Aug 2026· Organic and biomolecular chemistry· 0 citations
Medicine
TL;DR
This review systematically elucidates the structural characteristics, classification, and diverse reactions catalyzed by ThDP-dependent enzymes, with a primary focus on their potential for stereoselective C-C bond formation and cleavage.
Abstract
Over the past several decades, biocatalysis has become a valuable complement to synthetic chemistry due to its high efficiency, exceptional selectivity, and environmental compatibility. Thiamine diphosphate (ThDP), the biologically active form of vitamin B1, serves as an essential coenzyme for core metabolic processes in all organisms. This review systematically elucidates the structural characteristics, classification, and diverse reactions catalyzed by ThDP-dependent enzymes, with a primary focus on their potential for stereoselective C-C bond formation and cleavage. These enzymes are widely distributed across all domains of life and catalyze the formation and cleavage of C-C, C-N, C-S, and C-O bonds. The catalytic mechanism centers on the formation of the Breslow intermediate, which undergoes nucleophilic addition to various electrophiles. Despite considerable sequence diversity, all ThDP-dependent enzymes share two conserved domains-the pyrimidine (PYR) binding domain and the pyrophosphate (PP) binding domain-and are classified into five structural types and nine superfamilies. In terms of substrate scope, the decarboxylase family is predominantly R-selective, whereas the transketolase family is S-selective. These enzymes hold significant promise for biotechnological applications, particularly through protein engineering to tailor catalytic activity and stereoselectivity. Moreover, ThDP-dependent enzymes have been implicated in the pathogenesis of Alzheimer's disease, diabetes, and tumor proliferation. This review also summarizes relevant clinical applications and the use of competitive inhibitors. By integrating modular architectures, cofactor synergy mechanisms, regulatory networks, and emerging frontiers in photoelectrochemical biocatalysis, this review highlights the broad potential of ThDP-dependent enzymes in both fundamental research and translational applications.
ConspectusThiamine diphosphate (ThDP)-dependent enzymes are among nature's most elegant biocatalysts for C-C bond formation and cleavage, typically operating through a two-electron umpolung mechanism. Although a small subset of ThDP enzymes, such as pyruvate: ferredoxin oxidoreductase (PFOR), has been shown to proceed via single-electron radical pathways, this knowledge had not yet been harnessed for asymmetric radical reactions. Inspired by thiamine biocatalysis, chemists have developed biomimetic N-heterocyclic carbene (NHC) complexes since the 1950s, and more recently, established radical NHC catalysis as a powerful platform for radical transformations. In this approach, an NHC-stabilized ketyl radical undergoes cross-coupling with a free C-centered radical. However, achieving enantioselective cross-coupling involving prochiral C-center radicals had remained an unsolved challenge with chiral chemo-NHC catalysts. This difficulty stems from both the highly reactive nature of these open-shell intermediates and the inherent limitation of small organo-NHC complexes in creating a suitable and tunable chiral environment.To address the above challenges, we drew inspiration from nature's ThDP-dependent biocatalysis and biomimetic chemo-NHC-based radical pathways, developing a synergistic photoredox-/thiamine radical biocatalysis for asymmetric radical acylations. In this system, the thiamine-dependent benzaldehyde lyase from Pseudomonas fluorescens PfBAL activates aldehyde substrates to form catalytically active enzymatic Breslow intermediates. Visible-light-driven photoredox catalysis enables the concurrent formation of prochiral C-centered radicals and enzyme-bound thiamine-derived ketyl radicals. Through protein engineering, the evolved active site of PfBAL variant precisely orchestrates the enantioselective cross-coupling of the two radical intermediates. Overall, this combination of chemomimetic and biomimetic strategies repurposes ThDP-dependent PfBAL into a radical acyltransferase (RAT), expanding the reactivity repertoire of enzymes and overcoming the challenges in stereochemical control of free prochiral C-centered radicals.We first established this synergistic photoredox/thiamine radical catalysis through a decarboxylative radical acylation, in which prochiral C-centered radicals generated from N-(acyloxy)phthalimides under Eosin Y-mediated photoredox conditions were selectively acylated by PfBAL variants with