Then composite Qmean score was calculated using linearcombination of these four zscore (shown in different colour).


(a) J3M9Z7_Oryza brachyantha (b) J3MF16_Oryza brachyantha

Structural variations among:Dimer interfaceProduct binding siteMalonyl CoA binding site

(c) F6HH63_Vitis vinifera

Fig. 7 Modeled structure of F6HH63 VITVI and J3M9Z7 ORYBR: Structural variation predicted among dimer interface(green), product binding site (blue) and malonyl Co-A binding sites (light orange) are shown in 3 keto acyl CoAsynthase like proteins that are vary with each other.

identical to 2 pyrone synthase (1ee0) but these aminoacids have similar properties. The GRA site is absent inprotein J3M9Z7 ORYBR. Other maolonyl CoA bindingsite in this protein are F-288, V-259, R-32, and N-35andC-36 (Fig. 7).

V-451 and GRA 515-517 is also predicted in KCS likeprotein J3MFI6 ORYBR at 414-416 and 353 positionsrespectively, other site are RC-131, T-128, F291, V-353, and GRA-414 (Table 3). This analysis providesannotation of biologically active sites.

In J3MF16, LR is present and LN is present in othertwo proteins F6HH63 VITVI and J3M9Z7 ORYBR.GNIISYNL is present in F6HH63 VITVI . GNV, ISYNand GNV, VSYN are present in J3MF16 ORYBRand J3M9Z7 ORYBR proteins. QVH is common inF6HH63 VITVI and J3MF16 ORYBR.

Product binding site in F6HH63 VITVI consist of

R, VGV, GV, which shows slight variations withJ3MFI6 ORYBR protein at sites IGV, AV. In place of‘V’, ‘I’ is present and A is present in place of G and R isabsent. In J3M9Z7 ORYBR protein R, TGV, AV, RFwere predicted as product binding sites. T is present inplace of V/I.

Mutated Malonyl CoA binding sites inF6HH63 VITVI are I, DQ, GRA and in J3MFI6ORYBR In other proteins, these sites are RC, TRA,GRA. GRA is absent in J3M9Z7. Malonyl CoAbinding sites in J3M9Z7 ORYBR) are R, N, Cwhich are different from other two proteins (Table4).

When biological active sites of three protein werecompared with A9PIU6 9ROSI protein of Populus tri-chocarpa. All the active site and product binding sitesare common in J3MFI6 ORYBR and J3M9Z7 ORYBR


Table 3 Results on structure sequence alignment: Domain related to fatty acid synthesis pathway are shownbelow, these domain are predicted in KCS like protein of Vitis vinifera and Oryza brachyantha(J3MFI6 ORYBR and J3M9Z7 ORYBR) with 13.08, 17.74 and 15.81 z score. For putative PKSz score is 14.23 which s only predicted in Vitis vinifera. Structural & functional annotation isobtained from PDB and PDBe database

S.N Profile hit/PDB Code(HOMSTRAD)

Source Name Functional characterization (PDBe; knowl-edgebase)

1 hs3v7ia putativepolyketide synthase(3v7i) Germicidinsynthase

Streptomycescoelicolor

Germicidinsynthase (Gcs)

Catalytic activity, transferase sctivity(Only predicted in Vitis vinifera)

2 KAS2-pyrone synthase(1ee0)

Gerberahybrida

PKSs Catalytic activity, Transferase activity, biosyn-thesis of antimicrobial phytoalexins, antho-cyanin floral pigments

KAS CHS 2. (1i88) Medicagosativa

CHS Catalytic activity, Naringenin-chalcone syn-thase activity,

KASβ−ketoacylsynthaseIII.(1mzj)

Streptomycessp.r1128

ZhuH isa primingketosynthase

Catalytic activity, 3-oxoacyl-ACP synthase ac-tivity, B-ketoacyl-ACP synthase III activity.

KAS3-oxoacyl-ACPsynthase III. (1hzp)

Mycobacteriumtuberculosis

An usual β-ketoacyl ACP synthaseIII (mtFabH)

Fatty-acyl-coa binding, Catalytic activity, 3-oxoacyl-ACP synthase activity, Protein bind-ing, B-keto acyl-ACP synthase III activity,Protein homodimerization activity

KASoxoacyl-ACPsynthase (1ub73)

Thermusthermophilus

oxoacyl-ACPsynthase

Catalytic activity, 3-oxoacyl-ACP synthase ac-tivity, B-ketoacyl-ACP synthase III activity

KAS. (1hnj) Escherichiacoli

B−ketoacyl-acylcarrier proteinsynthase III

Catalytic activity, 3-oxoacyl-ACP synthase ac-tivity, B-ketoacyl-ACP III activity.

