Functional Characterization and Structural Modeling of a Novel Glycine Oxidase from Variovorax paradoxus Iso1.

The N -acyl-d-amino acid amidohydrolase ( N -d-AAase) of Variovorax paradoxus Iso1 can enantioselectively catalyze the zinc-assisted deacetylation of N -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 N-d-AAase 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 N -d-AAase were expressed on the same mRNA and could be transcriptionally induced by various N-acetyl-d-amino 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 N -d-AAase in an operon, and metabolizes N -acyl-d-amino acids in the metabolically versatile V. paradoxus Iso1. IMPORTANCE 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 N -acyl-d-amino-acid amidohydrolase ( N -d-AAase) gene from the strain of V. paradoxus Iso1. The aim of this study was to isolate and characterize (both in vitro and in vivo ) another potential gene found in the promoter region of this N -d-AAase gene. The protein was identified as a glycine oxidase, and the gene existed in an operon with N -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.