Glyphosate Resistance by Engineering the Flavoenzyme Glycine Oxidase

Abstract

Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and d-isomer of amino acids to yield the corresponding α-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the kcat/Km ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the α2-α3 loop (comprising residues 50–60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg54 could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide. Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and d-isomer of amino acids to yield the corresponding α-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the kcat/Km ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the α2-α3 loop (comprising residues 50–60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg54 could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide. IntroductionGlycine oxidase (GO) 2The abbreviations used are: GOglycine oxidaseEPSPs5-enolpyruvylshikimate-3-phosphate synthaseAMPAaminomethylphosphonate. from Bacillus subtilis (EC 1.4.3.19) is a flavoprotein oxidase consisting of four identical subunits; each subunit (≈42 kDa) contains one molecule of noncovalently bound FAD (1.Job V. Marcone G.L. Pilone M.S. Pollegioni L. J. Biol. Chem. 2002; 277: 6985-6993Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 2.Job V. Molla G. Pilone M.S. Pollegioni L. Eur. J. Biochem. 2002; 269: 1456-1463Crossref PubMed Scopus (41) Google Scholar). This enzyme catalyzes the oxidation of glycine in the biosynthesis of the thiazole ring of thiamine. Its crystal structure was solved as a complex with the inhibitor glycolate up to 1.8 Å resolution (3.Settembre E.C. Dorrestein P.C. Park J.H. Augustine A.M. Begley T.P. Ealick S.E. Biochemistry. 2003; 42: 2971-2981Crossref PubMed Scopus (97) Google Scholar, 4.Mörtl M. Diederichs K. Welte W. Molla G. Motteran L. Andriolo G. Pilone M.S. Pollegioni L. J. Biol. Chem. 2004; 279: 29718-29727Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). From a structural point of view, GO is classified as a member of the glutathione reductase family (subfamily GR2); monomeric sarcosine oxidase and d-amino acid oxidase represent closer structural neighbors (4.Mörtl M. Diederichs K. Welte W. Molla G. Motteran L. Andriolo G. Pilone M.S. Pollegioni L. J. Biol. Chem. 2004; 279: 29718-29727Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). GO and d-amino acid oxidase catalyze the oxidative deamination of amino acids to yield the corresponding α-imino acids and, after hydrolysis, α-keto acids, ammonia (or primary amines), and hydrogen peroxide (Scheme 1a), but they differ in substrate specificity. In addition to neutral d-amino acids (e.g. d-alanine and d-proline, which are also good substrates of d- amino acid oxidase), GO catalyzes the oxidation of primary and secondary amines (e.g. glycine, sarcosine) partially sharing substrate specificity with monomeric sarcosine oxidase (1.Job V. Marcone G.L. Pilone M.S. Pollegioni L. J. Biol. Chem. 2002; 277: 6985-6993Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 2.Job V. Molla G. Pilone M.S. Pollegioni L. Eur. J. Biochem. 2002; 269: 1456-1463Crossref PubMed Scopus (41) Google Scholar).Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid. This dominant herbicide (almost 90% of crops worldwide) inhibits the monomeric enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the penultimate step in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. Glyphosate is a competitive inhibitor with respect to phosphoenolpyruvate of EPSPs, which is more effective on the plant enzyme with respect to the bacterial homologues. The details of the structure-function relationships in EPSPs from Agrobacterium sp. strain CP4 have also been elucidated (5.Funke T. Han H. Healy-Fried M.L. Fischer M. Schönbrunn E. