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.
Scheme 2. Completion of (-)-7-Deacetoxyalcyonin Acetate Scheme 3. Overman's Completion of the Proposed Structure of Sclerophytin A
Enzymes are renowned for their catalytic e ciency and selectivity, but relatively few carbon-carbon bond forming enzymes have found their way into the biocatalysis toolbox. While engineering can overcome the challenges associated with C-C bond formation for some enzyme systems, the broader synthetic potential of biocatalysis is hindered by the lack of high-quality C-C bond forming transformations. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstD2.0, is e cient in a whole-cell biocatalysis format, which circumvents the need for enzyme puri cation, thereby facilitating its use in traditional organic settings. This new, highly stereoselective enzyme represents a unique expansion of the biosynthetic toolbox. The products are highly desirable, functionally rich bioactive γ-hydroxy amino acids that we demonstrate can be prepared stereoselectively on gram-scale. The X-ray crystal structure of UstD2.0 at 2.25 Å reveals the active site and the molecular basis for the remarkably promiscuity of this catalyst. Taking inspiration from the versatile reactivity of enamines in organic synthesis, we hypothesize that the enamine intermediate of UstD can be engineered to react with electrophiles other than aldehydes. The advent of structural information enabled by engineering of UstD2.0 provides a foundation for probing the unique mechanism of UstD and will guide efforts to expand the reactivity of this unique enzyme. Main TextMajor advances have been made in the practical use of enzymes for enantioselective functional group manipulations 9 . For example, enantioselective reduction of ketones or enantiospeci c hydrolysis of racemic esters are now routine in process chemistry. There have also been impressive strides made in enzymatic C-H activation 10 . However, the development of enzymes to form C-C bonds on preparative scale lags far behind traditional synthetic organic methodology 11 .To ll this gap, we were drawn to a recently described pyridoxal phosphate (PLP) dependent enzyme involved in the biosynthesis of Ustiloxin B, an inhibitor of microtubilin polymerization (Fig. 1A) 12 . This enzyme, UstD, decarboxylates the side chain of l-aspartate (1), forming a putative nucleophilic enamine intermediate (Fig. 1B). This enamine then attacks an aliphatic aldehyde appended to a cyclic tetrapeptide, resulting in the formation of a γ-hydroxy amino acid side chain. The loss of CO 2 renders this enantioselective C-C bond forming reaction irreversible. However, the native substrate for UstD is a complex, cyclic peptide, and it was unknown if this enzyme would react promiscuously with alternative substrates; if so, the enzyme would directly produce γ-hydroxy amino acids (Fig. 1B). Such non-standard amino acids (nsAAs) are found in bioactive natur...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.