Identification and biochemical characterization of threonine dehydratase from the hyperthermophile Thermotoga maritima

The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual component, d -lysine ( d -Lys), in addition to the typical d -alanine ( d -Ala) and d -glutamate ( d -Glu). In a previous study, we identified a Lys racemase that is presumably associated with d -Lys biosynthesis. However, our understanding of d -amino acid metabolism in T. maritima and other bacteria remains limited, although d -amino acids in the peptidoglycan are crucial for preserving bacterial cell structure and resistance to environmental threats. Herein, we characterized enzymatic and structural properties of TM0356 that shares a high amino acid sequence identity with serine (Ser) racemase. The results revealed that TM0356 forms a tetramer with each subunit containing a pyridoxal 5′-phosphate as a cofactor. The enzyme did not exhibit racemase activity toward various amino acids including Ser, and dehydratase activity was highest toward l -threonine ( l -Thr). It also acted on l -Ser and l - allo -Thr, but not on the corresponding d -amino acids. The catalytic mechanism did not follow typical Michaelis–Menten kinetics; it displayed a sigmoidal dependence on substrate concentration, with highest catalytic efficiency ( k cat / K 0.5 ) toward l -Thr. Interestingly, dehydratase activity was insensitive to allosteric regulators l -valine and l -isoleucine ( l -Ile) at low concentrations, while these l -amino acids are inhibitors at high concentrations. Thus, TM0356 is a biosynthetic Thr dehydratase responsible for the conversion of l -Thr to α-ketobutyrate and ammonia, which is presumably involved in the first step of the biosynthesis of l -Ile.