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ARTICLES A&EB

BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1

Keywords: EPSP synthase, glyphosate tolerance, Halomonas variabilis, structure prediction, error-prone PCR

IntroductionGlyphosate (N-(phosphonomethyl)glycine) is a popular herbicide, which is used to control grasses, herbaceous plants including deep rooted perennial weeds, brush, some broadleaf trees and shrubs, and some conifers. Glyphosate applied to foliage is absorbed by leaves and rapidly moves through the plant (http://infoventures.com/e-hlth/). It acts by inhibiting enolpyruvyl-shikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19), an enzyme in the pathway leading to biosynthesis of aromatic amino acids. This reduces the production of protein in the plant, and inhibits plant growth (8).

EPSPS, which is encoded by aro A, plays a crucial role in the glyphosate tolerance. Certain EPSPS enzymes are insensitive to glyphosate inhibition. The same mechanism of glyphosate resistance exists in insensitive aro A genes of bacteria, and plants (1, 5-7, 16, 18, 20-22). EPSPS converts shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form 5-enolpyruvylshikimate-3-phosphate (EPSP). Glyphosate, as an analog of PEP, is a competitive inhibitor of EPSPS by accessing to the PEP binding site in EPSPS to form a ternary complex of EPSPs·S3P·glyphosate (10). If some mutations within the EPSPS target site only allow S3P and PEP to form EPSP and avoid the binding of glyphosate, bacteria or plants can confer a modest degree of resistance to glyphosate. Subsequently there are two classes of glyphosate tolerant aro A genes that have been identifi ed. Class I EPSPS include those from Brassica napus Salmenella typhimurium E. coli.Class II EPSPS are those from Agrobacterium sp.CP4 and Pseudomonas sp. PG2982, and Class II EPSPS do not cross-react with the polyclonal antibodies against Class I EPSPS, and have less than 50% amino acid similarity to class I enzymes (7). When expressed in transgenic plants, the insensitive enzymes confer

tolerance to the herbicide. This mode of action accounts for all the commercial glyphosate-tolerant crops. The glyphosate tolerant genes also can be as a selectable marker in transplant transformation (23).

Previously we isolated a highly glyphosate tolerant bacterial strain HTG7 which has been identifi ed as Halomonas variabilis, and cloned a 1.35-kb aroA gene conferred glyphosate tolerance from a cosmid library (14).The plant expression vector pAroA harboring aro A was constructed, and leaf discs of tobacco (Nicotiana tabacum L. cv. NC89) were transformed with pAroA by Agrobacteria-mediated transformation. The transgenic plants tolerated a concentration 20-fold higher than those of the non-transgenic plants. Furthermore, the transgenic plants were morphologically normal, and showed no adverse symptoms (date not shown).All the results suggested that aro A might have a potential application in the genetic engineering of plants with enhanced glyphosate resistance. Therefore, the biochemical or molecular biological information was highly valuable for understanding of the aro A gene from Halomonas variabilis HTG7. Here some properties of EPSPS, including secondary structure, subcellular localization, phylogenetic analysis, and transmembrane segment, were predicted and detailed.

Some papers revealed that the resistance was due to multiple nucleotide changes conferring specifi c amino acid substitutions in this enzyme, and these cases were widely occurred especially in the virus (12, 15). In order to correlate the glyphosate resistance with amino acid changes in EPSPS, we performed error-prone polymerase chain reaction (PCR) to create mutants. The essence of error-prone PCR mutagenesis is the introduction of random base substitution into the target gene sequence. This approach has been applied successfully to engineer new functions and properties for enzyme (2). Two mutants, which possess enzyme activities while losing glyphosate tolerance, were selected. From comparing the

PROPERTIES OF ARO A FROM HALOMONAS VARIABILIS

Z. Liu1, 2, 3, W. Zhang1, S. Ping 1, Z. Yang2, M Lin 1

Biotechnology research Institute, Chinese Academy of Agriculture Science, China1

Sichuan University, College of Life Sciences, China2

University of Pittsburgh, Center for Biotechnology and Bioengineering, Pittsburgh, USA3

