Characterization of a novel cold-adapted phosphinothricin N-acetyltransferase from the marine bacterium Rhodococcus sp. strain YM12

Journal of Molecular Catalysis B: Enzymatic 104 (2014) 23–28

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Characterization of a novel cold-adapted phosphinothricin N-acetyltransferase from the marine bacterium Rhodococcus sp. strain YM12 Gaobing Wu a , Mingru Yuan a , Lu Wei a , Yi Zhang a , Yongjun Lin a , Lili Zhang b , Ziduo Liu a,∗ a

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production and Construction Corps, College of Life Science, Tarim University, Alar 843300, Xinjiang, China b

a r t i c l e

i n f o

Article history: Received 27 November 2013 Received in revised form 24 February 2014 Accepted 4 March 2014 Available online 13 March 2014 Keywords: Phosphinothricin Phosphinothricin-N-acetyltransferase Cold-adapted enzyme

a b s t r a c t Phosphinothricin (PPT) is a kind of non-selective, environmentally friendly herbicide. PPT-tolerance genes are vital in both plant biotechnology as selectable markers and the development of transgenic herbicideresistant crops. However, there are no other well-identified and commercially available PPT-resistance genes for use in plant genetic engineering besides two PPT N-acetyltransferase genes, which known as pat and bar derived from Streptomyces sp. Here, we isolated a novel PPT N-acetyltransferase gene from PPTresistant marine bacteria, Rhodococcus sp. strain YM12. The gene, designated as RePAT, encoded a protein (RePAT) of 162 amino acids, which showed 37% identity with that of PAT proteins. Key kinetic constants of RePAT were determined (Km = 0.076 mM, Kcat = 131 min−1 ) using PPT as a substrate, the enzyme retained considerable activity at pH 8.0 and had an optimum temperature of 35 ◦ C. Interestingly, it possessed over 50% of its maximal activity at temperature conditions between 0 and 10 ◦ C, suggesting that this enzyme is able to protect crop against PPT injury in cold environment. These results illustrated that RePAT could be a new resource for herbicide detoxification by transgenic crops. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Recently, much attention has been focused on the enzymatic modification of nonselective herbicides, owing to the potential application of the genes encoding such enzymes in agricultural biotechnology. Enzymes involved in amino acid biosynthesis in plant are thought to be desirable targets to kill the plants because some of these enzymes do not exist in animal cells [1]. Thereinto, glutamine synthetase (GS), which is an important enzyme in ammonia assimilation and nitrogen metabolism regulation in higher plants and microorganisms, is a target candidate. The inhibition of this factor could block an important pathway of glutamine biosynthesis, leading to a rapid depletion of glutamine in plants and accumulation of ammonia in photosynthetic tissues during photorespiration, causing cell death [2]. Phosphinothricin (also known as Glufosinate) is a broad spectrum, nonselective herbicide with good biodegradability. It specifically inhibits glutamine synthetase in plants [3] and

∗ Corresponding author. Tel.:.+86 15 927372165; fax: +86 27 87280670. E-mail address: [email protected] (Z. Liu). http://dx.doi.org/10.1016/j.molcatb.2014.03.001 1381-1177/© 2014 Elsevier B.V. All rights reserved.

functions well on perennial weeds. Creation of PPT-tolerant transgenic crops by the introduction of PPT-detoxifying gene has made PPT exert its advantages in weed control in a cultivated field. However, there are only two commercially available PPT-detoxifying genes for generation of herbicide-resistance crops, i.e., bar from Streptomyces hygroscopicus [4,5] and pat from Streptomyces viridochromogenes [6,7]. The two gene both encode phosphinothricin N-acetyltransferase (PAT)(EC2.3.1.183), which can detoxify PPT by acetylating the free amino group of PPT, and have been successfully used to engineer commercial transgenic crops. Moreover, phosphinothricin resistance gene is also an important tool in plant biotechnology as a selectable marker [8–12]. A safety assessment indicated that PAT had no side-effects on animals, feeds and food derived from transgenic crops expressing PAT and is therefore considered safe to use [13]. Considerable effort has been made to search new PPT-resistance gene from soil microbes. But little attention has been paid to the marine microbes, which has been proven to be a rich source of various novel and unique genes. In this work, a novel phosphinothricin N-acetyltransferase (RePAT) from the marine bacterium Rhodococcus sp. was obtained. The RePAT can utilize both PPT and l-methionine sulfoximine as substrates. The latter is also a

