title: Determination of L-methionine using methionine-specific dehydrogenase for diagnosis of homocystinuria due to cystathionine β-synthase deficiency.
abstract: To determine the L-methionine (L-Met) concentration in an extract from dried blood spots (DBSs) for newborn mass screening for homocystinuria (HCU) due to cystathionine β-synthase (CBS) deficiency, a new fluorometric microplate assay using a methionine-specific dehydrogenase (MetDH) and the diaphorase/reazusrin system was established. We created by directed mutagenesis an NAD⁺-dependent MetDH from phenylalanine dehydrogenase (PheDH) showing higher substrate specificity toward L-Met than L-phenylalanine (L-Phe). However, it also exhibited notable activity for branched-chain amino acids (BCAAs). BCAAs in blood clearly interfered with the determination of L-Met in the DBS specimens using a single application of MetDH. To measure L-Met selectively, we used a branched-chain amino acid transaminase (BCAT) to eliminate the BCAAs in the specimens and screened for a BCAT with low activity toward L-Met. In microplate assays using MetDH, pretreatment of specimens with the BCAT from Lactobacillus delbrueckii subsp. bulgaricus coupled with L-glutamate oxidase minimized the effects of BCAAs, and L-Met concentrations were determined with high accuracy even at elevated BCAA concentrations. This enzymatic end-point assay is suitable for determining L-Met concentrations in DBSs for neonatal screening for HCU due to CBS deficiency.
authors: Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors: Shino
authors: S
authors: Yamasaki-Yashiki
authors: Shinjiro
authors: S
authors: Tachibana
authors: Yasuhisa
authors: Y
authors: Asano
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D003541:Cystathionine beta-Synthase / Q000172:deficiency* / Q000378:metabolism; D059788:Dried Blood Spot Testing; D057075:Enzyme Assays; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods*; D006712:Homocystinuria / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology*; D006801:Humans; D008715:Methionine / Q000097:blood*; D015203:Reproducibility of Results; D000637:Transaminases / Q000378:metabolism
chemical_list: D008715:Methionine; D000594:Amino Acid Oxidoreductases; C442335:L-methionine dehydrogenase; D000637:Transaminases; D003541:Cystathionine beta-Synthase