Aug 2026· ACS Chemical Biology· 0 citations· 31 references
Medicine
TL;DR
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.
Abstract
β-Ketoacyl-ACP synthase (KAS) III enzymes play central roles in fatty acid biosynthesis and the production of various natural products. While their canonical function involves catalyzing C-C bond formation between acetyl-CoA and malonyl acyl-carrier-proteins (ACPs), an increasing number of non-canonical activities have been reported among KAS III homologues, including C-O and C-N bond formation. Here, we describe a KAS III family enzyme, CalO4, that catalyzes C-S bond formation during the biosynthesis of the potent antitumor agent calicheamicin. We successfully reconstituted its transacylation activities, demonstrated C-S bond formation, and assessed its substrate selectivity. Furthermore, X-ray crystallography combined with molecular docking and mutational analysis identified key residues likely involved in substrate selection and catalysis. This work not only expands the functional diversity of the KAS III family but also provides a potential biocatalyst for hindered C-S bond formation and a target for engineering novel calicheamicin-like or other bioactive compounds.
Nonheme iron/α-ketoglutarate-dependent dioxygenases (Fe/αKGDs) catalyze diverse oxidative transformations, including hydroxylation, desaturation, and ring expansion. Although these reactions are well established, Fe/αKGD-catalyzed epimerization remains rare and has not been characterized in ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report the RiPP-modifying enzyme YmI, a bifunctional Fe/αKGD that catalyzes Cβ-hydroxylation of Phe19 and Cα-epimerization of Ile20 on the precursor peptide YmA toward the formation of YM-216391. The hydroxylation generates (2S,3R)-β-hydroxyphenylalanine, whose l-threonine-like stereochemistry facilitates subsequent formation of an unusual 5-phenyloxazole moiety. Substrate profiling analyses reveal leader peptide dependence for catalytic activity. Structural and docking studies provide insights into the substrate binding and active site architecture. Deuterium labeling demonstrates that YmI catalyzes Ile epimerization by directly abstracting the Cα-hydrogen, generating a Cα radical that is subsequently reprotonated by a nonexchangeable hydrogen source within the enzyme. These results unveil an unprecedented mechanism and novel bifunctionality, markedly expanding the reaction landscape for αKG-dependent enzymes in RiPP biosynthesis.
Unknown authors· Journal of the American Chem...· 0 citations
Fub7 is a rare pyridoxal 5′-phosphate (PLP)-dependent enzyme that can subsequently catalyze the γ-elimination of O-acetyl-l-homoserine (OAH) and Michael addition with n-valeraldehyde (NVA) to synthesize 5-alkyl-pipecolic acid, which shows significant potential in synthetic chemistry. In this study, we constructed the computational models and performed MD and QM/MM calculations to illuminate the complete catalytic cycle of Fub7, which contains five reaction stages, including external aldimine formation, γ-elimination, Michael addition, reformation of the internal aldimine, and final cyclization and dehydration, of which γ-elimination and Michael addition correspond to relatively high energy barriers. In addition, according to our test calculations, Fub7 can also catalyze the β-elimination. During the catalysis, the terminal amino of Lys211 exhibits significant conformational changes and was found to play multiple roles. On one hand, Lys211 forms internal aldimine with the cofactor PLP. On the other hand, Lys211 functions as a catalytic base/acid or as a bridge to mediate a series of proton transfer in the γ-elimination and Michael addition. Tyr109 only participates in the Michael addition. The deprotonated Tyr109 acts as a base to abstract the α-proton of NVA to generate the carbanion nucleophile, which is the key for Michael addition. These findings may provide useful information for understanding the catalysis of PLP-dependent enzymes and designing biocatalysts for γ-substitution.
Unknown authors· Journal of Chemical Informat...· 0 citations
It is demonstrated that the cytochrome P450 monooxygenase CyaI catalyzes an oxidation reaction through a zwitterionic intermediate and facilitates a subsequent unusual C→N acetyl migration, which triggers a spontaneous intramolecular cyclization to forge the imidazolidin-4-one ring during 1 biosynthesis.
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.
Jinxi Huang, Letong Huang, Xuemei Wang et al.· Organic and biomolecular che...· 0 citations
Benzaldehyde is a widespread volatile compound produced by plants. Its final biosynthetic step is catalyzed by benzaldehyde synthase (BS), a peroxisomal enzyme composed of α and β subunits, both belonging to the short-chain dehydrogenase/reductase (SDR) family. Here, we report the crystal structure of Petunia hybrida BS, which reveals an α2β2 heterotetrameric arrangement. Structural and biochemical analyses show that the α subunits contain the canonical catalytic site, whereas the β subunits have lost catalytic activity but are essential for heterotetramer assembly. Notably, the C terminus of the β subunit extends into the diagonally positioned α subunit, contributing to the formation of the composite benzoyl-CoA substrate-binding pocket. Site-directed mutagenesis and subunit-mixing experiments support noncooperative, additive contributions of protomers within the heterotetramer. This work establishes BS as a rare heterotetrameric plant SDR and demonstrates how subunit specialization and intersubunit arrangement enable function, providing principles for understanding and engineering multimeric enzyme complexes.
Jason O. Matos, Jihee Lee, Ramasamy P. Kumar et al.· Science Advances· 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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