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glutamate analog that inhibits glutamine synthetase (GS) [14]. This new gene might provide us alternative genetic tool for use in agricultural biotechnology field for the creation of herbicide-resistant transgenic crops. 2. Materials and methods 2.1. Bacterial strains and chemicals Deep sea mud was obtained from the harbor of Xiamen city in China. Escherichia coli DH5␣ was used for DNA manipulation, and E. coli BL21 (DE3) was used as a host for gene expression. The plasmid pUC18 (TaKaRa, Japan) was used for construction of the genomic library, and the vector pGEX-6p-1 was used for the expression of RePAT (GE Healthcare). A GST-Bind Purification kit was purchased from Novagen (Germany). The DNA purification kit was purchased from Sangon Biotech (China). The DNA restriction endonucleases, T4 DNA ligase, DNA polymerase and protein markers were obtained from TaKaRa (Japan). All other reagents were purchased from Sigma (USA), unless otherwise specified. 2.2. Screening and identification of PPT-resistant strain For PPT-resistant strain screening, the marine bacterium, isolated from deep sea mud samples provided by Third Institute of Oceanography in China, were cultured at 28 ◦ C in M9 minimal medium supplemented with 0.4% (w/v) glucose as a carbon source and 50 mM PPT. After 5 to 7 days, colonies of the strains that grew rapidly were collected and further verified on M9 plates in the presence of 100 mM glyphosate PPT. After two round screening, one strainYM12 with high PPT-resistant obtained and its genomic DNA was prepared according to the methods described elsewhere [15]. To identify the strain, the 16S rRNA gene was amplified from the genomic DNA with the primers 16SF (5 -AGAGTTTGATCMT GGCTCAG-3 ) and 16SR (5 -TACGGYTACCTTGTTACGACTT-3 ), and the PCR fragments were sequenced by Genscript (Nanjing, China). 2.3. Isolated the gene involved in PPT-resistance The genomic DNA of YM12 was partially digested with BamHI. Fragments ranging from 3 to 9 kb were harvested by Gel Extraction Kit, and cloned into the same site of a calf intestinal alkaline phosphatase (CIAP)-treated pUC18 plasmid. The ligation products were transformed into E. coli DH5␣ using a heat shock process. The transformants were spread on LB plates containing 1 mM isopropyl-d-thiogalactopyranoside (IPTG) and 40 mg/ml of 5-bromo-4-chloro-3-indolyl-␤-d-galactopyranoside (X-gal) for a blue-white screening. The plates were then incubated overnight at 37 ◦ C. To identify the PPT resistance gene, all white colonies were picked and transferred to M9 minimal medium plates containing 5 mM PPT, followed by transferring the resistant colonies to the same medium containing 50 mM PPT. As a result, one colony harboring a recombinant plasmid pUC18-3k with about 3.0 kb insert fragment was obtained, and the interest fragment was sequenced by the Genscript (Nanjing, China). 2.4. Gene analysis and cloning DNA sequences were analyzed using the Softberry Gene Finding tool (http://linuxl.softberry.com/berry/). The DNA and protein sequence alignments were carried out using the Blast program (http://blast.ncbi.nlm.nih.gov/Blast). The encoding sequence was amplified using the primers patF (5 CGGAATTCATGCTGATCCGCGACGCC-3 , with EcoRI site underlined) and patR (5 -ATAAGAATGCGGCCGCCTAGAGGGTCAGCTGCAG-3 , with NotI site underlined). PCR conditions were as follows: one