benzyaldehyde as the acyl donor. Mechanistic studies confirmed the involvement of both ketyl and prochiral radical intermediates. Building on this discovery, we subsequently demonstrated the robustness of thiamine-dependent radical biocatalysis in diverse challenging transformations, including benzylic C(sp3)-H bonds acylation, C-C bonds acylation via nitrogen-centered radicals, three-component radical cross-coupling, and the construction of all-carbon quaternary stereocenter bearing minimally differentiated alkyl substituents.Across all these systems, a shared key mechanistic scheme is the cross-coupling of the enzymatic thiamine-derived ketyl radical with a prochiral C-centered radical, which could be assembled as the radical rebound pathway widely observed in P450 enzymes. Protein engineering campaigns and computational studies consistently suggest that residues 480 and 481 create differential steric environments, playing key roles in achieving high enantioselectivity.This Account describes our journey in repurposing PfBAL from a natural lyase to a versatile radical acyltransferase. The synergistic integration of photoredox catalysis, directed evolution, and enzymology establishes a generalizable platform for controlling free fleeting radical intermediates for enzymatic acylations. Looking forward, deeper mechanistic mimic, artificial intelligence-assisted protein design and engineering promise to further expand the chemical space and applicability accessible to thiamine-dependent enzymes.
Yuanyuan Xu, Xichao Peng, Xiaoqiang Huang· Accounts of Chemical Researc...· 0 citations
Non-canonical amino acids (ncAAs) are privileged building blocks for the synthesis of natural products, biocatalysts, and drug molecules. Among them, γ-tertiary-nitro-α-amino acids are highly valuable yet remain scarcely explored due to challenges in synthetic accessibility. Although pyridoxal-5′-phosphate (PLP)-dependent enzymes are powerful biocatalysts for ncAA production, no enzymatic platform has, to the best of our knowledge, enabled efficient access to γ-tertiary-nitro-α-amino acids. Here, we report a PLP-dependent enzymatic platform that selectively couples
O
-acetyl-serine with secondary nitroalkanes to yield γ-tertiary-nitro-α-amino acids. Through reaction design, enzyme screening, and directed evolution, we repurpose SidM, an enzyme that catalyzes pyrrole ring formation in the siderochelin biosynthetic pathway, into a highly active and stereoselective biocatalyst, affording γ-tertiary-nitro-α-amino acids in up to 99% yield and 99% diastereomeric excess. This enzyme exhibits broad substrate scope and good evolvability, allowing enhancement of catalytic efficiency or inversion of diastereoselectivity at the Cγ nitro center, and is readily scalable to gram synthesis. Mechanistic analyses combining computation and mutagenesis elucidate the structural determinants of activity and stereocontrol. This work establishes a general, tunable and scalable biocatalytic platform for the synthesis of γ-tertiary-nitro-α-amino acids, expanding the synthetic repertoire of PLP-dependent enzymes for asymmetric C–C bond formation.
Wenping Ding, Shenggan Luo, Yike Zou et al.· Nature Communications· 0 citations
Developing enzymatic strategies to selectively construct C–C and C–heteroatom bonds is a major objective in modern biocatalysis. Here we combine genetic code reprogramming and laboratory evolution to establish a family of allylic transferase enzymes that can achieve a remarkable breadth of chemistry, enabling the generation of valuable motifs including γ‑butenolides, chiral amines and all-carbon quaternary centres. Our enzymes operate through the formation of electrophilic imidazolium intermediates that can be generated from reagents equipped with a para-nitrophenol leaving group to facilitate high-throughput evolution. These intermediates can be intercepted with diverse carbon and nitrogen nucleophiles to generate densely functionalized products featuring Cβ or Cγ stereocentres. Interestingly, structural analysis suggests that catalysis proceeds through an unanticipated ternary complex involving para-nitrophenol and the incoming substrate nucleophile. This study adds a family of C–C and C–N bond-forming enzymes to the biocatalytic repertoire and illustrates how artificial enzymes can achieve precise control over challenging chemical conversions. Small catalytic nucleophiles can promote allylic transfer processes, but achieving regio- and stereocontrol is challenging. Here the authors develop a family of allylic transferase enzymes to mediate diverse C–C and C–N bond formations through the confinement of key intermediates within proteins.
