Aug 2026· Journal of the American Chemical Society· Vol 148 34, pp.
37144-37153
· 0 citations· 31 references
Medicine
Abstract
Nucleoside natural products exhibit diverse chemical architectures and potent biological activities, yet the biosynthetic strategies that generate their structural diversity remain incompletely understood. Here, we elucidate the early stage biosynthetic pathway of the cytidyl-cyclitol natural product K-563 and its derivatives. The cyclitol component is generated by the myo-inositol-1-phosphate synthase (MIPS) family enzyme KesM and is subsequently coupled to the cytidine moiety by KesL using cytidine 5'-triphosphate (CTP), followed by further modifications catalyzed by the phosphatase KesI and the unique dehydrogenase complex KesJ/KesK. The X-ray crystal structure and mutagenesis analyses reveal that KesL adopts the canonical fold of S-adenosyl-l-methionine (SAM) synthetases, which catalyze the adenosylation of l-methionine with adenosine 5'-triphosphate (ATP) in primary metabolism, while the KesL active site is extensively remodeled to accept the cyclitol phosphate with CTP as an atypical substrate pair, thereby generating the cytidyl-cyclitol core structure. This work not only expands the chemical logic of nucleoside biosynthesis but also demonstrates how the SAM synthetase-like protein scaffold is repurposed to catalyze an unusual nucleoside transfer reaction for specialized secondary metabolite assembly.
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.
S-adenosyl-L-methionine (SAM), an essential cofactor in all forms of life, is synthesized by the enzyme methionine adenosyltransferase (MAT) from methionine and ATP. The adenine moiety in SAM appears to have no direct function in catalysis, and some MAT homologs can utilize natural nucleotide triphosphates in vitro, producing the corresponding SAM nucleobase analogues. However, the molecular determinants of nucleotide choice of the MAT enzyme and the cellular significance of the nucleobase in SAM are unclear. In this study, using structure- and bioinformatics-guided mutagenesis, we identify a flexible active-site loop as a major determinant of nucleotide specificity in MAT. Loop mutations and loop swaps convert ATP-selective Escherichia coli MAT into variants that accept GTP, CTP, and UTP, enabling enzymatic synthesis and purification of S-guanosyl-, S-cytosyl-, and S-uracyl-L-methionine. Further, we show that these analogues partially rescue the growth of an E. coli SAM auxotroph under SAM-limited growth conditions. Biochemical assays show that the analogues bind the tested SAM-utilizing enzymes; they serve as substrates for E. coli SAM decarboxylase but do not support detectable methyl transfer by E. coli DNA adenine methyltransferase. These results establish the flexible loop as a gatekeeper of MAT nucleotide specificity and show that this loop can be engineered to produce SAM analogues which can selectively participate in downstream cellular metabolism. Graphical Abstract/ Table of contents only
In vitro and in vivo analyses support a recent finding that the defining carbon-carbon bond is formed not by a long-hypothesized PLP-dependent process, but by a vitamin B12-dependent radical SAM enzyme, and expand upon the current knowledge of radical-mediated C-C bond formation and PEARL enzyme catalysis.
Chi-Fang Lee, T. Zhou, Songyi Xue et al.· Journal of the American Chem...· 0 citations
In vitro approaches elucidated the modifications installed by the std biosynthetic enzymes and expand the known substrate scope of YcaO-TfuA enzymes and MNIOs and identify new roles for carbamoyltransferases in these pathways.
Dayna P. Hebron, Tucker J. Shriver, Joshua J. Ziarek et al.· Journal of the American Chem...· 0 citations
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
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
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