cycle of 4 min at 94 ◦ C, followed by 30 cycles of 30 s at 94 ◦ C, 30 s at 55 ◦ C, and 30 s at 72 ◦ C, with a final cycle of 10 min at 72 ◦ C. The PCR products were then digested by EcoRI and NotI, and then purified by agarose gel electrophoresis. The purified products were ligated into the corresponding sites of the vector pGEX-6p-1. The recombinant plasmid pGEX-6p-RePAT was confirmed by DNA sequencing, and then transformed into E. coli BL21 (DE3) for protein expression and purification. 2.5. Expression and purification of RePAT To prepare the recombinant RePAT, the bacteria were cultured overnight in LB medium supplemented with 100 ␮g/ml of ampicillin at 37 ◦ C. The culture was then inoculated into 1L fresh LB medium (1:100 dilution) containing 100 ␮g/ml ampicillin and grown at 37 ◦ C to an OD600 of 0.5–0.6. Expression was induced by the addition of IPTG to a final concentration of 0.1 mM, and the culture was incubated for 8 h at 18 ◦ C. The cells were then collected by centrifugation, washed and resuspended in phosphate buffer (pH 7.4, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 and 1.8 mM KH2 PO4 ). The cells were subjected to high pressure (1000 bar) at 4 ◦ C. The lysates were centrifuged at 12,000 × g for 30 min at 4 ◦ C and the supernatant was collected. The protein RePAT was purified using glutathione-S-transferase (GST) [16]. The GST tag was removed by digestion with a 3 C protease solution (10 U/␮l, PreScission, Pharmacia), and the purified protein was eluted with phosphate buffer [16]. All purification operations were conducted at 4 ◦ C. The protein was quantified using the Bradford reagent (Sigma, USA), and the purity of the extracted proteins was analyzed by sodium dodecyl sulfate denatured polyacrylamide gel electrophoresis (SDS-PAGE). 2.6. Enzyme assay Assays for N-acetyltransferase activity were assayed by measuring the rate of formation of the enzymatic product, CoASH, which was determined by its reaction with 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB). The yielding yellow 5-thio-2-nitrobenzoicacid was determined by spectrophotometry at 412 nm [17]. Reactions were conducted at 25 ◦ C for 30 min in a volume of 0.2 mL system containing 50 mM Tris buffer (pH = 8.0) and 0.2 mM DTNB, and then terminated by adding 6.0 M guanidine hydrochloride. The absorbance changes were measured at 412 nm with Thermo scientific multiscan spectrophotometer. One unit of RePAT activity was defined as the amount of enzyme liberating 1 ␮mol/min of CoASH. To determine the optimum condition for the activity of RePAT, the relative activity was determined at several temperatures and several pH levels. The effect of the temperature on RePAT activity was analyzed by testing the RePAT activity at a range of temperatures (0 to 70 ◦ C) by using the standard assay procedure. The optimal pH was determined by assaying the enzyme at 25 ◦ C with the following buffer systems: 0.2 M Na2 HPO4 -citric acid buffer (pH 4.0–8.0) and Gly–NaOH buffer (pH 9.0–11.0). Enzyme activities are compared to the largest activity value attained (i.e., 100% relative activity). For the thermal stability assay, the purified RePAT was incubated at 30, 40 and 50 ◦ C for different time, and the remaining activity was measured under standard conditions at 25 ◦ C. The protein treated at 4 ◦ C was used as control. To determine apparent Km and Kcat of the RePAT, the initial reaction velocitiesat at various concentrations of PPT (ranging from 0.05 to 4 mM) and l-methionine sulfoximine (ranging from 0.01 to 4 mM) were assayed, respectively. For both substrates, a near-saturating acetyl-CoA (0.3 mM) was used, and each assay was performed in triplicate. The Kinetic constants (Km and Vmax ) were determined by Lineweaver–Burk plots constructed from the initial velocities at various substrate concentrations. The Kcat was obtained with the following

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Fig. 1. Multiple alignment of the amino acid sequences of BAR (GenBank accession NO: CAA29262), PAT (GenBank accession NO: ZP 07302142), ScPAT (GenBank accession NO: NP 627417), MAT (GenBank accession NO: BAG06876), RePAT (this study), and Pita (GenBank accession NO: GAA20671). The amino acids involved in the binding of acetyl coenzyme A are indicated with filled inverted triangles.

equation: Kcat = Vmax /[E], where [E] is the enzyme concentration in the reaction mixture. 2.7. Evaluation of phosphinothricin tolerance in E. coli The E. coli DH5␣ was transformed with pGEX-6p-1 or pGEX-6pRePAT and grown by shaking at 37 ◦ C in liquid M9 minimal medium supplemented with PPT at concentrations of 0 mM, 50 mM and 100 mM. The growth of the cell with or without RePAT gene was observed by measured the OD600 values at 10-h intervals.

from S. viridochromogenes (GenBank accession NO: AAA72709.1) and BAR from S. hygroscopicus (GenBank accession NO: CAA29262). RePAT also had 33% identity with ScPAT (GenBank accession NO: NP 627417), 59% identity with MAT (GenBank accession NO: BAG06876) and 58% identity with (GenBank accession NO: 2J8M A). Based on the protein sequence alignment (Fig. 1), RePAT was high conserved at Glu82, Tyr86, Gly95, Lue100, Ala118, 124Asn and 127Ser, which are putatively to form a binding pocket for the substrate acetyl coenzyme A. Phylogenetic analysis (Fig. 2) indicated that RePAT and MAT belong to the same family, and RePAT is evolutionary distant from the Bar and Pat from Streptomyces sp.

