L-Threonine Transaldolase Activity Is Enabled by a Persistent Catalytic Intermediate

L-Threonine transaldolases (LTTAs) are a poorly characterized class of pyridoxal-5'-phosphate (PLP) dependent enzymes responsible for the biosynthesis of diverse beta-hydroxy amino acids. Here, we study the catalytic mechanism of ObiH, an LTTA essential for biosynthesis of the beta-lactone natural product obafluorin. Heterologously expressed ObiH purifies as a mixture of chemical states including a catalytically inactive form of the PLP cofactor. Photoexcitation of ObiH promotes the conversion of the inactive state of the enzyme to the active form. UV-vis spectroscopic analysis reveals that ObiH catalyzes the retro-aldol cleavage of L-threonine to form a remarkably persistent glycyl quinonoid intermediate, with a half-life of similar to 3 h. Protonation of this intermediate is kinetically disfavored, enabling on-cycle reactivity with aldehydes to form beta-hydroxy amino acids. We demonstrate the synthetic potential of ObiH via the single step synthesis of (2S,3R)-beta-hydroxyleucine. To further understand the structural features underpinning this desirable reactivity, we determined the crystal structure of ObiH bound to PLP as the Schiffs base at 1.66 angstrom resolution. This high-resolution model revealed a unique active site configuration wherein the evolutionarily conserved Asp that traditionally H-bonds to the cofactor is swapped for a neighboring Glu. Molecular dynamics simulations combined with mutagenesis studies indicate that a structural rearrangement is associated with L-threonine entry into the catalytic cycle. Together, these data explain the basis for the unique reactivity of LTTA enzymes and provide a foundation for future engineering and mechanistic analysis.

Automated summary

L-Threonine transaldolases (LTTAs) are a class of enzymes dependent on PLP, responsible for synthesizing diverse beta-hydroxy amino acids. ObiH, an essential LTTA for the biosynthesis of the beta-lactone natural product obafluorin, has been studied to understand its catalytic mechanism and structural features. The data explain the unique reactivity of LTTA enzymes and provide a basis for future engineering and mechanistic analysis.