Correspondence to: Lin Min E-mail: linmin57@vip.163.com

ABSTRACTThe aro A gene encoding enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) of Halomonas variabilis HTG7 and conferring glyphosate tolerance, was cloned from the cosmid library. The secondary structure, subcellular localization, transmembrane region, and phylogenetic analysis were predicted and analyzed according to the deduced amino acid sequence of EPSPS respectively. Two mutants, which have EPSPS enzyme activity while losing the glyphosate tolerance, were selected using error-prone PCR. By comparison with the sequence of native and mutated aro A genes, we found that the tertiary structure and backbone were the same. The torsion angles of the variant residues were discussed with respect to their accessibility and their possible involvement in the catalytic process.

35BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1

tertiary structure, backbone and atoms mode of glyphosate-tolerant and glyphosate-sensitive EPSPS, we are trying to elucidate the mechanism of glyphosate-resistance in aro A from Halomonas variabilis HTG7.

Materials and MethodsBioinformatics toolsPotential secondary structure for EPSPS was determined using PREDATOR (9) at http://bioweb.pasteur.fr/seqanal/interfaces/predator-simple.html. The subcellular localization was predicted using Psort version II (11) at http://psort.nibb.ac.jp/form2.html.The prediction of transmembrane region was determined using TopPED2 (4) at http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html. The tertiary structure was predicted using Swiss-model (19) at http://swissmodel.expasy.org//SWISS-MODEL.html. The multiple sequences alignments and phylogenetic analysis were undertaken using DNAMan software. The tree was generated from aligned EPSPS sequences (Table l). The Backbone and Atoms were aligned using Vector NT I software.

TABLE 1The amino acid sequence of EPSPS used for phylogenetic analysis.Organism Accession NumberMicrobulbifer degradans 2-40 ZP_00065119Pseudomonas aeruginosa PA01 NP_251854Azotobacter vinelandii ZP_00091753Psychrobacter sp. 273-4 ZP_00146177Coxiella burnetii RSA 493 NP_819558Xylella fastidiosa 9a5c NP_299603Xanthomonas campestris pv. campestris str. ATCC 33913 NP_636962

Magnetococcus sp. MC-1 ZP_00043137Geobacter metallireducens ZP_00080040Oceanobacillus iheyensis HTE831 NP_692701Novosphingobium aromaticivorans ZP_00093085Dichelobacter nodosus Q46550Thermoanaerobacter tengcongensis NP_622666Sinorhizobium meliloti NP_384359Thermosynechococcus elongatus BP-1 NP_681133Bacillus cereus ATCC 14579 NP_832685Rhodospirillum rubrum ZP_00014193Synechocystis sp. PCC 6803 NP_441799Streptococcus pyogenes MGAS315 NP_664831Desulfi tobacterium hafniense ZP_00098418Listeria innocua NP_471371Agrobacterium sp. CP4 AAL67577Caulobacter crescentus CB15 NP_422383Magnetospirillum magnetotacticum]. ZP_00055501Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 ZP_00062987

Mesorhizobium loti NP_105914Rhodobacter sphaeroides ZP_00005386*Brassica napus sequence Salmenella typhimurium sequence and E. Coli sequencewere from US patent (NO. 6,225,114). Agrobacterium sp.CP4 sequence and Pseudomonas sp. PG2982 sequence were from US patent (NO. 6,248,876).

E. coli strain, plasmid, and reagentsE. coli strain ER2799 was used for cloning and for expression experiments of the mutated aro A gene. Plasmid pACYC-1.35 producing EPSPS was used as a template for error-prone PCR amplifi cation of the aro A gene (14). MOPS with 20 mM glyphosate or without glyphosate was used for selection medium. All the chemicals were obtained from Promega (Promega, Madison, Wisconsin). Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Biolabs, Ipswich, Massachusetts).The primers (F aro A and R aro

A) for mutation of glyphosate-tolerant gene were synthesized according to the sequence of aro A gene from Halomonas variabilis HTG7, and DNA sequencing was done by Shanghai GeneCore CO. (GeneCore, Shanghai, China).

