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

Abstract

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 (kcat/K0.5) 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.