Characterization of unique EDTA-insensitive methylthioalkylmalate synthase from Eutrema japonicum and its potential application in synthetic biology.

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.

Tuning of p values activates substrates in flavin-dependent aromatic hydroxylases.

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation.

Tyr217 and His213 are important for substrate binding and hydroxylation of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that contains FAD as a redox-active cofactor. The enzyme catalyzes para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on the enzyme crystal structure, residue His213 is located close to the hydroxyl moiety, whereas Tyr217 is close to the carboxylate group of 3HB.