F aro A 5-CTGGAATTCATGCAACCACAGGG-3 R aro A 5-CCGGAATTCGGCTGCGGCGCTTCG-3(EcoRI site was bold)

DNA manipulationsPCR was performed under error-prone conditions (13). The amplifi ed aro A gene was size-fractionated by agrose gel eletrophoresis, and then purifi ed and digested with EcoRI and subcloned into pACYC184, which was also treated with the same restriction enzyme.Selection of aro A mutants

The pACYC184 derivatives carrying mutated aro A genes were transformed into competent E.coli strain ER2799 (17).Transformants were grown on LB-plates supplemented with Tc (50mg/mL) at 37˚C overnight. Each colony was picked, and streaked on the MOPS agar and the MOPS agar supplemented with 20 mM glyphosate. Two clones, which can grow well at MOPS while can not grow at the MOPS agar containing glyphosate, were selected for further characterization.

Results and Discussion Secondary structureThe secondary structure of a protein includes regions of alpha helix,beta sheets,turns, and random coil, or a few less common structures.PREDATOR (9) showed that the EPSPS was a protein, having 30.7 % α-helix and 20.9% β-strand, as shown in Fig. 1. The redundant α-helixes and β-strands make the understanding of structure and function more complicated.

Subcellular localization PSORT II is the use of the k-nearest neighbor (k-NN) algorithm for assessing the probability of localizing at each candidate site (11). When our query EPSPS was submitted, the prediction was performed using the k-nearest data points. Currently, k1 and K2 were set to 9 and 23 respectively. The query was predicted to be localized to the cytoplasm with the possibility of 73.9 %, and to the mitochondria with the possibility of the 21.7 %, and to the nucleus with the probability of 4.3%. So the protein was located in the cytoplasm.

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Fig.1. Prediction of secondary structure of EPSPS by PREDATORH = 4-turn helix (alpha helix) E = beta sheet in parallel and/or anti-parallel sheet conformation (extended strand).

Prediction of transmembrane regionA consensus sequence for a potential transmembrane segment in EPSPS was determined using TopPED2 (4).The transmembrane region contained 21 amino acid residues, ranging from I240 to L260, as shown in Fig. 2.

Fig. 2. Prediction of transmembrane region using TopPED2

Comparative phylogenetic analysisWe further investigated the relationship of EPSPS by generating an alignment of the 32 identifi ed EPSPS amino

acid sequences followed by the generation of a phylogenetic tree (Fig. 3).Phylogenetic analysis showed that aro A genes were classifi ed into four subfamilies, and that Class I EPSPS and Class II EPSPS of glyphosate tolerant aro A genes belong to subfamily IV and II, respectively. However, aro A from Halomonas variabilis belong to subfamily I. It composed of a lineage with the aro A of Microbulbifer degradans 2-40.

Random mutagenesis of the aro A gene using error-prone PCROligonucleotides (F aro A and R aro A) were used as primers and pACYC-1.35 was used as template for error-prone PCR mutagenesis. Randomly mutated aro A gene fragments were prepared, and E. coli ER2799 colonies carrying plasmids expressing putative mutated EPSPS proteins were identifi ed by comparing the growth in the MOPS agar with or without 20 mM glyphosate. E. coli strain ER2799, which harbors a stable EPSPS mutation, was unable to grow in minimal medium unless tyrosine, tryptophan, phenylalanine, dihydroxybenzoic acid, and p-aminobenzoic acid were added. The EPSPS derived from other genus, or organism, can be complemented with the defi ciency (3). So only the ER2799 colonies conferring EPSPS enzyme activity survive in the MOPS agar. Originally the aro A from Halomonas variabilis HTG7 has two properties, which confer EPSPS activity and glyphosate tolerance. The two mutants, which only carrying EPSPS activity, has been selected and sequenced as summarized in Table 2. T G, A G or A T transitions were commonly obtained. The base substitution rate was calculated under these experimental conditions and shown to be 0.2% leading to 0.6% amino acid substitution.

TABLE 2 Mutations generated by error-prone PCR technique

Clone Base substitutions Amino acid substitutions1 T245 G245 V82 G82

2 A179 G179 Q60 R60

2 A802 T802 T268 S268

Numbering of nucleotide,amino acid substitutions are given according to the native aro A gene and EPSPS protein.