2.8. 3D molecular modeling To identify homologous structures of RePAT, the Protein Data Bank (PDB) had been searched. A multiple sequence alignment was carried out using the CLUSTALW program in Geneious. The crystal structure of RePAT was predicted using the Swiss-Model (http://swissmodel.expasy.org/), and Pita (PDB accession NO: 2BL1) was utilized as the template to generate this structure. The final models were analyzed using PROCHECK [18]. 3. Results 3.1. Strain Identification, cloning, and sequence analysis After two rounds of screening, one strain, YM12 with high PPTresistance was obtained from about 100 marine strains derived from deep sea mud. 16S rRNA gene sequence of YM12 showed a 99.629% identity with Rhodococcus equi DSM 20307(T) (GenBank accession NO: X80614), and therefore the strain was identified as Rhodococcus sp. YM12 (CCTCC AB2014018). To characterize the gene conferring Rhodococcus sp. strain YM12 with PPT-resistance, a genomic library was constructed and nearly 5000 transformants were screened. Consequently, a colony grew robustly on the screening plate containing 50 mM PPT was obtained. The 3.0 kb DNA fragment from YM12 chromosomal DNA was sequenced and analyzed. An open reading frame (ORF) of 489 bp with an initiation codon ATG and a termination codon TAG was founded. This ORF, named RePAT, encoding a 162 amino acids protein, named RePAT, with a calculated molecular mass of 18.0 kDa. The nucleotide sequence of the RePAT was submitted to the GenBank database under the accession number of JQ398613. Based on the BLASTP amino acid alignments, RePAT was classified into to the GCN5-related N-acetyltransferase (GNAT) family (PF00583). RePAT shared 37% identity with amino acids of PAT

3.2. Enzyme expression and purification The recombinant RePAT was expressed in E. coli BL21 (DE3). After induction, the supernatant from cell lysate of the E. coli harboring pGEX-6p-RePAT exhibited a thick band at approximately 44 kDa (Fig. 3, lane 5), consistent with the predicted molecular mass of RePAT (18 kDa) with a GST tag (26 kDa). This showed that the recombinant RePAT was overexpressed in soluble form. The supernatant showed a specific activity of 0.07 U/mg (Table 1), indicating that the fused protein GST-RePAT possessed PPT Nacetyltransferase activity. RePAT was purified to high homogeneity (Fig. 3, lane 6) by GSH-agarose affinity chromatography followed by on-column cleavage with PreScission protease to remove the GST tag. A negligible, minor band might be a protein co-purified with RePAT. As shown in Table 1, about 1.08 mg of RePAT was purified 334.3-fold with a yield of 19.6% from 1L of IPTG-induced cultures of E. coli BL21.

3.3. Properties of purified RePAT The pH and temperature dependence on the activity of RePAT is shown in Fig. 4. It retains considerable activity between pH 7.5 and 8.5 with a maximum activity at pH 8.0. The optimum temperature of the enzyme was 35 ◦ C, and it possessed relatively high activity at temperatures ranging from 0 to 10 ◦ C. Even when temperatures fell to 0 ◦ C, the enzyme retained over 50% of its maximal activity, indicating it is a cold adapted enzyme. Thermal stability assay was carried out by measured the residual activity of the purified RePAT after incubating it at different temperature. As shown in Fig. 4C, the enzyme lost less than 20% activity at 30 ◦ C for 80 min incubation, and retained about 60% activity after being treated for 1 h at 40 and 50 ◦ C.

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Fig. 2. Phylogenetic tree analysis of RePAT using Maximum-Parsimony method in MAGE (version 4.0). Bar 0.1 substitutions per amino acid site. Agrobacterium radiobacter (GenBank accession NO: YP 002543819), Rhizobium leguminosarum (GenBank accession NO:YP 766974), Agrobacterium tumefaciens (GenBank accession NO: NP 353926), Agrobacterium vitis (GenBank accession NO: YP 002548913), Escherichia coli (GenBank accession NO: ZP 08353722), Pseudomonas syringae (GenBank accession NO: ZP 04586796), Pseudomonas aeruginosa (GenBank accession NO: GAA20671), Nocardia sp. (GenBank accession NO: BAG06876), Acinetobacter sp. (GenBank accession NO: YP 046305), Streptomyces viridochromogenes (GenBank accession NO: ZP 07302142), Streptomyces hygroscopicus (GenBank accession NO: CAA29262).

