An insight into the sequential, structural and phylogenetic properties of ...

Properties of banana ACC synthase 1

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An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5′-phosphate and aminoethoxyvinylglycine SWARUP ROY CHOUDHURY1*, SANJAY KUMAR SINGH1*, SUJIT ROY2 and DIBYENDU N SENGUPTA1** 1

Department of Botany, Bose Institute, 93/1, APC Road, Kolkata 700 009 Protein Chemistry Laboratory, Department of Chemistry, Bose Institute, 93/1, A.P.C Road, Kolkata 700 009 *These authors have contributed equally to this work. **Corresponding author (Fax, +91 033 2350 6790; Email, [email protected])

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In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of S-adenosylL-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, bananaACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5′-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP. [Choudhury S R, Singh S K, Roy S and Sengupta D N 2010 An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5′-phosphate and aminoethoxyvinylglycine; J. Biosci. 35 281–294] DOI 10.1007/s12038-010-0032-4

1.

Introduction

Ethylene, a plant hormone that controls many aspects of plant growth and development, is synthesized in response to a wide range of stimuli including ripening, auxin treatment, wounding and anaerobiosis (Yang and Hoffman 1984). The ripening process in climacteric fruits is initiated by the

natural biosynthesis of endogenous ethylene as the fruit matures or by the application of exogenous ethylene (Wills et al. 2001). Ethylene binds to its receptor and subsequently initiates the ripening pathway that regulates fruit ripening. 1-Aminocyclopropane 1-carboxylic acid synthase (ACC synthase) (EC 4.4.1.14) catalyses the rate-determining step in the ethylene biosynthesis pathway. This enzyme is

Keywords. ACC synthase; aminoethoxyvinylglycine; ethylene; ligand binding; pyridoxal-5’-phosphate; S-adenosyl-L-methionine Abbreviations used: AATase, aspartate aminotransferase; ACC, 1-aminocyclopropane-1-carboxylate; AVG, aminoethoxyvinylglycine; CDD, Conserved Domain Database; DOPE, discrete optimized protein energy; MA-ACS1, Musa acuminata ACC synthase 1; MACC, N-manonyl ACC; MTA, 5’ methylthioadenosine; MVG, methoxyvinylglycine; PLP, pyridoxal-5’-phosphate; RMSD, root mean square deviation; SAM, S-adenosyl-L-methionine; TATase, tyrosine aminotransferase Supplementary figures and table pertaining to this article are available on the Journal of Biosciences Website at http://www.ias.ac.in/jbiosci/ June2010/pp281-294/suppl.pdf http://www.ias.ac.in/jbiosci

J. Biosci. 35(2), June 2010, 281–294, © Indian Academy Sciences 281 J. Biosci.of35(2), June 2010

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responsible for the conversion of S-adenosyl-L-methionine (SAM) to ACC (Kende 1989, 1993). ACC is eventually converted into ethylene, carbon dioxide and cyanide by the activity of ACC oxidase in an oxygen-dependent process (John 1991) or to N-manonyl ACC (MACC) by manonyl transferase. Positive and negative feedback regulation of ethylene biosynthesis has been analysed in different plant species (Barry et al. 2000; Nakatsuka et al. 1998). The enzyme ACC synthase was first identified from the homogenates of ripe tomato pericarp tissue (Boller et al. 1979). Since ACC synthase is a cytosolic enzyme with a very short half-life, its intracellular concentration is low and the active form is labile. The structure of ACC synthase shares a modest level of sequence similarity with other members of the family, such as aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase) (Alexander and Grierson 2002; Christen and Metzler 1985). ACC synthase has been shown to be active as a dimer or as a monomer (Satoh et al. 1993). The crystal structures of ACC synthase from apple (Capitani et al. 1999) and tomato (Huai et al. 2001) have revealed that the enzyme forms a homodimer. The dimeric form of ACC synthase utilizes pyridoxal 5′- phosphate (PLP) as a cofactor. PLP has been found in the pre-bound state at the active site of the enzyme (Mehta et al. 1993). ACC synthase is inactivated by its substrate SAM. SAM exists as diastereomeric isomers (S, S and R, S) and the R, S form is responsible for inactivation of ACS activity via a mechanism-based manner by an inappropriate β, γelimination from SAM to yield L-vinylglycine. Normal α, γelimination yields ACC and MTA (5′ methylthioadenosine) from SAM (S, S form) (Feng and Kirsch 2000). Several vinylglycine analogues (AVG or aminoethoxyvinylglycine and MVG or methoxyvinylglycine) have been shown to act as potent inhibitors of ACC synthase (Jakubowicz 2002). Besides MTA, polyamines have also been found to inhibit the enzymatic activity of ACS in an uncompetitive and noncompetitive fashion (Hyodo and Tanaka 1986). In banana as in tomato, the enzyme ACC synthase is encoded by multigene families. Recently, Huang et al. (2006) have shown the presence of many isoforms of ACS genes in the banana genome. In banana, Musa acuminata ACC synthase 1 (MA-ACS1) is encoded by a single gene and its transcript accumulates in the fruit only with the onset of ripening (Huang et al. 2006; Liu et al. 1999). Although earlier studies have demonstrated the structural features of ACC synthase from apple (Capitani et al. 1999) and tomato (Huai et al. 2001), adequate information to understand the structural features of ACC synthase in banana is still limited. Banana is now considered as one of the important model crops for studying the ripening mechanism at the molecular level. Since MA-ACS1 plays the key role in ethylene biosynthesis in banana fruit, knowledge about the structure–function relationship of the enzyme is of great J. Biosci. 35(2), June 2010