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13010-13015Crossref PubMed Scopus (237) Google Scholar). Its efficacy against all plant species, low cost, and low mammalian toxicity impact highly favor its use in crops that have a transgenic tolerance mechanism (6.Padgette S.R. Kolacz K.H. Delannay X. Re D.B. LaVallee B.J. Tinius C.N. Rhodes W.K. Otero Y.I. Barry G.F. Eichholz D.A. Peschke V.M. Nida D.L. Taylor N.B. Kishore G.M. Crop. Sci. 1995; 35: 1451-1461Crossref Scopus (459) Google Scholar, 7.Duke S.O. Powles S.B. Pest. Manag. Sci. 2008; 64: 319-325Crossref PubMed Scopus (1047) Google Scholar). The currently commercially used glyphosate-resistant mechanisms are based on the overproduction of the plant enzyme (8.Goldsbrough P.B. Hatch E.M. Huang B. Kosinski W.G. Dyer W.E. Herrmann K.M. Weller S.C. Plant Sci. 1990; 72: 53-62Crossref Scopus (66) Google Scholar) and on a transgene encoding a bacterial EPSPs that is insensitive to inhibition by glyphosate (6.Padgette S.R. Kolacz K.H. Delannay X. Re D.B. LaVallee B.J. Tinius C.N. Rhodes W.K. Otero Y.I. Barry G.F. Eichholz D.A. Peschke V.M. Nida D.L. Taylor N.B. Kishore G.M. Crop. Sci. 1995; 35: 1451-1461Crossref Scopus (459) Google Scholar, 7.Duke S.O. Powles S.B. Pest. Manag. Sci. 2008; 64: 319-325Crossref PubMed Scopus (1047) Google Scholar). Most recently, a transgene encoding a soil microorganism glyphosate oxidoreductase that cleaves the nitrogen-carbon bond in glyphosate yielding aminomethylphosphonic acid (AMPA) and glyoxylate (9.Castle L.A. Siehl D.L. Gorton R. Patten P.A. Chen Y.H. Bertain S. Cho H.J. Duck N. Wong J. Liu D. Lassner M.W. Science. 2004; 304: 1151-1154Crossref PubMed Scopus (242) Google Scholar, 10.Pline-Srnic W. Weed Technol. 2006; 20: 290-300Crossref Scopus (54) Google Scholar) and the N-acetylation of the herbicide by an evolved bacterial acetyltransferase were proposed as further mechanisms for glyphosate tolerance in crops (11.Siehl D.L. Castle L.A. Gorton R. Chen Y.H. Bertain S. Cho H.J. Keenan R. Liu D. Lassner M.W. Pest Manag. Sci. 2005; 61: 235-240Crossref PubMed Scopus (46) Google Scholar, 12.Siehl D.L. Castle L.A. Gorton R. Keenan R.J. J. Biol. Chem. 2007; 282: 11446-11455Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Currently, the only independent mechanism to give sufficient and stable resistance to glyphosate for commercialization of glyphosate-resistant crops has been glyphosate-resistant forms of EPSPs (10.Pline-Srnic W. Weed Technol. 2006; 20: 290-300Crossref Scopus (54) Google Scholar).The investigation of the crystal structure and of the substrate specificity of GO prompted us to consider this flavoenzyme as a starting point for the evolution of new functions of biotechnological interest. In particular, we report here on a protein engineering study aimed to develop the activity of GO on glyphosate with respect to glycine (the physiological substrate). We thus propose that introducing the gene coding for the evolved GO in plants is an effective alternative mechanism for glyphosate tolerance in transgenic crops. In addition, transgenic plants that are able to oxidize glyphosate may represent an innovative bioremediation system for the soil treated with this herbicide.DISCUSSIONIn this paper, we describe the evolution of B. subtilis glycine oxidase to obtain an enzyme with a high catalytic efficiency on the herbicide glyphosate. First, we analyzed the substrate preference of wild-type GO, highlighting its wide substrate specificity (Fig. 1). We also demonstrated that wild-type GO oxidizes the herbicide glyphosate, which is a synthetic amino acid largely used in agriculture and which presents structural features similar to those of glycine (Scheme 1c). GO catalyzes the deaminative oxidation of glyphosate because it yields glyoxylate, AMPA, and hydrogen peroxide using 1 mol of dioxygen/1 mol of herbicide (Scheme 1b). This reaction differs from that of glyphosate oxidoreductase because of a stoichiometry of two molecules of glyphosate oxidized per molecule of oxygen and the lack of hydrogen peroxide production (9.Castle L.A. Siehl D.L. Gorton R. Patten P.A. Chen Y.H. Bertain S. Cho H.J. Duck N. Wong J. Liu D. Lassner M.W. Science. 2004; 304: 1151-1154Crossref PubMed Scopus (242) Google Scholar, 10.Pline-Srnic W. Weed Technol. 2006; 20: 290-300Crossref Scopus (54) Google Scholar). Furthermore, the mechanism proposed for the oxidoreductase in which the reduced flavin obtained by oxidation of the first molecule of glyphosate catalyzes the oxygenation of a second molecule of glyphosate is profoundly different from the hydride transfer mechanism proposed for GO (3.Settembre E.C. Dorrestein P.C. Park J.H. Augustine A.M. Begley T.P. Ealick S.E. Biochemistry. 