l -Threonine Transaldolase Activity Is Enabled by a Persistent Catalytic Intermediate
| Autor: | Prasanth Kumar, Craig A. Bingman, Jonathan M. Ellis, Andrew R. Buller, Grace A Carlson, Anthony Meza |
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| Rok vydání: | 2020 |
| Předmět: |
Threonine
0301 basic medicine Light Protein Conformation Stereochemistry Protonation Molecular Dynamics Simulation Crystallography X-Ray 01 natural sciences Biochemistry Catalysis Article Cofactor 03 medical and health sciences chemistry.chemical_compound Protein structure Biosynthesis Catalytic Domain Reactivity (chemistry) Amino Acid Sequence Amino Acids Glycine Hydroxymethyltransferase chemistry.chemical_classification biology 010405 organic chemistry Chemistry Quinones Active site General Medicine 0104 chemical sciences Amino acid Kinetics 030104 developmental biology Catalytic cycle Pyridoxal Phosphate biology.protein Molecular Medicine Spectrophotometry Ultraviolet Crystallization |
| Zdroj: | ACS Chem Biol |
| ISSN: | 1554-8937 1554-8929 |
| DOI: | 10.1021/acschembio.0c00753 |
| Popis: | 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 A 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. |
| Databáze: | OpenAIRE |
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