Glycine oxidase from Bacillus subtilis: Role of Histidine 244 and Methionine 261

Boselli, Angelo; Rosini, Elena; Marcone, Giorgia Letizia; Sacchi, Silvia; Motteran, Laura; Pilone, Mirella S.; Pollegioni, Loredano; Molla, Gianluca

doi:10.1016/j.biochi.2007.04.019

Cited by 10 publications

(14 citation statements)

References 24 publications

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“…Highest homology at the 4 F. Jamil et al amino acid level was found with strains 168 (98.6%) and CU1065 (97.8%) of B. subtilis (Table 1). The amino acid residues comprising the active site of GO from the strain 168 (Boselli et al 2007) were completely conserved in GoxR. The amino acid contents of GoxR are shown in Table 2.…”

Section: Resultsmentioning

confidence: 99%

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Gene cloning and characterization of glycine oxidase from newly isolated Bacillus subtilis strain R5

et al. 2010

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The gene encoding the glycine oxidase from Bacillus subtilis strain R5 (goxR) was cloned and expressed in Escherichia coli. The gene consisted of 1,110 nucleotides that encoded a protein (GoxR) of 369 amino acid residues with a molecular mass of 40,761 Da. The GoxR exhibited 98.6% identity with glycine oxidase from B. subtilis strain 168. Gene expression and purification of the recombinant GoxR were performed. The recombinant GoxR existed in a homotetramer form. The recombinant protein effectively catalyzed the oxidation of glycine and D-alanine. The specific activity of the purified recombinant GoxR was 0.96 U/mg when glycine was used as a substrate and 1.0 U/mg when D-alanine was substrate. The enzyme displayed its highest activity at pH 8.0 and at a temperature of 50• C. The activation energy of the reaction catalyzed by the enzyme was calculated to be 26 kJ/mol. The enzyme activity was significantly inhibited in the presence of organic solvents. No enhancement of enzyme activity was observed in the presence of metal cations. The experimental results presented in this study demonstrate that the enzyme was a bonafide glycine oxidase.

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Section: Resultsmentioning

confidence: 99%

“…Glycine oxidase from Bacillus subtilis strain R5 7 & Imanaka 1998), 0.56 mM (Martínez-Martínez et al 2006), 0.7 mM (Boselli et al 2007), 0.6 mM . Similarly the K m values for this enzyme using d-alanine as the substrate are 81 mM (Nishiya & Imanaka 1998) The GoxR described in this study exhibited high enzyme activity at a broad temperature range (40 to 55…”

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Gene cloning and characterization of glycine oxidase from newly isolated Bacillus subtilis strain R5

et al. 2010

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“…The recombinant GO proteins were purified from the crude extracts by using HiTrap chelating affinity chromatography (GE Healthcare) as reported in (2, 13). The purified GOs were then equilibrated with 50 mM disodium pyrophosphate buffer, pH 8.5, and 10% glycerol (13).…”

Section: Methodsmentioning

confidence: 99%

Glyphosate Resistance by Engineering the Flavoenzyme Glycine Oxidase

Pedotti

Rosini

Molla

et al. 2009

Journal of Biological Chemistry

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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 ␣-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 ␣2-␣3 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.

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“…The recombinant GO proteins were purified from the crude extracts by using HiTrap chelating chromatography (GE Healthcare, Little Chalfont, UK) as reported previously [5,31]. The purified GO variants were then equilibrated with 50 mM disodium pyrophosphate buffer, pH 8.5, and 10% glycerol [24].…”

Section: Enzyme Expression and Purificationmentioning

confidence: 99%

“…2A) by decreasing the K m,app value (up to 5-fold for the H244K GO variant, Table 2). Previous site-directed mutagenesis studies showed that the H244F substitution increased product release and negatively affected the rate of flavin reduction with sarcosine, without affecting the turnover number [24]. Moreover, the presence of an alanine residue at site 244 had a synergistic effect on the activity of GO on glyphosate as substrate by increasing the stability of variants containing multiple replacements [11].…”

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Novel biosensors based on optimized glycine oxidase

et al. 2014

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Glycine is involved in several physiological functions, e.g. as a neurotransmitter in the central nervous system, and sarcosine has been identified as a differential metabolite greatly enhanced during prostate cancer progression and metastasis. Glycine oxidase from Bacillus subtilis (GO) was engineered with the final aim of producing specific analytical systems to detect these small achiral amino acids. Based on in silico analysis, site-saturation mutagenesis was independently performed at 11 positions: a total of 16 single-point GO variants were analyzed. Significantly improved kinetic parameters were observed on glycine for the A54R, H244K-N-Q-R, Y246W and M261R variants. The introduction of multiple mutations then identified the H244K/M261R variant showing a 5.4-fold increase in maximal activity on glycine. With sarcosine as substrate, a number of single-point variants showed increased maximal activity and/or affinity: the kinetic efficiency was increased 6-fold for the M49L variant. Two GO variants with a high substrate specificity ratio for glycine (versus sarcosine, i.e. H244K GO) or for sarcosine (versus glycine, i.e. M49L GO) combined with high substrate affinity were used to set up a simple fluorescence-based biosensor. This optical sensing assay represents a novel, inexpensive and fast tool to assay glycine or sarcosine concentrations in biological samples (detection limit ≤ 0.5 lM). IntroductionGlycine is the smallest and the only nonchiral natural a-amino acid used for protein biosynthesis. In addition to such a structural role, it possesses further physiological functions: it is an important biosynthetic precursor (i.e. it is used for de novo synthesis of porphyrins and purines) and it acts as a neurotransmitter in the central nervous system. Glycine is a coagonist, together with D-serine, of glutamate at glutamatergic N-methyl-Daspartate receptors (NMDARs) in the forebrain [1], thus serving both inhibitory and excitatory functions [2]. Due to this role, glycine is probably involved in the pathophysiology of schizophrenia and other neurological diseases. For example, a correlation exists between low levels of glycine and catatonia (a psychiatric disease), and the perturbation of the glycine deportation system is associated with ifosfamide encephalopathy and congenital nonketotic hyperglycinemia [2].Sarcosine, 2-(methylamino)acetic acid or N-methylglycine, is a natural, small, endogenous amino acid present in low concentrations in the blood and in urine. It has been proposed to play a role in the progression of prostate cancer, and assessing sarcosine concentrations in body fluids was suggested as a novel biomarker for prostate cancer [3]. With the final aim of developing a biosensor to assay glycine and sarcosine in biological fluids, we engineered glycine oxidase from Bacillus subtilis (GO) (EC 1.4.3.19) to improve its kinetic efficiency on these two compounds. GO is a homotetrameric flavoenzyme that contains one molecule of noncovalently bound FAD per 47 kDa protein monomer [4,5]. Purified recomb...

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Glycine oxidase from Bacillus subtilis: Role of Histidine 244 and Methionine 261

Cited by 10 publications

References 24 publications

Gene cloning and characterization of glycine oxidase from newly isolated Bacillus subtilis strain R5

Gene cloning and characterization of glycine oxidase from newly isolated Bacillus subtilis strain R5

Glyphosate Resistance by Engineering the Flavoenzyme Glycine Oxidase

Novel biosensors based on optimized glycine oxidase

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