L-Threonine transaldolases (LTTAs) are a poorly characterized class of pyridoxal-5′-phosphate (PLP) dependent enzymes responsible for the biosynthesis of diverse β-hydroxy amino acids. Here, we study the catalytic mechanism of ObiH, an LTTA essential for biosynthesis of the β-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 ∼3 h. Protonation of this intermediate is kinetically disfavored, enabling on-cycle reactivity with aldehydes to form β-hydroxy amino acids. We demonstrate the synthetic potential of ObiH via the single step synthesis of (2S,3R)-β-hydroxyleucine. To further understand the structural features underpinning this desirable reactivity, we determined the crystal structure of ObiH bound to PLP as the Schiff's base at 1.66 Å 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.
Multi-enzyme biocatalytic cascades are emerging as practical routes for the synthesis of complex bioactive molecules. However, the relative sparsity of water-stable carbon electrophiles limits the synthetic complexity of molecules made from such cascades. Here, we develop a chemoenzymatic platform that leverages styrene oxide isomerase (SOI) to convert readily accessible aryl epoxides into α-aryl aldehydes through Meinwald rearrangement. These unstable aldehyde intermediates are then intercepted with a C−C bond forming enzyme, ObiH, that catalyzes a transaldolase reaction with L-threonine to yield synthetically challenging β-hydroxy-α-amino acids. Co-expression of both enzymes in E. coli yields a whole-cell biocatalyst capable of synthesizing a variety of stereopure non-standard amino acids (nsAA) and can be produced on a gram scale. We used isotopically labeled substrates to probe the mechanism of SOI, which we show to catalyze a concerted isomerization featuring a stereospecific 1,2-hydride shift. The viability of in situ generated α-aryl aldehydes was further established by intercepting them with a recently engineered decarboxylative aldolase to yield γ-hydroxy nsAAs. Together, these data establish a versatile method of producing α-aryl aldehydes in simple, wholecell conditions and show that these intermediates are useful synthons in C−C bond forming cascades.
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