Novel method for l-methionine determination using l-methionine decarboxylase and application of the enzyme for l-homocysteine determination

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2020-05, Vol.84 (5), p.927-935
• Main Authors: Okawa, Atsushi, Hayashi, Masaya, Inagaki, Junko, Okajima, Toshihide, Tamura, Takashi, Inagaki, Kenji
• Format: Article
• Language: English
• Subjects: Amino Acid Metabolism, Inborn Errors - blood; Amino Acid Metabolism, Inborn Errors - diagnosis; Carbon-Sulfur Lyases - metabolism; Carboxy-Lyases - metabolism; Enzyme Assays - methods; Escherichia coli - genetics; Escherichia coli - metabolism; Glycine N-Methyltransferase - blood; Glycine N-Methyltransferase - deficiency; Homocysteine - blood; Homocystinuria - blood; Homocystinuria - diagnosis; Humans; hypermethioninemia; l-homocysteine; L-methionine; l-methionine decarboxylase; l-methionine γ-lyase; Methionine - blood; Plasmids - genetics; Pseudomonas putida - enzymology; Pseudomonas putida - genetics; Spectrophotometry - methods; Streptomyces - enzymology; Streptomyces - genetics
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1080/09168451.2020.1715781

For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10-200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine preincubated with MetDC. DTT: dithiothreitol; IPTG: isopropyl-β-d-thiogalactopyranoside; KPB: potassium phosphate buffer; MBTH: 3-methyl-2-benzothiazolinonehydrazone; mdc: the gene coding l-methionine decarboxylase; MetDC: l-methionine decarboxylase; mgl: the gene coding l-methionine γ-lyase; MGL: l-methionine γ-lyase; PLP: pyridoxal 5ʹ-phosphate Standard curve for the L-methionine determination using MetDC (A) and L-Homocysteine determination by MGL after pretreatment of MetDC (B).