interest to gain further insight into the regulation of ethylene production for ripening in this fruit. In this study, we have illustrated the sequential, structural and phylogenetic features of banana ACC synthase 1. We have predicted the active site of MA-ACS1 in the homology model and analysed the binding sites of PLP and AVG. To our knowledge, this is probably the first detailed computational analysis of banana ACS1 and provides new interesting information about the mechanistic analysis of the enzyme. 2. 2.1

Methods Plant material

The banana cultivar Giant governor (Musa acuminata, AAA group, subgroup Cavendish) was obtained from a banana farm (West Bengal State Council of Science and Technology, India) and was grown in soil at the Bose Institute experimental field. 2.2

RNA isolation, RT-PCR and molecular cloning of banana MA-ACS1 cDNA clone

Total RNA was extracted from post-climacteric (12 days after harvest, ~92 days post anthesis) banana pulp (cultivar Giant governor, subgroup Cavendish) by modification of the SDS phenol method as described previously (Roy Choudhury et al. 2008a). RNA isolation from root, stem, leaf and peel was carried out by the Tris-borate method (Clendennen and May 1997). Reverse transcriptase (RT) reactions were carried out with the Thermoscript RT kit (Invitrogen, USA) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification was performed by using gene-specific primers (HPLC purified synthetic oligos, Sigma). Banana MA-ACS1 cDNA clone was obtained from ripe pulp tissue by RT-PCR with the gene-specific oligos (MAC1/5: 5′GCATGAGCTCATAACGGGTCACATGAGGATCTAC-3′ and MAC1/3: 5′-GTTTCAGGTGGCGGCTTGAAC-3′) designed from the public database sequence of MA-ACS1 (AB021906). The amplified cDNA was cloned in the pCR4TOPO cloning vector (Invitrogen). The cloned fragment was subsequently sequenced and submitted to the GenBank database (AY702076). The coding sequence of MA-ACS1 cDNA appeared to be a full-length cDNA of 1458 bp encoding a protein of 486 amino acid sequences (predicted molecular mass of 55 kDa). 2.3

RNA gel blot analysis

Twenty microgram of total RNA was denatured, separated on 1.2% agarose gel containing 2.2 M formaldehyde and the RNA was then transferred onto a nytran membrane according

Properties of banana ACC synthase 1 to Sambrook et al. (1989). After prehybridization for 4 h at 42ºC, hybridization was performed overnight at 42ºC in a solution containing 50% formamide, 5X SSC, 5X Denhard solution, 0.1% SDS and 100 μg/ml denatured salmon sperm DNA. 32P-labelled 1.4 kb MA-ACS1 cDNA was used as a probe. The membrane was washed twice in 2X SSC, 0.1% SDS for 15 min at room temperature followed by two washes in 0.1X SSC, 0.1% SDS for 10 min. A hybridization signal was detected by exposing the membrane to X-OMAT X-ray film (Kodak) for 7 days at –80ºC. 2.4

Protein extraction and immunoblotting

Protein extracts were prepared by homogenizing banana tissues in a pre-chilled mortar and pestle with three volumes of ice-cold buffer as described previously (Roy Choudhury et al. 2008b). Equal amounts (30 μg) of protein extracts from each sample were analysed in 10% SDS-PAGE according to Laemmli (1970) and the proteins were then electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Amarsham Pharmacia) using a Bio-Rad mini transblot cell (Bio-Rad CA) according to the manufacturer’s instructions. Immunoblot analysis was carried out with anti-banana MA-ACS1 (prepared in our lab) polyclonal antibody. Primary antibody-recognized cross-reacting bands on the membrane were detected by following the enzymatic assay of alkaline phosphatase for colour development as described by Sambrook et al. (1989). The intensity of the immunoreactive bands was quantified by using a Bio-Rad Imaging Densitometer (GS-700). 2.5

Multiple sequence alignments were performed with the amino acid sequences in the CLUSTALW 1.83 program (Thompson et al. 1994). The alignment parameters used were: gap open penalty 10.00; gap extension 0.20; sequence >10% diverged delayed; BLOSSUM 62 matrix; residue-specific penalties on; and hydrophobic penalties on. The alignment was revised using the BioEdit program (Hall 1991). Residues of structural and evolutionary importance were calculated by the ConSeq server (Berezin et al. 2004). This program enabled us to calculate the conservation score in every alignment position, taking into account the relationships between the sequences and the physicochemical similarity between the amino acids. The conservation scores are represented on a 10-point scale and can be used to determine the functional relevance of a residue in evolution. 2.7

Phylogenetic analysis

Phylogenetic analysis of the ACS sequences was performed using MEGA version 3.1 (Tamura et al. 2007). Neighbour-joining and maximum parsimony algorithms were used for phylogenetic tree searching and inference. The phylogenetic trees were tested by bootstrap analysis with 1000 replications. Moreover, protein distances were calculated using the algorithm of p-distance. The complete deletion option was used in handling gaps or missing data obtained from the alignment. Since similar topologies were found for both the algorithms employed, only the tree by the neighbour-joining method is displayed in the manuscript.

