6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from Eutrema japonicum, commonly referred to as Japanese wasabi. Eutremajaponicum MAMs (EjMAMs) were expressed in an Escherichiacoli expression system, subsequently purified, and in vitro enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used in silico analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined in vivo biosynthesis in E. coli containing Arabidopsis thaliana branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting l-methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property in vitro and highest activity on converting l-methionine to 2-(2-MT)EM in vivo which displays high potential for isothiocyanate biosynthesis in E. coli platform.