{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown"],"volume":["88(23)"],"submitter":["Lo KY"],"pubmed_abstract":["The <i>N</i>-acyl-d-amino acid amidohydrolase (<i>N</i>-d-AAase) of Variovorax paradoxus Iso1 can enantioselectively catalyze the zinc-assisted deacetylation of <i>N</i>-acyl-d-amino acids to yield consistent d-amino acids. A putative FAD-binding glycine/d-amino acid oxidase was located immediately upstream of the <i>N-d-AAase</i> gene. The gene encoding this protein was cloned into Escherichia coli BL21 (DE3)pLysS and overexpressed at 25°C for 6 h with 0.5 mM isopropyl β-d-1-thiogalactopyranoside induction. After purification, the tag-free recombinant protein was obtained. The enzyme could metabolize glycine, sarcosine, and d-alanine, but not l-amino acids or bulky d-amino acids. Protein modeling further supported these results, demonstrating that glycine, sarcosine, and d-alanine could fit into the pocket of the enzyme's activation site, while l-alanine and d-threonine were out of position. Therefore, this protein was proposed as a glycine oxidase, and we designated it VpGO. Interestingly, VpGO showed low sequence similarity to other well-characterized glycine oxidases. We found that VpGO and <i>N</i>-d-AAase were expressed on the same mRNA and could be transcriptionally induced by various <i>N-acetyl-d-amino</i> acids. Western blotting and zymography showed that both proteins had similar expression patterns in response to different types of inducers. Thus, we have identified a novel glycine oxidase that is co-regulated with <i>N</i>-d-AAase in an operon, and metabolizes <i>N</i>-acyl-d-amino acids in the metabolically versatile V. paradoxus Iso1. <b>IMPORTANCE</b> The Gram-negative bacterium Variovorax paradoxus has numerous metabolic capabilities, including the association with important catabolic processes and the promotion of plant growth. We had previously identified and characterized an <i>N</i>-acyl-d-amino-acid amidohydrolase (<i>N</i>-d-AAase) gene from the strain of V. paradoxus Iso1. The aim of this study was to isolate and characterize (both <i>in vitro</i> and <i>in vivo</i>) another potential gene found in the promoter region of this <i>N</i>-d-AAase gene. The protein was identified as a glycine oxidase, and the gene existed in an operon with <i>N</i>-d-AAase. The structural basis for its FAD-binding potential and substrate stereo-specificity were also elucidated. This study first reported a novel glycine oxidase from V. paradoxus. We believe that our study makes a significant contribution to the literature, because this enzyme has great potential for use as an industrial catalysis, as a biosensor, and in agricultural biotechnology."],"journal":["Applied and environmental microbiology"],"pagination":["e0107722"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC9746326"],"repository":["biostudies-literature"],"pubmed_title":["Functional Characterization and Structural Modeling of a Novel Glycine Oxidase from Variovorax paradoxus Iso1."],"pmcid":["PMC9746326"],"pubmed_authors":["Lee CY","Tsai YF","Lo KY","Hsu CH"],"additional_accession":[]},"is_claimable":false,"name":"Functional Characterization and Structural Modeling of a Novel Glycine Oxidase from Variovorax paradoxus Iso1.","description":"The <i>N</i>-acyl-d-amino acid amidohydrolase (<i>N</i>-d-AAase) of Variovorax paradoxus Iso1 can enantioselectively catalyze the zinc-assisted deacetylation of <i>N</i>-acyl-d-amino acids to yield consistent d-amino acids. A putative FAD-binding glycine/d-amino acid oxidase was located immediately upstream of the <i>N-d-AAase</i> gene. The gene encoding this protein was cloned into Escherichia coli BL21 (DE3)pLysS and overexpressed at 25°C for 6 h with 0.5 mM isopropyl β-d-1-thiogalactopyranoside induction. After purification, the tag-free recombinant protein was obtained. The enzyme could metabolize glycine, sarcosine, and d-alanine, but not l-amino acids or bulky d-amino acids. Protein modeling further supported these results, demonstrating that glycine, sarcosine, and d-alanine could fit into the pocket of the enzyme's activation site, while l-alanine and d-threonine were out of position. Therefore, this protein was proposed as a glycine oxidase, and we designated it VpGO. Interestingly, VpGO showed low sequence similarity to other well-characterized glycine oxidases. We found that VpGO and <i>N</i>-d-AAase were expressed on the same mRNA and could be transcriptionally induced by various <i>N-acetyl-d-amino</i> acids. Western blotting and zymography showed that both proteins had similar expression patterns in response to different types of inducers. Thus, we have identified a novel glycine oxidase that is co-regulated with <i>N</i>-d-AAase in an operon, and metabolizes <i>N</i>-acyl-d-amino acids in the metabolically versatile V. paradoxus Iso1. <b>IMPORTANCE</b> The Gram-negative bacterium Variovorax paradoxus has numerous metabolic capabilities, including the association with important catabolic processes and the promotion of plant growth. We had previously identified and characterized an <i>N</i>-acyl-d-amino-acid amidohydrolase (<i>N</i>-d-AAase) gene from the strain of V. paradoxus Iso1. The aim of this study was to isolate and characterize (both <i>in vitro</i> and <i>in vivo</i>) another potential gene found in the promoter region of this <i>N</i>-d-AAase gene. The protein was identified as a glycine oxidase, and the gene existed in an operon with <i>N</i>-d-AAase. The structural basis for its FAD-binding potential and substrate stereo-specificity were also elucidated. This study first reported a novel glycine oxidase from V. paradoxus. We believe that our study makes a significant contribution to the literature, because this enzyme has great potential for use as an industrial catalysis, as a biosensor, and in agricultural biotechnology.","dates":{"release":"2022-01-01T00:00:00Z","publication":"2022 Dec","modification":"2025-04-19T10:24:23.781Z","creation":"2025-02-19T02:37:59.405Z"},"accession":"S-EPMC9746326","cross_references":{"pubmed":["36377957"],"doi":["10.1128/aem.01077-22"]}}