Measurement of ACC content

For determination of the ACC content, banana tissues were homogenized in 80% ethanol and then centrifuged at 12 000× g for 12 min at 4ºC. The supernatant was assayed for the estimation of ACC content by the method of Lizada and Yang (1979). 2.6

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Protein data mining and sequence analysis

Homologous protein sequences were obtained from Gene Bank hosted on the webpage of the National Centre for Biotechnological Information (NCBI) (http: //www.ncbi.nlm.nih.gov/, validated 22 April 2008). By BLAST and PSI-BLAST programs (Altschul et al. 1997) using ACC synthase 1 of Musa acuminata as an initial query, we searched for possible ACC synthase proteins. Domain positions were confirmed using the simple modular architecture research tool (SMART) (Schultz et al. 1998) and Conserved Domain Database v2.11 (CDD) (MarchlerBauer et al. 2003).

2.8

3-D model building and ligand-binding analysis

Template selection was carried out as described previously (Singh et al. 2008). Target (MA-ACS1, accession number AAU09672) and template sequences ([PDB code 1YNU, Malus domestica ACS] [Capitani et al. 1999], [PDB code 1IAX and 1IAY, Solanum lycopersicum ACS] [Huai et al. 2001]) were aligned in the CLUSTALW 1.83 program. Overhanging regions of MA-ACS1 were edited using the BioEdit program. Finally, homology modelling of the core structure was done using the model-multiple.py script of MODELLER9v2, which included more than one template for model building (Marti-Renom et al. 2000). Initially, the quality of the model was determined from the root mean square deviation (RMSD) value of the position of the amino acids in the modelled protein to the paired amino acids in the template and the discrete optimized protein energy (DOPE) score. For further refinement of protein structures (monomer and dimer), molecules were energy-minimized by 10 000 steps of conjugate gradients using NAMD J. Biosci. 35(2), June 2010


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(Kale et al. 1999) with the CHARMM22 force field. After the refinement process, final validation of the model was carried out using PROCHECK (Laskoswki et al. 1993), ERRAT server (Colovos and Yeates 1993), WHAT-IF server (Vriend and Sander 1993) and Verify3D (Luthy et al. 1992). These servers apply different criteria to check the quality of a protein model. AVG and PLP were manually docked into the models by superposition of the built model with the crystal structure of Solanum lycopersicum ACC synthase (PDB 1IAY). Binding pocket and ligand–protein interactions were analysed by Pocketpicker 1.0 (Weisel et al. 2007) and the LPC server (Sorokine et al. 1999). All the figures were generated using either PyMOL (http://www.pymol.org) or Swiss-PDBviewer (Guex and Peitsch 1997). 3.

Results and discussion

3.1 Analysis of tissue-specific expression of MA-ACS1 We investigated the expression of MA-ACS1 in root, stem, leaf, peel and pulp tissues of banana by northern blot analysis using 20 μg of total RNA. MA-ACS1 mRNA was not detected in root, stem and leaf tissues, whereas a very

low level of the transcript was found to accumulate in ripe fruit peel (figure 1a, lanes 1–4). The maximum signal for MA-ACS1 mRNA was detected in ripe fruit pulp (figure 1a, lane 5). Immunodetection of MA-ACS1 protein level in the indicated tissues using anti-banana MA-ACS1 polyclonal antibody also revealed maximum expression of the 55 kDa band of banana MA-ACS1 in the ripe pulp tissue (figure 1c), indicating a tissue-specific expression of MA-ACS1 in the fruit. The expression of MA-ACS1 in ripe pulp tissue correlated well with the transcript level. A similar set of proteins was probed with rabbit preimmune serum, which did not detect the MA-ACS1 protein (figure 1d), thus indicating the specificity of recognition of the MA-ACS1 protein by the anti-banana MA-ACS1 antibody. To correlate the expression profile of MA-ACS1, we next estimated the relative ACC content in various tissues. ACC content was found to be considerably high in ripe pulp tissue as compared to other tissues and this finding was thus consistent with the enhanced expression level of MA-ACS1 in ripe fruit pulp (figure 1e). Taken together, these results indicate the key role of MA-ACS1 in the formation of ACC and thus ethylene biosynthesis in banana fruit during ripening.

