3if9
Crystal structure of Glycine Oxidase G51S/A54R/H244A mutant in complex with inhibitor glycolate
Structural highlights
FunctionGLYOX_BACSU Catalyzes the FAD-dependent oxidative deamination of various amines and D-amino acids to yield the corresponding alpha-keto acids, ammonia/amine, and hydrogen peroxide. Oxidizes sarcosine (N-methylglycine), N-ethylglycine and glycine (PubMed:9827558, PubMed:11744710, PubMed:19864430). Can also oxidize the herbicide glyphosate (N-phosphonomethylglycine) (PubMed:19864430). Displays lower activities on D-alanine, D-valine, D-proline and D-methionine (PubMed:9827558, PubMed:11744710). Does not act on L-amino acids and other D-amino acids (PubMed:9827558). Is essential for thiamine biosynthesis since the oxidation of glycine catalyzed by ThiO generates the glycine imine intermediate (dehydroglycine) required for the biosynthesis of the thiazole ring of thiamine pyrophosphate (PubMed:12627963).[1] [2] [3] [4] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedGlycine 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 alpha-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 k(cat)/K(m) ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the alpha2-alpha3 loop (comprising residues 50-60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg(54) could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide. Glyphosate resistance by engineering the flavoenzyme glycine oxidase.,Pedotti M, Rosini E, Molla G, Moschetti T, Savino C, Vallone B, Pollegioni L J Biol Chem. 2009 Dec 25;284(52):36415-23. Epub 2009 Oct 28. PMID:19864430[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
| ||||||||||||||||||||