2003; 42: 2971-2981Crossref PubMed Scopus (97) Google Scholar, 4.Mörtl M. Diederichs K. Welte W. Molla G. Motteran L. Andriolo G. Pilone M.S. Pollegioni L. J. Biol. Chem. 2004; 279: 29718-29727Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar).The efficient oxidation of glyphosate by wild-type GO is limited by the low apparent affinity (Km,app = ≈90 mm; Table 2). To understand the putative mode of glyphosate binding at the GO active site, an in silico molecular docking analysis was carried out. In the best solutions glyphosate is bound in the same orientation inferred for its natural substrate glycine and with the phosphonate moiety pointing toward the entrance of the active site. The docking analysis identified the residues potentially involved in glyphosate binding (Fig. 2B). A number of GO variants with improved activity on glyphosate were recognized after site saturation mutagenesis on 11 positions of the active site. Finally, we combined the information gathered from the site saturation mutagenesis approach, producing a GO variant containing multiple mutations (G51S/A54R/H244A) and possessing an apparent specificity constant (the ratio between kcat/Km for the glyphosate and glycine) up to 15,000-fold higher than that for the wild-type enzyme. A total of three amino acid replacements introduced into wild-type GO suffice to provide a 13.2 kJ/mol decrease in the free energy of activation for the reaction on glyphosate and a 11.1 kJ/mol increase of the same value for the oxidation of glycine (ΔΔG values were calculated from the data in Table 2).The crystal structure of the best GO variant in complex with glycolate enables us to pinpoint structural features that affect the evolution of its substrate preference. The substitutions introduced in the evolved G51S/A54R/H244A GO appear to modify its substrate preference acting in different ways: (i) The α2-α3 loop (comprising residues 50–60 and possessing a high mobility) assumes a different conformation in the G51S/A54R/H244A variant as compared with wild-type GO. This rearrangement is mainly due to the introduction of the bulky side chain of arginine at position 54, which appears to locate close to the phosphonate group of glyphosate. This mutation replaces a neutral residue with a positively charged amino acid; new electrostatic interactions in G51S/A54R/H244A GO allow a tighter binding of the herbicide, maintaining the optimal positioning for catalysis (Fig. 4B). (ii) The introduction of arginine residue(s) at the active site entrance (positions 51 and 54) improves the kinetic efficiency of GO on glyphosate by decreasing the Km,app value (up to 20-fold in the G51R/A54R GO), whereas the maximal activity was only slightly affected (maximal change was a 2-fold increase in the G51R variant compared with the wild-type GO; Table 2). On the other hand, this substitution(s) negatively affects the protein stability; the G51R/A54R double variant shows a drastically lower stability than the wild type, and the other evolved GO variants (its activity is fully lost after 50 h of incubation at room temperature; Table 3), thus limiting its biotechnological use. Instead, the G51S/A54R/H244A GO shows stability, which is suitable for its utilization at room temperature; 60 and 70% of residual activity after 3 days at room temperature was measured for this variant and wild-type GO, respectively. (iii) The dramatic increase in specificity constant observed for the best GO variant is also due to the decrease in kinetic efficiency on glycine. This effect, too, is mainly due to a decrease in binding energy (Km,app for glycine is ≈150-fold higher in G51S/A54R/H244A than for the wild-type GO), whereas the maximal activity on glycine is only marginally altered in the evolved GO variants (Table 2). (iv) The presence of an alanine residue at position 244 only marginally affects the kinetic properties of GO when it is the only mutation introduced in the protein, but it had a synergistic effect both on the activity on glyphosate as substrate (Table 2) and the level of protein expression (supplemental Table S1) when it is introduced in the G51S/A54R GO. It appears that the smaller alanine residue at position 244 eliminates steric clashes with Glu55, for example, thus facilitating the interaction between Arg54 and glyphosate in the evolved GO variants.The observation that the same products are produced by glyphosate oxidation using GO and glyphosate oxidoreductase (i.e. AMPA and glyoxylate) might suggest a close similarity between these