Figure 1. (a) Expression pattern of Musa acuminata ACC synthase 1 (MA-ACS1) mRNA in different tissues (root, stem, leaf, peel and pulp) of the banana plant. Northern blot was carried out using 20 μg total RNA samples electrophoresed and probed with radiolabelled MA-ACS1 probe. (b) Ethidium bromide staining of gel is shown below the blot as loading control. (c) Protein gel blot analysis with total protein extracts (25 μg) prepared from different tissues by using anti-banana MA-ACS1 antibody. The position of the immunoreactive band is indicated by an arrowhead. (d) Similar analysis with pre-immune serum used as control. (e) Determination of 1-aminocyclopropane-1carboxylate (ACC) content from the indicated tissues. Error bar indicates the ±1 SE of five independent replicates. J. Biosci. 35(2), June 2010

Properties of banana ACC synthase 1 3.2

MA-ACS1 sequence analysis

Twenty-five ACS sequences from various plant species and Penicillium citrinum were obtained from GenBank hosted on the NCBI webpage. The Blast and PSI-Blast programs (Altschul et al. 1997) were used for initial analyses and alignments (supplementary figure 1). Comparison of fruit-

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specific ACC synthase sequences from different plant species revealed a more or less similar length of proteins comprising approximately 440–496 amino acid residues. The 486 amino acid residue-long MA-ACS1 protein showed seven conserved domains, which were similar to citrus ACC synthase (Wong et al. 1999) (figure 2). Multiple sequence analysis of most of the ACS sequences available in GenBank

Figure 2. Prediction of residue conservation and location based on ConSeq. Different secondary structural elements, as explained in the text, are depicted above the sequence. Different conserved residues are shown in boxes. Residues that are more conserved are shown in an increasingly darker background with white text; more variable sequences are shown in black text. Below the sequence, ‘e’ suggests a residue predicted to be exposed (surface) and ‘b’ indicates a buried residue; ‘s’ and ‘f’ represent ConSeq’s prediction of residues of ‘structural’ or ‘functional’ importance. J. Biosci. 35(2), June 2010

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revealed that ACS is a highly conserved protein except for the extreme N- and C-terminal residues and there were five main insertion sites within the sequences (supplementary figure 1). Analysis using ConSeq showed that most of the exceptionally conserved amino acid residues were either in the α-helix region or β-sheets (figure 2). Furthermore, an insignificant number of structurally or functionally important residues were present at the extreme N- or Cterminal position, which supported the previous suggestion that these regions have no functional role in enzyme activity (Huai et al. 2001). The biological importance of a residue often correlates with its level of evolutionary conservation within the protein family (Berezin et al. 2004). We analysed a broad range of ACC synthase sequences and determined highly conserved residues that are potentially necessary for either structural integrity or biological functionality. Different degrees of darkness and colour of amino acid residues illustrate the degree of conservation and this conservation was mapped onto the sequence of MA-ACS1 (figure 2). The most highly conserved residues of functional importance were found to be exposed, while the structurally important highly conserved residues were buried. Among the seven most conserved regions, ~44% of the residues were predicted to be exposed (hydrophilic) and ~56% residues were buried (hydrophobic). The putative MA-ACS1 polypeptide appeared to be composed of a highly hydrophilic amino and carboxy terminus end that showed the characteristics of cytosolic protein, as detected in PEPPLOT, GCG 10.0 (Gribskov et al. 1986). The two predicted O-glycosylated residues, which were detected at Thr329 and Thr338 by the OGPET program (http://ogpet.utep.edu/OGPET/), were different from other fruit-specific ACC synthase sequences. Glyprot (Bohne-Lang and Vonder Lieth 2005) recognized the same N-glycosylation sites (Thr329 and Thr338) in the modelled structure of MA-ACS1. The existence of O-glycosylated residues appears to probably be responsible for intercellular transportation or maintaining protein folding. The theoretical isoelectric point and molecular weight of MA-ACS1 were calculated to be 7.60 and 54 877.60, respectively (http: //www.us.expasy.org). However, due to phosphorylation at multiple sites (Net phos 2.0) (Blom et al. 1999), the actual Mw of MA-ACS1 was found to be approximately 55 kDa. CDD analysis (Marchler-Bauer et al. 2003) detected the main domain from this MA-ACS1 (50-431: aminotransferase classes I and II). Proscan analysis (http://npsa-pbil.ibcp.fr/ cgi-bin/npsa_automat.pl?page=npsa_prosite.html) detected an aminotransferase class-I pyridoxal-phosphate attachment site: Ser-Leu-Ser-Lys-Asp-Leu-Gly-Val-Pro-Gly-Phe-ArgVal-Gly (276–289). Mutagenesis analysis clearly depicted the role of principal amino acid residues at the active site of ACC synthase of apple and tomato. Sequence analysis distinguished those J. Biosci. 35(2), June 2010

