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Phylogenetic Based Characterization of HC Toxin Reductase from Zea mays Md. Munan Shaik, Mst. Monira Khaton Islamic University, Kushtia, Bangladesh.

ABSTRACT HC toxin produces by pathogenic fungus Cochliobolus carbonum can kill susceptible maize plants. Zea mays produces NADPH-dependent HC-toxin reductase (ZmHCTR), that can detoxify the HC toxin. ZmHCTR protein was characterized phylogenetically. A three-dimensional model of this enzyme was built and the ZmHCTR protein structure was found to belong to Rossmann fold (Nucleotide binding protein super family). The active site and the NADP binding site were characterized and similar structure in the protein data bank was identified. Members of this family of protein were identified based on (Point Accepted Mutation) PAM alignment of amino acids sequence from many distant species even in bacteria. The ZmHCTR protein was bioinformatically characterized at the molecular level. Key word: Zea mays, Helminthosporium carbonum (HC) toxin reductase, Rossmann fold, NADP. Manuscript Submitted: July 9, 2011 ______________________________________________________________________________________________________

INTRODUCTION Cochliobolus carbonum race 1 (CCR1) is a pathogenic fungus that causes leaf spot and ear mold on maize (Zea mays). It is among the most destructive pathogens of maize, whose asexual form (anamorph) is known as Helminthosporium carbonum (HC). It can kill susceptible maize plants at any stage of development (Ullstrup, 1941) and produces the HC- toxin. In fungus the synthesis of the HC-toxin is controlled by the Tox2 locus, which encodes two enzymes (HTS-1 and HTS-2) of a cyclic peptide synthetase (ScottCraig et al. 1992). This gene was cloned by transposon tagging (Johal and Briggs 1992). In response to this fungus the maize produced NADPH-dependent HC-toxin reductase (HCTR), encoded by Hm1 gene, inactivates HC-toxin produced by the fungus the thus confers resistance to the pathogen. The homologous sequence of the Hm1 gene was also found with rice, barley wheat, oats, and sorghum (Meeley and Walton, 1993). Unlike most other plant pathogens, CCR1 can invade every part of the host, causing blight of the leaves, rot of the roots and the stalk, and mold of the ear (Sindhu et al., 2008). CCR1 is strictly a pathogen of maize, but

the orthologs of Hm1 and the HC-toxin reductase activity are present in the grass family, proved that an ancient and evolutionarily conserved role of this DR trait in plants. For determination of the biological function of the Hm1 homologue HCtoxin reductase- like gene in rice (the YK1 gene) (Hayashi et al, 2005) established transgenic rice plants with ectopic expression of YK1. The YK1overexpressing rice plants showed enhanced resistance to rice blast disease and to abiotic stresses like UV radiation, increased salinity, and submergence (Uchimiya et al., 2002). NADPHdependent HC-toxin reductase is a member of nucleotide binding family of protein and shares a common catalytic and substrate binding mechanism. Anthocyanins and tannins are two important flavonoid compounds importance for plant survival and human nutrition. The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzyme dihydroflavonol 4reductase (DFR) catalyzes the last step in the biosynthesis of anthocyanins and condensed tannins. This enzyme has been widely investigated in many plant species, and the crystal structure was also solved (Petit et al., 2007) which is in fact very similar in respect of sequence similarity. But there is no crystal 13