Tertiary structure and alignment of backbone modeBoth amino acid sequences of native and mutated EPSPS were submitted to a Swiss-model (19) , and the results showed that they have the same tertiary structure (Figure not shown). We further aligned the backbone mode using Vector NT I, and the results showed they have same backbone mode (Fig. 4).

37BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1Fig. 4 The same backbone mode using Vector NT I analysis

Alignment of atoms modeAlthough EPSPS has the same tertiary structure and backbone, some changes can be measured from the native and mutated amino acids. The torsion angles of changeable amino acids are different (Fig. 5).The amino acid contains the atoms O=C-N-C where N-H lies out of the peptide plane. The torsion angle is represented νO. In clone 1, the νO of V82 was 152.19deg by compared with that of native G82 was -139.06deg. In clone 2, the νO of Q60 was 135.17deg by compared with that of native R60 was 138.56deg; the νO of T268 was 132.88deg by compared with that of native S268 was 155.98deg.

Fig. 3 Phylogenetic analysis based on EPSPS amino acid sequences using undertaken using DNAMan software. Support for individual nodes was shown as percent values of 1000 replications of bootstrap analysis. The scale bar represents a 5% estimated difference in nucleotide sequences

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Fig. 5 The changed torsion angle between native and mutated amino acids

In our previous study, a 1.35 kb DNA fragment, which encoding EPSPS and conferring glyphosate tolerance, was cloned from the cosmid library of Halomonas variabilis strain HTG7. The sequence of the aroA gene was now deposited under the GenBank accession number AY573186.The predicted amino acid sequence of aroA has less than 30% amino acid similarity with class I enzymes. It also has less than 46% amino acid similarity with class II EPSPS. Sequence analysis revealed that the molecular weight of the deduced protein was 47,062 Da, and the G+C content of the aroA coding region was 54% (14).

Here some properties of the novel aro A gene have been predicted and analyzed. The prediction of secondary structure showed that the EPSPS has 30.7 % α-helix and 20.9% β-strand. The redundant α-helix and β-strand making the fold of a three-dimensional structure become more fl exible on different conditions. The protein was located in the cytoplasm. However, the EPSPS from higher plants were located in the chloroplasts. This reminds us that aro A should be fused to the chloroplast transit peptide portion which carries EPSPS protein to where the enzyme is supposed to function, in this case chloroplast, so that the protein can function effi ciently when we constructed the plant expression vector. We assume that the transmembrane-segment architecture is assumed evoking potential high-level EPSPS expression or accumulation levels via distinct intracellular mechanisms. How the transmembrane-segment is working for the glyphosate tolerance needs to be researched further. Phylogenetic analysis showed that aro A genes were classifi ed into four subfamilies, and that Class I EPSPS and

Class II EPSPS of glyphosate tolerant aro A genes, which have been defi ned by previous researchers, belong to different subfamilies. However, aro A from Halomonas variabilis belongs to another subfamily. We postulate that there is a new class EPSPS of glyphosate tolerant aro A genes in nature. We are doing a cross-reaction with the polyclonal antibodies against Class I EPSPS and Class II EPSPS to see what it is happened. The alignment of tertiary structure and backbone showed that conservation of the 3D structure and backbone of EPSPS was important for its enzyme activity. A conformational change did occur in the replacement of V82, Q60, or T268

with G82, R60, S268, respectively. The substitutions of V82, Q60, or T268

with G82, R60, S268 elicited the loss of glyphosate tolerance of EPSPS. So V82, Q60, or T268 played an important role in glyphosate tolerance of EPSPS. The replacement of these amino acid residues was not shown to affect this enzyme activity, suggesting that they

do not play a major role in the enzyme activity of EPSPS. In brief, the enzyme activity of EPSPS was decided by the 3D structure and backbone. Eventually the EPSPS lost the glyphosate tolerance in accordance with the replacement of these amino-acid residues, which leading to conformational change, especially the torsion angle change.

Acknowledgements This work was supported by High-technology Research and Development Program of China, and in part fi nancial support from Program on Special Problems and Specifi c Project for Gene-transferring of China .We thank Charlie Davis for generously reviewing this work.

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