amino acid residues in case of MA-ACS1 (figure 2). The numbers in brackets indicate equivalent residues in ACS from apple and tomato (Capitani et al. 1999; Huai et al. 2001). The active site Lys279 (273, 278) was found to form a Schiff base (internal aldimine) with the bound PLP in the unligated enzyme, while the Arg388 (407, 386) guanidino moiety was associated with the carboxylate group of SAM by making an ion pair. The conserved Tyr95 (85, 70) residue was probably involved in substrate recognition and related to the aromatic character of its side chain. Asn197 (202, 209) was found to form hydrogen bonds with the pyridine ring of PLP, whereas Asp240 (230, 237) also formed hydrogen bonds with the pyridine ring of PLP. The formation of external aldimine was promoted by the Tyr253 (233, 240) residue due to attraction of the proton of Lys279. In addition, the Tyr155 (145, 152) residue may be involved in maintaining the pyridine ring of PLP. Thr131 (121, 128), Ser276 (270, 275), Ser278 (272, 277), Arg287 (266, 281) and Ala130 (120, 127) appeared to be required for proper orientation of PLP in the active site during the formation of a hydrogen bond with the phosphate oxygen of PLP. The guanidine group of Arg160 (150, 157) formed a hydrogen bond with the sugar of SAM, whereas the α-carboxylate group of SAM also formed a hydrogen bond with the nitrogen of Ala56 (46, 54). The guanidium moiety of Arg413 (407, 412), Tyr29 (19, 27), Phe30 (20, 28) and Pro154 (144, 153) residues remained within a hydrophobic pocket of the adenine ring of SAM. 3.3

Phylogenetic analyses

An evolutionary comparison of MA-ACS1 across different kingdoms was performed to understand the phylogenetic relationship of MA-ACS1 with 29 other ACS sequences (figure 3). Phylogenetic analyses revealed the existence of four main clades, among which one clade (1) represented the animal protein sequences, which diverged much earlier from that of the plant ACC synthase sequences. However, fungal protein acquired a different clade (2) stemming from the same node from which animal ACS proteins actually originated. ACC synthase of Arabidopsis thaliana and Triticum aestivum remained within the same clade (3), which was closely related with other plant ACS proteins. Most of the plant ACS proteins belonged to the same clade (4) and, within the same clade, there were several subclades. The ACS sequences of Solanaceae (Nicotiana tabacum, Solanum tuberosum, Solanum lycopersicum), Cucurbitaceae (Cucumis melo, Momordica charantia), Rosaceae (Pyrus communis, Malus domestica), Leguminosae (Glycine max, Pisum sativum, Medicago truncatula) remained within the different subclades. MA-ACS1 was found to be tightly clustered with ACC synthase sequences from other plant species with a topology that was supported by a significantly


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Figure 3. A consensus bootstrap neighbour-joining tree based on the CLUSTALW alignments of the amino acid sequences of 1aminocyclopropane-1-carboxylate synthase (ACS) from different organisms has been constructed. Musa acuminata ACC synthase 1 (MAACS1) is indicated by a rectangle. Sequences with their Gene Bank accession numbers in parentheses are as follows: Musa acuminata (AAU09672); Diospyros kaki (BAB89349); Malus domestica (BAA92351); Momordica charantia (AAQ14268); Medicago truncatula (AAL35745); Glycine max (CAA47474); Populus canadensis (BAA94600); Pyrus communis (AAL66205); Cucumis melo (BAB18464); Solanum Lycopersicum (AAF97614); Persea americana (AAM21683); Pisum sativum (AAD04199); Solanum tuberosum (BAB16433); Gossypium hirsutum (ABC75833); Citrus sinensis (CAB60831)); Lactuca sativa (AAP14019); Carica papaya (AAC98809)); Betula pendula (AAM80888); Pelargonium hortorum (ABQ51839); Nicotiana tabacum (ABW97851); Rumex palustris (AAB96658); Camellia sinensis (ABM88785); Arabidopsis thaliana (NP_199982); Triticum aestivum (AAB18418), Penicillium citrinum (BAA92149); Canis familiaris (XP_854150); Homo sapiens (EAW68073); Mus musculus (A2AIG8); Pan troglodytes (XP_508382).

high bootstrap value (>70%), which always acquired an independent subclade. The overall results indicated that

further divergence probably occurred after the split of monocots and dicots. J. Biosci. 35(2), June 2010


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Amino acid compositional distances were calculated in order to study the difference in amino acid composition of the sequences used for phylogenetic analysis. Compositional distance demonstrated the number of differences between sequences even if the substitution patterns were homogeneous among lineages. Analysis of compositional distances suggested that compositionally MA-ACS1 is closer to Pyrus communis and Glycine max (0.223), while it is most distantly related to Mus musculus (1.170) (supplementary table 1). 3.4

