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.

Translation-dependent bioassay for amino acid quantification using auxotrophic microbes as biocatalysts of protein synthesis

“…The slope of this curve indicated that almost all of the added Arg was recovered by the assay, estimating the recovery rate to be 98%. Chromatographic analysis (Kameya et al 2013) estimated the Arg concentration in the same casamino acid solution to be 133 nM. This value is comparable to that estimated by the previous bioassay (140 nM), demonstrating the successful measurement of an amino acid in crude samples by this bioassay.…”

“…A variety of methods have been developed to meet these demands, such as instrumental methods (Kaspar et al 2009) and enzymatic assays Kameya et al 2013;Liu et al 2014;Matsui et al 2015). Among these analytical methods, amino acid bioassays have been popular over the past 70 years because of their low cost, easy manipulation, coverage of wide range of amino acids, and applicability to high-throughput analysis (Cardinal and Hedrick 1948;Steele et al 1949;Tamura et al 1952).…”

Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum

“…VioA enzyme kinetics were determined in a coupled peroxidase assay mainly as described previously (7,9). A reaction mixture of 600 l containing 50 mM Tris-HCl, pH 9.0, 0.2 M VioA, 1 mM 4-aminoantipyrine, 1 mM phenol, and 15 units/ml horseradish peroxidase was pre-incubated at 30°C.…”

“…1) (7)(8)(9). Subsequently, oxidative coupling of two imines by VioB, RebD, or StaD results in the formation of a short-lived compound that was proposed to be an IPA imine dimer (7,10).…”

“…Inhibitor concentrations of up to 10 mM were analyzed in the coupled VioA activity assay (9) or alternatively in VioA substrate depletion kinetics (compare Fig. 3, A-C).…”