two FAD-containing flavoenzymes. The reaction catalyzed by the two enzymes strongly differs concerning the oxygen utilization and hydrogen peroxide production. Furthermore, the two enzymes show a low sequence identity (18.1%); a Blast analysis identifies a number of d-amino acid dehydrogenase as the most related proteins for glyphosate oxidoreductase. A further main difference is related to the kinetic properties of the two glyphosate-converting enzymes; although the apparent maximal activity reported for glyphosate oxidoreductase is not easily comparable with the value determined for GO (it was measured at higher temperature and on crude extracts, determining the amount of enzyme by means of Western blot analysis), the evolved GO we describe here shows a 5-fold lower Km for glyphosate and a kinetic efficiency (kcat/Km ratio) 1 order of magnitude higher than that of the best variant obtained for glyphosate oxidoreductase (2.1 versus 0.3 mm−1 s−1, respectively) (9.Castle L.A. Siehl D.L. Gorton R. Patten P.A. Chen Y.H. Bertain S. Cho H.J. Duck N. Wong J. Liu D. Lassner M.W. Science. 2004; 304: 1151-1154Crossref PubMed Scopus (242) Google Scholar, 10.Pline-Srnic W. Weed Technol. 2006; 20: 290-300Crossref Scopus (54) Google Scholar). This higher catalytic efficiency is most apparent when comparing recombinant proteins expressed in E. coli. The specific activity in the crude extract of the evolved GO described here is 20 milliunits/mg of protein, whereas that for glyphosate oxidoreductase is ≤0.015 milliunit/mg of protein. The low level of activity observed for the oxidoreductase might explain the limitations encountered to develop commercially available crops based on this enzyme. Concerning the toxicity of GO-produced glyphosate products, AMPA has been shown to be mildly toxic to soybean (24.Hoagland R.E. Weed Sci. 1980; 28: 393-400Crossref Google Scholar, 25.Reddy K.N. Rimando A.M. Duke S.O. WSSA Abstr. 2004; 44: 116Google Scholar); this potential for toxicity has not been reported in other glyoxylate oxidoreductase-containing plants treated with glyphosate.In conclusion, our work shows that evolved GO may be a novel system for the development of glyphosate-tolerant transgenic plants and may represent an effective supplement or alternative to the transgene currently being used that is based on an EPSPs insensitive to inhibition by glyphosate. The kcat/Km of G51S/A54R/H244A GO enzyme for glyphosate is ≈2 s−1, lower than maximal value the dramatic in kinetic efficiency with glyphosate, GO is from the of IntroductionGlycine oxidase (GO) 2The abbreviations used are: GOglycine oxidaseEPSPs5-enolpyruvylshikimate-3-phosphate synthaseAMPAaminomethylphosphonate. from Bacillus subtilis (EC 1.4.3.19) is a flavoprotein oxidase consisting of four identical subunits; each subunit (≈42 kDa) contains one molecule of noncovalently bound FAD (1.Job V. Marcone G.L. Pilone M.S. Pollegioni L. J. Biol. Chem. 2002; 277: 6985-6993Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 2.Job V. Molla G. Pilone M.S. Pollegioni L. Eur. J. Biochem. 2002; 269: 1456-1463Crossref PubMed Scopus (41) Google Scholar). This enzyme catalyzes the oxidation of glycine in the biosynthesis of the thiazole ring of thiamine. Its crystal structure was solved as a complex with the inhibitor glycolate up to 1.8 Å resolution (3.Settembre E.C. Dorrestein P.C. Park J.H. Augustine A.M. Begley T.P. Ealick S.E. Biochemistry. 2003; 42: 2971-2981Crossref PubMed Scopus (97) Google Scholar, 4.Mörtl M. Diederichs K. Welte W. Molla G. Motteran L. Andriolo G. Pilone M.S. Pollegioni L. J. Biol. Chem. 2004; 279: 29718-29727Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). From a structural point of view, GO is classified as a member of the glutathione reductase family (subfamily GR2); monomeric sarcosine oxidase and d-amino acid oxidase represent closer structural neighbors (4.Mörtl M. Diederichs K. Welte W. Molla G. Motteran L. Andriolo G. Pilone M.S. Pollegioni L. J. Biol. Chem. 2004; 279: 29718-29727Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). GO and d-amino acid oxidase catalyze the oxidative deamination of amino acids to yield the corresponding α-imino acids and, after hydrolysis, α-keto acids, ammonia (or primary amines), and hydrogen peroxide (Scheme 1a), but they differ in substrate specificity. In addition to neutral d-amino acids (e.g. d-alanine and d-proline, which are also good substrates of d- amino acid oxidase), GO catalyzes the oxidation of primary and secondary amines (e.g. glycine, sarcosine) partially sharing substrate specificity with monomeric sarcosine oxidase (1.Job V. Marcone G.L. Pilone M.S. Pollegioni L. J. Biol. Chem. 2002; 277: 6985-6993Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 2.Job V. Molla G. Pilone M.S. Pollegioni L. Eur. J. Biochem. 