Publications

• Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein.

  Job V., Marcone G.L., Pilone M.S., Pollegioni L.

  Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) and d-amino acids (d-alanine, d-proline). The products of GO reaction ... >> More

  Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) and d-amino acids (d-alanine, d-proline). The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase. GO shows properties that are characteristic of the oxidase class of flavoproteins: it stabilizes the anionic flavin semiquinone and forms a reversible covalent flavin-sulfite complex. The approximately 300 mV separation between the two FAD redox potentials is in accordance with the high amount of the anionic semiquinone formed on photoreduction. GO can be distinguished from d-amino acid oxidase by its low catalytic efficiency and high apparent K(m) value for d-alanine. A number of active site ligands have been identified; the tightest binding is observed with glycolate, which acts as a competitive inhibitor with respect to sarcosine. The presence of a carboxylic group and an amino group on the substrate molecule is not mandatory for binding and catalysis. << Less

  J. Biol. Chem. 277:6985-6993(2002) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Structure and Enzymatic Properties of an Unusual Cysteine Tryptophylquinone-Dependent Glycine Oxidase from Pseudoalteromonas luteoviolacea.

  Andreo-Vidal A., Mamounis K.J., Sehanobish E., Avalos D., Campillo-Brocal J.C., Sanchez-Amat A., Yukl E.T., Davidson V.L.

  Glycine oxidase from Pseudoalteromonas luteoviolacea (PlGoxA) is a cysteine tryptophylquinone (CTQ)-dependent enzyme. Sequence analysis and phylogenetic analysis place it in a newly designated subgroup (group IID) of a recently identified family of LodA-like proteins, which are predicted to posses ... >> More

  Glycine oxidase from Pseudoalteromonas luteoviolacea (PlGoxA) is a cysteine tryptophylquinone (CTQ)-dependent enzyme. Sequence analysis and phylogenetic analysis place it in a newly designated subgroup (group IID) of a recently identified family of LodA-like proteins, which are predicted to possess CTQ. The crystal structure of PlGoxA reveals that it is a homotetramer. It possesses an N-terminal domain with no close structural homologues in the Protein Data Bank. The active site is quite small because of intersubunit interactions, which may account for the observed cooperativy toward glycine. Steady-state kinetic analysis yielded the following values: k_cat = 6.0 ± 0.2 s^-1, K_0.5 = 187 ± 18 μM, and h = 1.77 ± 0.27. In contrast to other quinoprotein amine dehydrogenases and oxidases that exhibit anomalously large primary kinetic isotope effects on the rate of reduction of the quinone cofactor by the amine substrate, no significant primary kinetic isotope effect was observed for this reaction of PlGoxA. The absorbance spectrum of glycine-reduced PlGoxA exhibits features in the range of 400-650 nm that have not previously been seen in other quinoproteins. Thus, in addition to the unusual structural features of PlGoxA, the kinetic and chemical reaction mechanisms of the reductive half-reaction of PlGoxA appear to be distinct from those of other amine dehydrogenases and amine oxidases that use tryptophylquinone and tyrosylquinone cofactors. << Less

  Biochemistry 57:1155-1165(2018) [PubMed] [EuropePMC]

• Purification and characterization of a novel glycine oxidase from Bacillus subtilis.

  Nishiya Y., Imanaka T.

  The open reading frame yjbR which had been sequenced as a part of the Bacillus subtilis genome project encodes a putative 40.9-kDa protein. The yjbR-coding sequence was slightly similar to those of bacterial sarcosine oxidases and possibly compatible with the tertiary structure of the porcine kidn ... >> More

  The open reading frame yjbR which had been sequenced as a part of the Bacillus subtilis genome project encodes a putative 40.9-kDa protein. The yjbR-coding sequence was slightly similar to those of bacterial sarcosine oxidases and possibly compatible with the tertiary structure of the porcine kidney D-amino acid oxidase. The yjbR gene product was overproduced in Escherichia coli, purified to homogeneity from the recombinant strain, and characterized. This protein effectively catalyzed the oxidation of sarcosine (N-methylglycine), N-ethylglycine and glycine. Lower activities on D-alanine, D-valine, and D-proline were detected although no activities were shown on L-amino acids and other D-amino acids. Since glycine is a product and not a substrate for sarcosine oxidase, this protein is not a type of demethylating enzymes but a novel deaminating oxidase, named glycine oxidase as a common name. Several enzymatic properties of the B. subtilis glycine oxidase were also investigated. << Less

  FEBS Lett. 438:263-266(1998) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Characterization and directed evolution of BliGO, a novel glycine oxidase from Bacillus licheniformis.

  Zhang K., Guo Y., Yao P., Lin Y., Kumar A., Liu Z., Wu G., Zhang L.

  Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtil ... >> More

  Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40°C. Interestingly, BliGO retained 60% of the maximum activity at 0°C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (Km, kcat and k(cat)/K(m)) of BliGO were 11.22 mM, 0.08 s(-1), and 0.01 mM(-1) s(-1), respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM(-1) s(-1)) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency. << Less

  Enzyme Microb. Technol. 85:12-18(2016) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Characterization and structural modeling of a novel thermostable glycine oxidase from Geobacillus kaustophilus HTA426.

  Martinez-Martinez I., Navarro-Fernandez J., Garcia-Carmona F., Takami H., Sanchez-Ferrer A.

  Glycine oxidase from Geobacillus kaustophilus HTA426 (GOXK) is a 43 kDa monomer flavoenzyme containing noncovalently bound FAD. The induction of the enzyme resulted in the expression of a fully soluble protein with higher specific activity than those previously reported for GOX from B. subtilis (G ... >> More

  Glycine oxidase from Geobacillus kaustophilus HTA426 (GOXK) is a 43 kDa monomer flavoenzyme containing noncovalently bound FAD. The induction of the enzyme resulted in the expression of a fully soluble protein with higher specific activity than those previously reported for GOX from B. subtilis (GOXB). A study of the kinetic properties of this novel GOXK revealed the lowest KM values for most of the substrates analyzed, with the exception of D-proline which kept a similar value and had the highest Vmax value reported. The Vmax/KM ratio maintained a substrate preference of GOXK for amines of small size, like glycine, sarcosine, N-ethyl-glycine, and glycine-ethyl-ester. GOXK presented good stability at 60-70 degrees C and in alkaline media (pH 6-9.5). The putative tridimensional structure was modeled by sequence alignment and by comparing the changes between GOXK and GOXB, and the residues that could be responsible for the substrate specificity as well as those essential for the catalytic activity were found. The comparison between the possible topology of GOXK with that of GOXB showed changes at the putative interactions between monomers for the building of the tetrameric oligomerization. << Less

  Proteins 70:1429-1441(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Glyphosate resistance by engineering the flavoenzyme glycine oxidase.

  Pedotti M., Rosini E., Molla G., Moschetti T., Savino C., Vallone B., Pollegioni L.

  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 alpha-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate ... >> More

  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 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. << Less

  J. Biol. Chem. 284:36415-36423(2009) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.