In silico three-dimensional modelling of MA-ACS1 and validation

Supplementary figure 2a and b illustrates the homology modelling of the monomer and homodimer of MA-ACS1, respectively. The monomeric structure is coloured by a secondary structure, while the dimer is coloured by a chain. After seven iterations of PSI-BLAST, we detected only three Protein Data Bank entries, 1IAX, 1M7Y and 1YNU, with significant similarity. Based on the length, per cent sequence identity, percentage gaps and the e-values, the X-ray crystallographic structure of ACC synthase from Malus domestica (PDB 1YNU) and Solanum lycopersicum (PDB 1IAX) were selected as template sequences for model building of the monomer and further analysis. The targets showed 57% and 67% sequence identities, respectively, with the template sequence. Such values were well above the 30% threshold value for a more precise structure prediction (Baker and Sali 2001). The dimer of MA-ACS1 was modelled onto the crystal structure of the Solanum lycopersicum ACC synthase homodimer (PDB 1IAX) (Capitani et al. 1999). The Swiss-Pdb viewer was used to calculate the root mean square deviation (RMSD) between the backbone atoms of the template and those of the model. Here, the RMSDs between the monomer and dimer models and their respective templates were found to be 0.60Å and 0.36Å, respectively. The quality of both the monomer and dimer of MA-ACS1 was checked by different programs. The first validation was

carried out using MODELLER’s DOPE score. DOPE has been shown to have a statistical potential to assess homology models in protein structure prediction (Shen and Sali 2006). DOPE confirmed that reasonable models were obtained with a suitable energy score comparable to that of the templates (table 1). The models with the lowest objective function values were further analysed for violations of main chain Phi/Psi dihedral bond angle ratios and backbone/side chain and side chain/side chain steric conflicts by using the Ramchandran plot with the PROCHECK program (Laskoswki et al. 1993). The Phi and Psi distributions of the Ramachandran plot of non-glycine, non-proline residues are summarized in table 1. ERRAT is a so-called ‘overall quality factor’ for non-bonded atomic interactions, and higher scores mean higher quality. Practically, the normally accepted range is >50 for a high-quality model (Colovos and Yeates 1993). In the current case, the ERRAT score for the monomer model was 96.651 and 80.332 for the dimer, while the ERRAT scores for the templates were 93.41 and 94.132, respectively (table 1). The values were clearly well within the range of a high-quality model. Taken together, the above analyses suggested that the backbone conformation and non-bonded interactions of the homology models were all reasonable within a normal range. Additionally, WHAT-IF is commonly used to check the normality of the local environment of amino acids (Vriend and Sander 1993). Thus, for the WHAT-IF evaluation, the quality of the distribution of atom types was determined around amino fragments. We found that the quality control value for most of the residues in the predicted model agreed well with the threshold value (–2.0) and only a few residues lay within –2.0 and –3.0. Besides these, none of the scores for each residue in the homology model was less than –3.0 as depicted in figure 4a, b and c. Therefore, WHAT-IF evaluation also indicated that the homology model structure was very reasonable. Verify3D analysis (Luthy et al. 1992) indicated that 90.87% and 96.83% of the residues of the monomer and dimer structures had a 3D-1D averaged score >0.2, which again proved the good quality of the predicted structures.

Table 1. Quality of structures checked by PROCHECK, ERRAT and MODELLER’s DOPE score. PROCHECK Ramachandran plot quality (%)

ERRAT score

DOPE score

Goodness factor

Core

Allowed

General

Disallowed

Dihedrals

Single chain model

94.7

5.3

0.0

0.0

–0.06

–0.33

–0.15

96.651

–53543.52

Template

85.9

14.1

0.0

0.0

0.10

0.53

0.27

93.41

–53148.94

Both chain model

92.8

6.7

0.3

0.3

0.08

–0.19

–0.02

80.332

–56211.87

Template

85.5

13.8

0.5

0.1

0.10

0.51

0.27

94.132

–58987.95

J. Biosci. 35(2), June 2010

Covalent Overall

Properties of banana ACC synthase 1 In brief, the geometrical quality of the backbone conformation, residue interaction, residue contact and

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energy profile of the structure were all well within the limits established for reliable structures. Therefore, all

Figure 4. The WHAT-IF packing quality scores calculated for the template and the homology model. Modelled structures are coloured in red while templates are coloured in black. (a) Single chain model without ligands is compared with template 1YNU. (b) Dimer model without any ligand is compared with its template 1IAX. (c) Musa acuminata ACC synthase 1 (MA-ACS1) monomer model with aminoethoxyvinylglycine (AVG) and pyridoxal-5’-phosphate (PLP) is compared with template 1IAY. J. Biosci. 35(2), June 2010

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these evaluations suggested for generation of reasonable homology models for the monomer and dimer of MA-ACS1 have a high degree of similarity with the chosen templates to allow for further examination of protein–substrate interactions.

3.5

Characteristics of the structure of MA-ACS1 protein and analysis of protein–ligand interactions

There is an expected similarity in the relative abundance of α-helix and β-sheet motifs between the MA-ACS1 model and

Figure 5. Comparison of the modelled Musa acuminata ACC synthase 1 (MA-ACS1) binding site in pyridoxal-5'-phosphate (PLP) and aminoethoxyvinylglycine (AVG) with the experimentally determined site in Solanum lycopersicum ACS (PDB id 1IAY). LE-ACS is depicted in yellow, while MA-ACS1 is coloured in red. Residue labels for MA-ACS1 appear in blue. For the sake of clarity, PLP is not shown with AVG and vice versa, in spite of the fact that their binding pockets lie within 6.5 Å. (a) Residues within 6.5 Å of the cofactor (PLP) are shown. PLP from both model and template structures are shown in red and pink, respectively. (b) Residues within 6.5Å of the inhibitor (AVG) are shown. AVG from both model and template structures are shown in blue and violet, respectively. J. Biosci. 35(2), June 2010