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structure for the NADPH-dependent HC-toxin reductase (HCTR) reported until now, in the present investigation the three dimensional homology model structure of ZmHCTR built and the NADP and substrate binding site as well as the reaction mechanism was explored bioinformatically. MATERIALS AND METHODS Bioinformatics The protein sequence of Zea mays HC toxin reductase (ZmHCTR) was retrieved from NCBI database accession no. AAC04335.1. The protein properties and amino acid sequence analyses were performed using Protparam (Gasteiger et al., 2005) tools present in the ExPASy Proteomics Server. Searches for sequences similar to ZmHCTR were done with the BLAST tool available at National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov). The conserved domains in the enzymes were analyzed using the Conserved Domain Database (www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) (Marchler-Bauer et al., 2011). The presence of signal peptides sequence was predicted from Signal IP 3.0 (Bendtsen et al., 2004). Multiple Sequence alignment and Phylogenetic Analysis The sequence obtained from NCBI database was subjected to NCBI–BLAST against nonredundant protein sequence and also against PDB to extract information about suitable structural templates as well as secondary structure elements. The protein sequence of ZmHCTR was aligned using multiple sequence alignment tool COBALT present in NCBI. PDB ID: 2P4H, 2C29, 1R6D, 1Y1P and 2Z1M were selected from the blast result against PDB for further analysis. ClustalW (Li, 2003) from EMBL-EBI server was used to perform multiple alignments. Aligned sequence were edited manually and visualized by Espript (Gouet et al., 1999). Based on the alignment, phylogenetic tree was constructed according to the neighbor-joining method (Saitou and Nei, 1987) and the resulting unrooted tree was visualized as radial tree or polar tree layout with FigTree v1.3.1. program (Papadopoulos and Agarwala, 2007). The coordinates and sequence file of template protein structure (PDB ID: 2P4H; Shao et al., 2007) was obtained from the RCSB Protein Data Bank (PDB, http://www.pdb.org/pdb/home/home.do)

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Secondary structure prediction The FASTA sequences of ZmHCTR protein were used to predict the secondary structure using GOR IV (Garnier et al., 1996), SOPMA (Geourjon, C. and Deléage, 1995) and predict protein (Rost et al., 2004). Homology Modeling and Quality Validation of Models FFAS server, a profile–profile alignment and fold-recognition tool for identifying the templates for modeling and generating the alignments, was used to identify template (Rychlewski et al., 2000). The homology model of ZmHCTR was modeled using I-TASSER server (Roy et al., 2010) with selecting the template PDB option and putting all other option as default. An initial structural model was generated for ZmHCTR and subjected to energy minimization to improve the van der Waals contacts and correct the stereochemistry of the model with YASARA Energy Minimization Server (Krieger et al., 2009). The qualities of the model were checked using the SAVES server (http://nihserver.mbi.ucla.edu/SAVES_3/saves.p hp), PSQS server (http://www1.jcsg.org/psqs/psqs.cgi). Overall and local model quality was analyzed with ProSAWev server (Wiederstein and Sippl, 2007). Predicted local error, residue error, geometric inaccuracies, stereochemistry, torsion and absolute quality were evaluated by QMEAN Server (Benkert et al., 2008). The quality scores of the selected structural models (Table 1) suggested that the model of ZmHCTR was reliable. Theoretical model was visualized with graphical software PyMOL (Delano, 2008) and superimposed with the template PDB ID: 2C29 for comparison. Residues involved in the catalysis and the cofactor-binding groove were identified manually comparing the superimposed structure in the PyMOL. Structure Comparison Comparison of the three dimensional structure of ZmHCTR was made with DaliLite v. 3 (Holm and Rosenstrom, 2010). RESULT AND DISCUSSION Sequence analysis The maize Hm1 gene was the first plant disease resistance gene to be cloned and is an example of the class of plant disease resistant 14

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genes that encode a detoxifying enzyme. Sequence analysis of ZmHCTR sequence (Accession no. AAC04335.1) shows that it encodes a full-length cDNA, 1071 bp in size. The ZmHCTR ORF encodes a protein of 356 amino acid residues with calculated molecular mass of the matured protein is approximately 38.51 kDa with a predicated isoelectric point of 5.87 using Protparam (Gasteiger et al., 2005) tools present in the ExPASy Proteomics Server. The instability index (II) is computed to be 48.65; this classifies the protein as unstable. In the deduced amino acid sequence of ZmHCTR protein, total number of negatively charged residues (Asp + Glu) were 43, total number of positively charged residues (Arg + Lys) were 38. Conserved domain search with ZmHCTR sequence reveal that this protein belongs to super family cl09931, Rossmann-fold NAD (P) (+)-binding proteins; A large family of proteins that share a Rossmann-fold NAD (P) H/NAD (P) (+) binding (NADB) domain. The NADB domain containing protein is found in numerous dehydrogenases of metabolic pathways such as glycolysis, and many other redox enzymes. NAD binding involves numerous hydrogen bonds and van der Waals contacts, in particular H-bonding of residues in a turn between the first strand and the subsequent helix of the Rossmann-fold topology. Characteristically, this turn exhibits a consensusbinding pattern similar to GXGXXG, in which the first 2 glycines participate in NAD (P)binding, and the third facilitates close packing of