Zachary Birch-Price, Amy E Hutton, Fei Zhao et al.· Nature Catalysis· 1 citation
A KAS III family enzyme, CalO4, is described that catalyzes C-S bond formation during the biosynthesis of the potent antitumor agent calicheamicin and provides a potential biocatalyst for hindered C-S bond formation and a target for engineering novel calicheamicin-like or other bioactive compounds.
Fang Pang, Yu-Ju Peng, Srinivas Thadkapally et al.· ACS Chemical Biology· 0 citations
Fluoroacetate dehalogenase (FAcD) is a unique, nonmetal-dependent enzyme capable of cleaving the inert C-F bond of fluoroacetate (FA) under mild conditions, making it an important enzymatic model for organofluorine degradation. This study investigated the catalytic degradation mechanism of FA by FAcD using density functional theory calculations with an active-site cluster model. The model included the FA substrate, two crystallographic water molecules, and the key amino acid residues surrounding the active site. An integrated energy profile connecting defluorination and hydrolysis was obtained within a single computational framework. The calculated reaction pathway consists of four elementary steps: (I) C-F bond activation, (II) nucleophilic attack, (III) C-O bond cleavage, and (IV) proton transfer. Structural analyses showed that Arg111, Arg114, and Tyr219 stabilize the carboxylate group of FA and His155, Trp156, and Tyr219 stabilize the fluoride anion during C-F bond cleavage. Furthermore, His155 was suggested to activate the hydrolytic water molecule and participate directly in the nucleophilic attack step. These findings provide a unified quantum-chemical description of FAcD-catalyzed defluorination and hydrolysis and offer insights into the design of engineered enzymes and biomimetic catalysts for organofluorine degradation.
Manussada Ratanasak, Yuta Hori, Kohei Sato et al.· Journal of Physical Chemistr...· 0 citations
Iron(II)/2-oxoglutarate-dependent (Fe/2OG) enzymes catalyze consecutive C-C bond formations to assemble complex heterobicyclic ring systems and generate three new stereocenters in piperazine alkaloids helvamide B and the arizonamides through C(sp3)-H activation─a transformation that remains challenging in synthetic chemistry. Here, we report a comprehensive mechanistic study of this unique transformation catalyzed by the Fe/2OG enzyme Hvm1, using a combination of deuterated substrates, substrate analogs bearing electron-withdrawing substituents, and multiple spectroscopic methods (LC-MS, X-ray crystallography, CD spectroscopy, and NMR). The reaction proceeds via consecutive radicaloid C-C (C3'-C2 and C3-C3″) couplings, involving sequential radical attack on the olefin and benzoyl group (Minisci variant), initiated by a C3' radical generated through C3' pro-S hydrogen atom transfer (HAT). The first C-C bond (C3'-C2) is formed on the Si-face of C2 with retention of C3' configuration. The second C-C bond (C3-C3″) formation can proceed with either of two stereochemical senses─antarafacial or suprafacial─relative to the first newly formed C3'-C2 bond: the antarafacial pathway leads to helvamide B, while the suprafacial pathway affords the previously unreported epimer, helvamide A. Crystal structure analysis identifies Y67 as a key residue governing the partitioning of stereochemical outcomes in the second C-C bond formation. Furthermore, the conclusive stereochemical assignment of helvamide B corrects the prior misassignment of the C3' configuration in the arizonamides.
Shengbin Zhou, Jia-Peng Zhang, Jia-Yu Zuo et al.· Journal of the American Chem...· 0 citations
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