Table 4 Amino acids at different active site related to fatty acid synthesis are illustrated. Common sitesare shown in bold

Source Organism/Protein name

Activesite

Product bindingsite

Malonyl CoAbinding site

Dimer interface

Vitis viniferaF6HH63|F6HH63 VITVI

C-344,H-512,N-545

ENI-373-375,R-395, VGV-445-447, GV-455,S-545, S-575

I-229, DQ-232-233, F-392, V-451, GRA-515

LN-68, P-72, PT-317, A-323, N-327, GNIISYNL-333-340, MG-343-344, DL-353-354, D-357, QVH-360-362, T-420, RTH-422-424, VSLS-447-450, K-580

Oryza brachyanthatr|J3MFI6|J3MFI6 ORYBR

C-244,H-411,N-442

ENI-272, IGV-344, AV-354, S-444, S-474

RC-131, T-128F291, V-353,GRA-414

LR-167-168, P-171, PT-215-216,A221, N225, GNV-231-233, ISYN-235-238, MG-242-243, DL-252-253,D-256, QVH-259-262, T-319, RTH-321-323, VSLS-344-347, K-479

Oryza brachyanthaJ3M9Z7 ORYBR

C-240,H-407,N-447

ENI-176-178,R-198, TGV-338-340, AV -348-349,S-450, RF-345

F-288, V-259,R-32, N-35and C-36

LN-163-164, P-167, A-211, N221,GNV-227-229, VSYN-231-234, MG-238-239, DL-248-249, D252,QW-255-256, T315, RTH-317-319,VSLS-342-345, K475

and A9PIU6 9ROSI, few product binding sites and mal-ony CoA binding sites were present in F6HH63 VITVIbut not present in A9PIU6 9ROSI protein. There areno common residues in malonyl CoA binding site and

dimer interface of A9PIU6 9ROSI, J3MF16 ORYBRand J3M9Z7 proteins. Proteine F6HH63 VITVI of vitisvinifera shows common site in dimer interface and twomalonyl CoA binding site (Table 5).


(a) (b)

Fig. 8 Structure- structure alignment of modeled structure obtained from homology modeling (a) putative PKS (pdb-3v7i;gray colour) of Streptomyces coelicolor is aligned (green)with structure (shown in cornflower blue colour) of Vitisvinifera (b) Structurally aligned and conserved amino acids are shown in peach colour for Putative PKS (pdb-3v7i)of Streptomycin coelicolor, aligned with structure model of Vitis vinifera.

(a) (b)

(c) (d)Fig. 9 (a) KAS chalcone synthase 2(1i88 pdb) structure of Medicago sativa is aligned with J3M9Z7 protein structure of

Oryza brachyantha. (b) Structurally aligned and conserved amino acids are shown in peach colour for domainKAS chalcone synthase 2(1i88 pdb) structure of Medicago sativa aligned with J3M9Z7 protein structure of Oryzabrachyantha. (c) KAS 2-pyrone synthase (1ee0; gray colour) of Gerbera hybrida is aligned with J3MF16 proteinstructure model (shown in cornflower blue colour) of Oryza brachyantha. (d) Structurally aligned and conservedamino acids are shown in peach colour for KAS 2-pyrone synthase (pdb-1ee0; gray colour) of Gerbera hybrid, alignedwith J3MF16 protein structure model (shown in cornflower blue colour) of Oryza brachyantha.


Table 5 Comparative account of biologically active sites of three proteins with A9PIU6 9ROSI protein ofPopulus trichocarpa

Source Organism/

Protein name

Activesite

Productbinding site

Malonyl CoAbinding site

Dimer Interface

Populus trichocarpaA9PIU6 9ROSI

C-252H-419N-452

ENI-280, R-302, VGV-352,AV-362, S-454,S-484

T-137, ET-140,F-301, V-363,GRA-422

PR-179, M-183, PT-224, A-230, N-234,GNILSYNL-240, MG-250, DL-260, Q-264,QVH-267, C-338, CV-341, VSLS-354, K-487

Vitis viniferaF6HH63 VITVI

C-344,H-512,N-545

ENI-373-375, R-395,VGV-445-447, GV-455,S-545, S-575

I-229, DQ-232-233, F-392, V-451, GRA-515

LN-68, P-72, PT-317, A-323, N-327, GNIISYNL-333-340, MG-343-344, DL-353-354, D-357, QVH-360-362, T-420, RTH-422-424, VSLS-447-450, K-580