2002; 269: 1456-1463Crossref PubMed Scopus (41) Google Scholar).Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid. This dominant herbicide (almost 90% of crops worldwide) inhibits the monomeric enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the penultimate step in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. Glyphosate is a competitive inhibitor with respect to phosphoenolpyruvate of EPSPs, which is more effective on the plant enzyme with respect to the bacterial homologues. The details of the structure-function relationships in EPSPs from Agrobacterium sp. strain CP4 have also been elucidated (5.Funke T. Han H. Healy-Fried M.L. Fischer M. Schönbrunn E. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13010-13015Crossref PubMed Scopus (237) Google Scholar). Its efficacy against all plant species, low cost, and low mammalian toxicity impact highly favor its use in crops that have a transgenic tolerance mechanism (6.Padgette S.R. Kolacz K.H. Delannay X. Re D.B. LaVallee B.J. Tinius C.N. Rhodes W.K. Otero Y.I. Barry G.F. Eichholz D.A. Peschke V.M. Nida D.L. Taylor N.B. Kishore G.M. Crop. Sci. 1995; 35: 1451-1461Crossref Scopus (459) Google Scholar, 7.Duke S.O. Powles S.B. Pest. Manag. Sci. 2008; 64: 319-325Crossref PubMed Scopus (1047) Google Scholar). The currently commercially used glyphosate-resistant mechanisms are based on the overproduction of the plant enzyme (8.Goldsbrough P.B. Hatch E.M. Huang B. Kosinski W.G. Dyer W.E. Herrmann K.M. Weller S.C. Plant Sci. 1990; 72: 53-62Crossref Scopus (66) Google Scholar) and on a transgene encoding a bacterial EPSPs that is insensitive to inhibition by glyphosate (6.Padgette S.R. Kolacz K.H. Delannay X. Re D.B. LaVallee B.J. Tinius C.N. Rhodes W.K. Otero Y.I. Barry G.F. Eichholz D.A. Peschke V.M. Nida D.L. Taylor N.B. Kishore G.M. Crop. Sci. 1995; 35: 1451-1461Crossref Scopus (459) Google Scholar, 7.Duke S.O. Powles S.B. Pest. Manag. Sci. 2008; 64: 319-325Crossref PubMed Scopus (1047) Google Scholar). Most recently, a transgene encoding a soil microorganism glyphosate oxidoreductase that cleaves the nitrogen-carbon bond in glyphosate yielding aminomethylphosphonic acid (AMPA) and glyoxylate (9.Castle L.A. Siehl D.L. Gorton R. Patten P.A. Chen Y.H. Bertain S. Cho H.J. Duck N. Wong J. Liu D. Lassner M.W. Science. 2004; 304: 1151-1154Crossref PubMed Scopus (242) Google Scholar, 10.Pline-Srnic W. Weed Technol. 2006; 20: 290-300Crossref Scopus (54) Google Scholar) and the N-acetylation of the herbicide by an evolved bacterial acetyltransferase were proposed as further mechanisms for glyphosate tolerance in crops (11.Siehl D.L. Castle L.A. Gorton R. Chen Y.H. Bertain S. Cho H.J. Keenan R. Liu D. Lassner M.W. Pest Manag. Sci. 2005; 61: 235-240Crossref PubMed Scopus (46) Google Scholar, 12.Siehl D.L. Castle L.A. Gorton R. Keenan R.J. J. Biol. Chem. 2007; 282: 11446-11455Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Currently, the only independent mechanism to give sufficient and stable resistance to glyphosate for commercialization of glyphosate-resistant crops has been glyphosate-resistant forms of EPSPs (10.Pline-Srnic W. Weed Technol. 2006; 20: 290-300Crossref Scopus (54) Google Scholar).The investigation of the crystal structure and of the substrate specificity of GO prompted us to consider this flavoenzyme as a starting point for the evolution of new functions of biotechnological interest. In particular, we report here on a protein engineering study aimed to develop the activity of GO on glyphosate with respect to glycine (the physiological substrate). We thus propose that introducing the gene coding for the evolved GO in plants is an effective alternative mechanism for glyphosate tolerance in transgenic crops. In addition, transgenic plants that are able to oxidize glyphosate may represent an innovative bioremediation system for the soil treated with this herbicide.