Table 1 Purification summary of the recombinant RePAT. Purification step

Total protein (mg)

Specific activity (U/mg)

Total act (U)

Yield (%)

Purification fold

Crude extract Affinity purification

1840 1.08

0.07 23.40

128.8 25.27

100 19.60

1.0 334.3

RePAT was able to utilize not only PPT but also l-methionine sulfoximine. The Km and Kcat values (Table 2) of purified enzyme with PPT and l-methionine sulfoximine suggested that PPT was the natural substrate. We also tried to use glyphosate as a substrate, but it had no effect (data not shown).

3.4. PPT-resistance assay in vitro To further evaluate the phosphinothricin-resistance of RePAT, the E. coli DH5␣ haboring plasmid pGEX-6p-RePAT was grown under various concentrations of PPT and the growth curves were monitored. As shown in Fig. 5, the cells containing RePAT grow well at 50 mM or 100 mM PPT. In contrast, the growth of the cells harboring no RePAT was strongly inhibited by 50 mM or 100 mM PPT. This result suggests that the gene RePAT can confer E. coli with high resistance to PPT. 3.5. 3D molecular modeling The structural information about the family of phosphinothricin N-acetyltransferase is limited. Among the RePAT homologues, only Pita, which shares 58% identity at amino acid level with RePAT, had a known crystal structure [19]. So it was chosen as the template for RePAT modeling using the software Molecular Operating Environment (MOE). The quality of the modeling was analyzed using PROCHECK [18]. Similar to Pita, the modeled structure of RePAT existed as a homodimer (Fig. 6), and was arranged in a similar manner to other dimeric GNAT superfamily members, such as the Salmonella typhimurium RimL N␣ -acetyltransferase [20],

Fig. 3. SDS-PAGE analysis of the overexpression and purification of RePAT. The soluble fragment from the cell lysate was used for each assay. Lanes: (1) protein marker; (2) uninduced cell lysate of E. coli BL21 (DE3) harboring pGEX-6p-1; (3) IPTG-induced cell lysate of E. coli BL21 (DE3) harboring pGEX-6p-1; (4) uninduced cell lysate of E. coli BL21 (DE3) harboring pGEX-6p-RePAT; (5) induced cell lysate of E. coli BL21 (DE3) harboring pGEX-6p-RePAT, the fusion protein GST-RePAT was marked by a white arrow; (6) purified RePAT.

Table 2 Kinetic Parameters for the RePAT. Kcat (min−1 )

Substrate

Km (mM)

Phosphinothricin Methionine sulfoximine

0.076 ± 0.005 0.156 ± 0.003a

a

Is the mean ± SEM.

a

131.4 ± 1.643a 29.8 ± 2.354a

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Fig. 4. Effects of temperature and pH on enzyme activity and thermal stability. (A) The effect of temperature on RePAT. The relative activity of RePAT was measured at temperatures ranging from 0 to 70 ◦ C. (B) The pH effect on RePAT. 0.2 M Na2 HPO4 -citric acid buffer (pH 4.0–8.0) and Gly–NaOH buffer (pH 9.0–11.0) were used. (C) Thermal stability assay. After incubation at 30, 40 and 50 ◦ C for different time, the residual activity was measured.

the interface between the two subunits and high closed to its counterpart. Presumably, ring stacking and salt bridge derived from His114-Trp152 and Arg72-Glu82, respectively, are important forces for the RePAT subunit interaction.

4. Discussion

Fig. 5. Growth curve of E. coli DH5␣ harboring either pGEX-6p-RePAT. or pGEX6p-1 in liquid M9 minimal medium supplemented with PPT at concentrations of 0 mM, 50 mM and 100 mM. filled circle, E. coli growing without PPT; filled square, E. coli harboring RePAT growing without PPT; filled inverted triangle, E. coli harboring RePAT growing in 50 mM PPT; blank circle, E. coli harboring RePAT growing in 100 mM PPT; filled triangle, E. coli growing in 50 mM PPT; filled rhombus, E. coli growing in 100 mM PPT.

Saccharomyces cerevisiae glucosamine-6-phosphate Nacetyltransferase GNA1 [21], and Enterococcus faecium aminoglycoside acetyltransferase AAC(6 ) [22]. As compared to Pita, in RePAT the sites Arg72, Glu82, His114 and Trp152 were conserved (Fig. 1). A previous report has illustrated these residues are important for formation of the homo-dimer in Pita. As shown in Fig. 6, the site Arg72, Glu82, His114 and Trp152 located at

Fig. 6. The homology model of RePAT. The structure of RePAT was modeled using MOE with the PPT N-acetyltransferase like protein, Pita [19], as a template. The residues Arg72, Glu82, His114 and Trp152 locate at the interface between the two subunits.