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the helix to the beta-strand. Typically, proteins in this family contain a second domain in addition to the NADB domain, which is responsible for specifically binding a substrate and catalyzing a particular enzymatic reaction. Homology analysis A Blast search with ZmHCTR with PDB revealed that the deduced amino acid of ZmHCTR shares a high degree of sequence homology with Vestitone Reductase from Alfalfa (Medicago Sativa, PDB: 2P4H, 33% identity), Dihydroflavonol reductase (Vitis Vinifera, PDB: 2C29, 31% identity) and Dtdp-Glucose 4, 6Dehydratase (Streptomyces Venezuelae, PDB: 1R6D, 31% identity). The red colour in the alignment region indicated that amino acid residues are highly conserved in particular with NADP binding consensus sequence GXGXXG (Figure 1). A phylogenetic tree with genetic diversity was constructed based on the amino acid sequence also indicate the high degree of sequence similarity (Figure 2). To investigate the sequence conservation of ZmHCTR in many other crop species and bacteria found that many species contain similar protein of this known disease resistance protein, even they are phylogenetically far. The conservation of the same protein may help in the near future to identify and detoxify genes in many other species and characterize them. The conservation of the HCTR gene among the grass family was already reported (Sindhu et al., 2007).

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Figure 1. Alignment of ZmHCTR amino acid sequence with similar PDB structure: PDB: 2P4H, Vestitone reductase from Alfalfa (Medicago Sativa); PDB: 2C29, Dihydroflavonol reductase (Vitis Vinifera); PDB: 1R6D, Dtdp-Glucose 4, 6Dehydratase (Streptomyces Venezuelae); PDB ID: 1Y1P, carbonyl reductase (Sporobolomyces salmonicolor); PDB: 2Z1M, Gdp-D-Mannose Dehydratase (Aquifex aeolicus). Colour coding of conserved residues are denoted by boxed red text and invariant residues by red highlight. Secondary structure elements of ZmHCTR are shown on the top.

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Figure 2. Phylogenetic tree based on the amino acid sequence, showing the phylogenic relation between ZmHCTR with a similar sequence derived from blast. Sequences were aligned by the neighbor-joining method using ClustalW. Based on the alignment, the resulting unrooted tree was visualized as a Polar tree layout. The distance scale represents evolutionary distance as indicated by the scale bar representing Point Accepted Mutation (PAM).

Secondary structure prediction The secondary structure of the protein ZmHCTR was predicted with primary sequence. This protein is supposed to fold as globular protein and the predicted secondary structure are helix 37.9%, beta strand=11.5%, loop=50.6% and reasonably no regular secondary structure (NORS) region. ZmHCTR is localized in cytoplasm, as there is no signal sequence for localization in nucleus or periplasm. Secondary Structure Elements identified from the PSVS structure validation server were 11  helices: 1(19-30), 2 (46-49), 3 (58-61), 4 (76-79), 5 (103-119), 6 (164-167), 7(176-190), 8 (255-267), 9 (285-295), 10 (316-321), 11 (329-343) and beta strands: 1 (10-13), 2 (34-

37), 3 (62-64), 4 (82-87), 5 (127-133), 6 (202-207), 7 (209-211), 8 (252-254); (amino acids residues number are in the parentheses). Homology model FFAS server find two protein structure (PDB ID: 2P4H, Petit et al., 2007; PDB ID: 2C29, Shao et al., 2007) with similar score (-103.00) as the best template for model building for ZmHCTR. Based on the automated structure alignment generated I-TASSER server, the same server was used to derive the theoretical structure of ZmHCTR using the crystal structure of Vestitone Reductase from Alfalfa (PDB code: 2P4H) of Medicago sativa the monomer as the template (Figure 3A). The EBI-DaliLite server was used to compare the model with template indicating a 17