Properties of banana ACC synthase 1 the template. The monomer of MA-ACS1 has been found to be a two-domain protein as in tomato and apple (Capitani et al. 1999; Huai et al. 2001). The large domain (αβα) includes a central eight-strand β-sheet flanked by nine α-helices, whereas the small domain (αβ) contains seven α-helices and nine βstrands, and each domain is composed of a central sheet of βstrands connected by α-helices that pack on both sides. In the case of tomato (Huai et al. 2001), 11−19 amino acid residues are mainly responsible for dimer formation; subsequently, in banana, the existence of more or less the same amino acid residues in near about similar locations (13−21) indicates that probably this region may be responsible for dimer MA-ACS1 formation in banana. The contents of α-helices, β-strands, turns and coils constituted 56.27%, 19.22%, 24.51% and 18.66% of the total amino acids, respectively. The overall structure of the modelled binding site for PLP and AVG in MA-ACS1 was very similar to the known structure for the binding site in apple and tomato ACC synthase (Capitani et al. 1999; Huai et al. 2001). Superposition of the unliganded MA-ACS1 onto the complex with PLP and AVG indicated no significant conformational change in the three-dimensional structure of MA-ACS1 upon binding to the ligand (figure 5a and b). The RMSD between the two PDB files was found to be 0.61Å. PLP interacted with Ala127, Thr128, Tyr152, Asn209, Asp237, Tyr240, Ser275, Ser277, Lys278 and Arg286 of LE-ACS (Huai et al. 2001). The residues from the PLP-binding pocket, essential for ligand binding, have been found to be mostly conserved across the structure of MA-ACS1 (Ala130, Thr131, Tyr155, Asn212, Asp240, Ser276, Ser278, Lys279 and Arg287 except at the Phe243, which replaced the Tyr240 of LE-ACS). The structures showed good conservation of the catalytic residues, suggesting a similar catalytic mechanism for ACS and other PLP-dependent enzymes. The competitive inhibitor AVG has been shown to bind at a location adjacent to PLP. Both Ala56 and Arg413, which have been shown to be the binding residues for AVG, were well conserved in MA-ACS1. 4.

Conclusion

ACC synthase is a dimeric enzyme that utilizes PLP as a cofactor and belongs to a fold type I PLP-dependent enzyme. The PLP-dependent enzymes catalyse a vast array of reactions in the metabolism of amino acids including racemization, transamination, deamination, decarboxylation and deletion or substitution of β and γ carbons. These enzymes share little sequence similarity with diverse catalytic specificities (Mehta et al. 1993). Significant progress has been made in the past decade in determining the three-dimensional structures of PLPdependent enzymes and, to date, more than 200 crystal structures are available in the Protein Data Bank. Extensive

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biochemical and structural studies have classified the PLP-dependent enzymes into four types of fold (Jansonius 1998) and such analyses have revealed that PLP-dependent enzymes share a common catalytic mechanism (Jansonius 1998), while the determinant for defined specificities of PLP-enzymes remains unclear. Three crystal structures of apple ACC synthase are now available, including the unliganded enzyme (Capitani et al. 2002) and those as complexes with the potential inhibitor L-AVG (Capitani et al. 2003) and an aminooxy analogue of the substrate SAM (Capitani et al. 2002). In tomato, crystal structures of both unliganded and AVG-complexed ACC synthase have been demonstrated (Huai et al. 2001). Such structural information has provided remarkable support for the construction of a plausible model for the complex with the natural substrate SAM, with the aim of understanding the mechanistic detail of catalysis carried out by ACC synthase. In contrast to this, structural features of ACC synthase in banana, which is one of the most widely cultivated economically important fruit crops, remain mostly unknown. In the present study, we have described an in silico analysis to unravel the important sequential, structural and phylogenetic features of ACC synthase 1, the key enzyme involved in ethylene biosynthesis in banana fruit during ripening. Our results provide insight into the threedimensional structural features of MA-ACS1 along with the structural conservation of PLP-dependent enzymes in higher plant genomes. The data presented here reveal important features regarding the composition of enzyme active sites. Comparison of the detailed structural features of the modelled proteins with their respective templates confirms the reliability of the model. The ligands were placed in a near-native orientation in the consensus-binding site of the modelled protein. The presence of the ligands was included in the homology modelling process in terms of user-defined restraints. Manual docking has recently been used for homology and knowledge-based models to find the position of ligands in the binding pockets, which would agree with known ligand–receptor interactions (Ballesteros et al. 2001; Yu et al. 2008).. Structural genomics is augmenting the functional assignment of proteins by determining the corresponding three-dimensional structures. This permits a functional assignment by identifying proteins of known structure and function, which exhibit a similar overall fold to the protein of unknown structure. The three-dimensional structure of protein is of major importance in providing insights into its molecular functions. Further analysis of threedimensional structures will help in the identification of activator- and inhibitor-binding sites. Homology modelling helps in predicting the three-dimensional structure of a macromolecule with unknown structure (target) by J. Biosci. 35(2), June 2010