Oryza brachyanthaJ3MFI6 ORYBR

C-244,H-411,N-442

ENI-272,IGV-344,AV-354,S-444, S-474

RC-131, T-128F291, V-353,GRA-414

LR-167-168, P-171, PT-215-216, A221,N225, GNV-231-233, ISYN-235-238, MG-242-243, DL-252-253, D-256, QVH-259-262, T-319, RTH-321-323, VSLS-344-347,K-479

Oryza brachyanthaJ3M9Z7 ORYBR

C-240,H-407,N-447

ENI-176-178,R-198, TGV-338-340, AV-348-349, S-450, RF-345

F-288, V-259,R-32, N-35andC-36

LN-163-164, P-167, A-211, N221, GNV-227-229, VSYN-231-234, MG-238-239,DL-248-249, D252, QW-255-256, T315,RTH-317-319, VSLS-342-345, K-475

4 Conclusion

This study provides functional characterization ofthree uncharacterised protein 3 ketoacyl CoA syn-thase like protein in Vitis vinifera and Oryza brachyan-tha with 93.7% and two 3 keto acyl Co synthase inOryza brachyantha with 87.0 and 97.2 similarities to3 keto acyl CoA synthase of Populus hybrid. Se-quence analysis reveals uncharacterized proteins in Vi-tis Vinifera and Oryza brachyantha. One protein inOryza brachyantha is recommended as 3 keto acyl CoAsynthase, Although 3 keto acyl CoA synthase has beencharacterised in several plants and Oryza brachyan-tha; still there is need of potential research to func-tionally characterise of 3 keto acyl CoA synthase pro-tein in Oryza brachyantha. Conserved domains analy-sis reveals presence of pfam08392, cd00830, cd00827,COG3424, PLN03169, cd00831 which are related toflavonoid pathways. This study provides structuraland functional annotation of 3 keto acyl CoA synthaseby comparative analysis of protein sequences in plantsand provides annotation of proteins in Vitis viniferaand Oryza brachyantha. These proteins show pres-ence of Nineteen (19) domains in these three organ-isms. Protein J3MFI6 ORYBR of Oryza brachyanthashow presence of chalcone and stilbene synthase do-main. All three proteins were found to have C ter-minal domain which is similar to domain of thiolaseand β keto acyl synthase. N terminal domain is absentin J3M9Z7 protein of Oryza brachyantha and F6HH63pritein of Vitis vinifera. Differences in N-terminal

domain is responsible for distinguish activity. Max-imum homologs of 3 keto acyl CoA synthase of Vi-tis vinifera and Oryza brachyantha were predicted inGlycine max, Vitis vinifera and Cucumus sativus. KCSHomologs of Oryza brachyantha in Brachypodium dis-tachyon are more similar than other homologs. Gcs(hs3v7ia), a type III PKS putative PKS from strepto-myces coelicolor and Chalcone and stilbene synthases(KAS) from 2-pyrone synthase (Gerbera hybrid) do-main were predicted.Similarities to CHS 2 and 2 py-rone synthase suggest their role in chalcone, naringe-nine synthase activity and anthocyanin biosynthetic ac-tivity respectively. Similarities to these structures maybe valuable information for characterisation of 3 ketoacyl CoA synthase in Vitis viifera and Oryza brachyan-tha. This study confirms that both N and C terminaldomain of chalcone and stilbene synthase are present inprotein J3MFI6 ORYBR, thus it is confirmed that thisprotein perform functions of Chalcone and stilbene syn-thase. All though full domain is absent in this protein.The C-terminal domain of CHS is reported to be struc-turally similar to domains in thiolase and β ketoacylCoA synthase.

In all these sites GRA and V is present in J3MF16and F6HH63 protein at various positions of these threeplant species, but GRA is absent in J3M9Z7 Activesite amino acids Cysteine, Histidine and Asparagine areconserved in protein of three plant species. Cysteine isnecessary to load the starter molecule in 3 keto acylCoA synthase. These sites are not identical but similarto 2-pyrone synthase structure (1ee0) of Gerbera hybrid.


These sites may be modified by site directed mutagene-sis and genetic engineering approach to enhance the bi-ological activity in KCS like protein of Oryza brachyan-tha and Vitis vinifera. Molecular dynamic simulationmight be useful approach to define precisely the struc-tural and functional behavior of product binding andmalonyl CoA binding sites.

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