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strong structural conservation seen among them and similarity in the structural folds. Most similar proteins structures were found VESTITONE REDUCTASE, PDB: 2P4H (Petit et al., 2007), Z-score 42.9; r.m.s.d 1.1 Å, which is infact the template of the model. Other structures similar to ZmHCTR are Dihydroflavonol reductase PDB ID: 2C29, Z-score 36.6; r.m.s.d 2.3 Å, aldehyde reductase 2 from Sporobolomyces salmonicolor PDB ID: 1UJM, Z-score 30.0; r.m.s.d 2.8 Å. Overall and local model quality was analyzed with ProSA-Wev server. Predicted local error, residue error, geometric inaccuracies, stereochemistry, torsion and absolute quality were evaluated by QMEAN Server and the value for ZmHCTR model was reasonably good when compared to that of the template structure that supports the reliability of the theoretical model (Table 1). The major segment of the ZmHCTR sequence was in good consensus with the template. Characterization of ZmHCTR active site The active site of ZmHCTR consists of the two pockets: NADP binding pocket and the substrate

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binding pocket (and is very commonly seen in almost all the members of this family of proteins. The structure was aligned to identify the active site and the catalytic residues. As the theoretical model of ZmHCTR was built based on the template 2P4H, but identification of catalytic residues failed as the crystal structure is without cofactor and/ substrate. No molecule is bound in the substrate-binding site of crystal structure Vestitone Reductase from Alfalfa (PDB code: 2P4H. A superposition of the monomer of the structure of PDB: 2C29, Dihydroflavonol Reductase (Vitis Vinifera), which has an NAD molecule bound, allows a discussion of the active site residues. The region of binding of the NADP (GxGxxG motif) and the groove for substrate binding is very well conserved, with all the side chain residues involved in some interaction present in both enzymes in the same positions (Figure 3B). There is some small change also present in the shift of the chain around the substrate binding chain; all these differences may contribute to the different substrate specificities of these enzymes.

Table 1 PROCHECK (Ratio of residues in the core region of the Ramachandran plot) Most favoured regions Additional allowed regions Generously allowed regions Disallowed regions

(257) 86.5% (36) 12.1% (1) 0.3% (3) 1.0%

Errat (Ratio of residues with an average three- to one-dimensional score 0.2)

90.789

Verify 3D (Ratio of residues with error value below the 95% rejection limit. Good high resolution structures generally produced values around 95% or higher. For lower resolution structures (2.5–3 Å), the average overall quality factor was around 91%) Prove (Ratio of residues not classified as outliers)

92.65%

PSQS (Average PSQS for PDB structures was 0.3)

-0.1514

ProSA Server (Z-Score - Overall model quality indicates by the z-score, which measures the deviation of the total energy of the structure with respect to an energy distribution derived from random conformations) Close Contacts (within 2.2 Å): Ideal Geometry (PDB validation software) RMS deviation for bond angles RMS deviation for bond lengths

94%

-8.51

0 2.6 ° 0.011 Å

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Figure 3. Cartoon view of ZmHCTR three-dimensional structure. A. ZmHCTR model showing typical rossman fold. B. ZmHCTR with NADP in the nucleotide-binding pocket.

Figure 4. Enlarged view of ZmHCTR nucleotide-binding site with the GxGxxG motif (green) coordinates with NADP (magenta).

CONCLUSION Fungal disease causes a huge loss of crop every year. Development of disease resistant variety is a top priority in agriculture biotechnology. Exploring intrinsic resistance genes is a milestone as transgenic crops still face much criticism. ZmHCTR is an intrinsic fungal disease resistance gene, that is able to detoxify fungal toxin. Selective breeding or molecular marker aided breeding can help to develop the resistant variety of maize for this fungal pathogen. On the other hand genetic transformation of this gene to other important

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