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comparing it with a known template from another. Several studies have been described in the literature that apply homology models of proteins to explain putative protein–ligand interactions (Bourdon et al. 1997; Escherich et al. 2000; Garcia-Nieto et al. 2000; Le Novere et al. 2002). In some cases, the models were also subsequently used for designing new potent inhibitors (Kiyama et al. 1999; Rong et al. 2002; Tiraboschi et al. 1999). Structural and biochemical data are necessary for designing ACC synthase inhibitors, whose applications are expected to have immense agricultural effects on controlling banana fruit ripening. Acknowledgements SRC is the recipient of a Research Associate Fellowship from the Council of Scientific and Industral Research (New Delhi) project. SKS is the recipient of a Senior Research Fellowship from the University Grants Commission, New Delhi. The Department of Science and Technology, New Delhi provided the Young Scientist Fellowship Grant to SR. References Alexander L and Grierson D 2002 Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening; J. Exp. Bot. 53 2039–2055 Altschul S F, Madden T L, Schaffer A A, Zhang J, Zhang Z, Miller W and Lipman D J 1997 D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs; Nucleic Acids Res. 25 3389–3402 Baker D and Sali A 2001 Protein structure prediction and structural genomics; Science 294 93–96 Ballesteros J A, Shi L and Javitch J A 2001 Structural mimicry in G protein coupled receptors: implications of the high-resolution structure of rhodopsin for structure–function analysis of rhodopsin-like receptors; Mol. Pharmacol. 60 1–19 Barry C S, Llop-Tous M I and Grierson D 2000 The regulation of 1aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato; Plant Physiol. 123 979–986 Berezin C, Glaser F, Rosenberg Y, Paz I, Pupko T, Fariselli P, Casadio R and Ben-Tal N 2004 ConSeq: the identification of functionally and structurally important residues in protein sequences; Bioinformatics 20 1322–1324 Blom N, Gammeltoft S and Brunak S 1999 Sequence and structurebased prediction of eukaryotic protein phosphorylation sites; J. Mol. Biol. 294 1351–1362 Bohne-Lang A and Vonder Lieth C W 2005 GlyProt: in silico glycosylation of proteins; Nucleic Acids Res. 33 W214–W219 Boller T, Herner R C and Kende H 1979 Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1carboxylic acid (tomatoes); Planta 145 293–303 Bourdon H, Trumpp-Kallmeyer S, Schreuder H, Hoflack J, Hibert M and Wermuth C G 1997 Modelling of the binding site of the J. Biosci. 35(2), June 2010

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MS received 27 August 2009; accepted 22 February 2010 ePublication: 28 April 2010 Corresponding editor: MARÍA ELIANO LANIO



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An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5′-phosphate and aminoethoxyvinylglycine SWARUP ROY CHOUDHURY, SANJAY KUMAR SINGH, SUJIT ROY and DIBYENDU N SENGUPTA J. Biosci. 35(2), June 2010, 281–294 © Indian Academy of Sciences Supplementary figures and table


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Supplementary figure 1.




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Supplementary figure 1.


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Supplementary figure 1. Alignment of the deduced full-length amino acid sequence of Musa acuminata ACC synthase 1 (MA-ACS1) with other 1-aminocyclopropane-1-carboxylate (ACC) synthase sequences. Gaps are shown as dashes; letters on a black background indicate identical amino acids, while boxes are used to show the conserved regions. ClustalW (1.83) performed multiple sequence alignment. Five main insertion sites are indicated by ‘’.



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Supplementary figure 2. (a) A ribbon rendering of the Musa acuminata ACC synthase 1 (MA-ACS1) monomer. The secondary structures, α-helices are shown in red, β-sheets in yellow, others in green. (b) Ribbon representation of the proposed dimeric MA-ACS1. Subunits are coloured by chain . J. Biosci. 35(2), June 2010


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Supplementary table 1. Estimates of differences in base composition bias between sequences Plant name Musa acuminate Diospyros kaki

0.463

Malus domestica

0.333

Momordica charantia

0.383

Medicago truncatula

0.263

Glycine max

0.223

Populas canadensis

0.450

Pyrus communis

0.223

Cucumis melo

0.497

Lycopersicon esculentum

0.327

Persea americana

0.423

Pisum sativum

0.283

Solanum tuberosum

0.513

Gossypium hirsutum

0.530

Citrus sinensis

0.307

Lactuca sativa

0.523

Carica papaya

0.430

Betula pendula

0.357

Pelargonium hortorum

0.363

Nicotiana tabacum

0.427

Rumex palustris

0.297

Camellia sinensis

0.373

Arabidopsis thaliana

0.397

Penicillium citrinum

0.783

Canis familiaris

1.057

Homo sapiens

1.117

Mus musculus

1.170

Pan troglodytes

1.133

Triticum aestivum

0.460