PPT N-acetyltransferase gene is a kind of herbicide-resistant gene resources that are indispensable elements in the field of crop genetic engineering. Many efforts have been made to explore new PPT resistant gene from the soil microorganisms [5–7,23,24], but little attention is paid to the marine environment, where rich unique enzyme and gene resources exist [25]. Here, to the best of our knowledge RePAT was the first characterized phosphinothricin-resistant gene from marine microorganism. RePAT showed low identity (37%) with the predominantly used PPT N-acetyltransferases PAT and BAR [5,6]. ScPAT from S. coelicolor and MAT from a Nocardia sp. are another two well-characterized PPT N-acetyltransferases (Table 3). RePAT displays 33% and 59% identity with ScPAT and MAT, respectively. Like MAT, RePAT can use both PPT and l-methionine sulfoximine as substrate [24], and the optimum substrate for both RePAT and MAT is PPT. Pita from P. aeruginosa had 58% sequence identity to RePAT, but it’s optimum substrate methionine sulfoximine has no activity toward PPT [19]. This data might provide new insight into functional diversity of the PPT-acetyltransferase homologues, which are widespread in microbial ecology [19,26]. Glutamine synthetase is the target of the herbicide PPT, and it localizes in the chloroplast and cytosol. The pH environment in the chloroplast stroma fluctuates with illumination, and is boosted to 8.0 by illuminated condition due to a photosynthetic CO2 fixation and proton flux from the thylakoid space into stroma [27,28]. Here, the optimum pH of RePAT was 8.0, and like other PPT Nacetyltransferases (Table 3), had considerable activity between pH 7.0 and 8.5, suggesting that the RePAT can function well in both chloroplast and cytosol. During the crop-growing season, the air temperatures are often as low as 10 ◦ C in night and as high as 40 ◦ C. It means that the ideal PPT-detoxifying enzyme for use in transgenic crop should work well in this temperature range. As shown in Table 3, the optimum temperature for the well characterized PPT N-acetyltransferases are 30 ◦ C However, Bar and Pat, which have been successfully used for the creation of PPT-tolerant crops, lose their activity by 80% at 10 ◦ C [29,30], and thus can’t adequately detoxify PPT under cooler conditions. Wendy has investigated the response of PPT-resistant soybeans containing bar gene to PPT, and found that Bar could not protect the soybeans against PPT injury at 15 ◦ C [31]. Here, the new

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Table 3 The comparison between the RePAT and other well characterized PPT N-acetyltransferases. Parameters

RePAT

Bar [5,29]

Pat [7,29]

Mat [24]

ScPat [30]

Amino acid identity to (%) Km for PPT (mM) Topt (◦ C) pHopt Proline content (%)

100 0.076 35 8.0 3.1

37 0.06 30 7.0 8.7

37 0.06 30 7.0 7.7

59 2.20 30 8.5 3.4

33 1.00 – – 8.8

Note: Topt , optimum temperature; pHopt , optimum pH.

PPT N-acetyltransferases (RePAT) isolated from the marine bacteria remains more than 80% of the original activity even at 15 ◦ C, and is moderately stable at temperature around 40 ◦ C. This means that the cold-adaptive RePAT may provide transgenic crop complete protection against PPT injury, and its encoding gene is a good candidate for use in developing PPT-resistant corps. Cold-adapted enzyme is believed to have an increased flexible structure, which may enhance substrate accessibility in cold environment [32,33]. Proline can reduce conformational flexibility of protein structure because its side chain is able bind to the N atom of the peptide backbone [32]. Here, RePAT has lower proline content than its mesophilic counterparts (Table 3), which might be a factor conferring more flexibility to the structure of RePAT. In summary, we identified a novel phosphinothricin Nacetyltransferase from the deep-sea mud bacterium Rhodococcus sp. strain YM12. Given the findings about the cold-adapted activity of the enzyme, it will be provide us a new genetic resource for creating herbicide-resistant crops. Efforts are underway to engineer PPT-resistant crops by using this new gene. Acknowledgments This work was supported by grants from the China National Natural Sciences Foundation (u1170303), the Genetically Modified Organisms Breeding Major Projects of China (2011zx08001-001), and the opening project of the Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Corps (BRZD1101). References [1] [2] [3] [4]

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