title: Selective tryptophan determination using tryptophan oxidases involved in bis-indole antibiotic biosynthesis.
abstract: A novel tryptophan assay was developed using tryptophan oxidases. Although many l-amino acid oxidases (LAAOs) have been reported to catalyze tryptophan oxidation, most of them have broad substrate specificity and oxidize multiple amino acids besides tryptophan. To obtain a tryptophan-specific LAAO, we focused on bis-indole antibiotic biosynthesis, a bacterial secondary metabolic pathway. A putative LAAO from Streptomyces sp. TP-A0274, StaO involved in staurosporine biosynthesis, was heterologously expressed, biochemically characterized, and shown to serve as a selective tryptophan oxidase for the first time. In addition, another LAAO, VioA involved in violacein biosynthesis in Chromobacterium violaceum, was characterized for comparison with StaO. Interestingly, StaO and VioA share similar properties, namely narrow substrate specificity and high affinity for l-tryptophan, despite the phylogenetic distance between these enzymes. Owing to these features, uncommon among known LAAOs, StaO and VioA assays can be used for selective and accurate quantification of l-tryptophan via a coupled colorimetric reaction. Indeed, StaO and VioA assays provided tryptophan concentrations in human plasma as accurately as those obtained by high-performance liquid chromatography. Therefore, these enzymes were clearly shown to offer an effective method for determining tryptophan in biological samples rapidly, inexpensively, and accurately. The results shown here also suggest the possibility of metabolism-oriented screening as a strategy to obtain enzymes highly selective for individual biomolecules.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Kameya
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Onaka
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000145:classification / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001419:Bacteria / Q000201:enzymology; D003001:Cloning, Molecular; D006801:Humans; D015394:Molecular Structure; D010088:Oxidoreductases / Q000378:metabolism*; D010802:Phylogeny; D013045:Species Specificity; D013379:Substrate Specificity; D014364:Tryptophan / Q000097:blood / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D014364:Tryptophan; D010088:Oxidoreductases; D000594:Amino Acid Oxidoreductases