This study identifies Bg-3βHSD2 as an efficient and versatile steroid-transforming enzyme, expands the knowledge of functional diversity within the amphibian 3βHSD family, and provides insights into the enzymatic basis of steroid and bufadienolide metabolism in B. bufo gargarizans.
Abstract
Abstract 3β-Hydroxysteroid dehydrogenases (3βHSDs) are key enzymes in steroid metabolism, catalyzing C3 oxidation–reduction and Δ5→Δ4 isomerization reactions that govern metabolic flux across multiple steroidogenic pathways. However, the functional diversity of 3βHSDs involved in bufadienolide metabolism in amphibians remains poorly explored. Here, we systematically characterized the 3βHSD gene family in the Asian toad (Bufo bufo gargarizans) using integrated transcriptomic, biochemical, and metabolomic analyses. Seven Bg-3βHSD genes were identified from multi-tissue transcriptomes generated under control and Pb2+ exposure conditions, and six were heterologously expressed for functional evaluation. In vitro assays revealed pronounced functional divergence among Bg-3βHSD isoforms. Bg-3βHSD1 primarily catalyzed bidirectional C3 redox reactions of C21 steroids and bile acid-related substrates, consistent with canonical steroidogenic roles. In contrast, Bg-3βHSD2 enzyme exhibited broad substrate specificity and high catalytic efficiency toward hormones, bile acids, and bufadienolides. In addition to canonical C3 redox reactions and Δ5→Δ4 isomerization, Bg-3βHSD2 also displayed additional oxidation activity at the C17 position for several steroid substrates. A third homolog, Bg-HSD3B7 (GenBank accession no. XM 044303756.1), selectively converted 7α-hydroxylated sterols, suggesting a potential role in classical bile acid metabolism. Integration of tissue-specific expression profiles with bufadienolide distribution patterns suggests that Bg-3βHSD2 may contribute to connecting classical steroid metabolism with bufadienolide biosynthesis in adrenal tissue. Together, the present study identifies Bg-3βHSD2 as an efficient and versatile steroid-transforming enzyme, expands our knowledge of functional diversity within the amphibian 3βHSD family, and provides insights into the enzymatic basis of steroid and bufadienolide metabolism in B. bufo gargarizans.
Mycolicibacterium
strains are among the most effective biofactories for converting phytosterols into active pharmaceutical intermediates. Despite the main route resembling fatty acid β-oxidation, the specific enzymes and their precise roles are poorly defined. In this study, we performed a comprehensive gene knock-out analysis in the industrial 4-AD-producing strain,
Mycolicibacterium neoaurum
HGMS9 strain, a variant of
M. neoaurum
B-3805. Our results demonstrate that the three β-oxidation cycles responsible for phytosterol side-chain degradation share a suite of core enzymes. Specifically, seven enzymes, including the acyl-CoA dehydrogenase ChsE1/E2, ChsE4/E5, the hydratase, ChsH1/H2 and the aldolase Ltp2, function in both the second and third β-oxidation cycles to generate the final products, 4-AD. Importantly, we identified five potential secondary routes that divert metabolism to produce eight off-route C22, C23 and C24 intermediates. These compounds represent valuable precursors for the synthesizing advanced steroids like corticosteroids and bile acids. This study not only clarifies the enzymatic steps of phytosterol side-chain degradation but also establishes a metabolic blueprint for engineering high-yielding
Mycolicibacterium
strains.
Our previous study demonstrated that (Z)-3-hexenyl vicianoside and (Z)-3-hexenyl primeveroside display antiherbivore activity against tea gray geometrids, yet their biosynthetic glycosyltransferases (GTs) remained uncharacterized. Here, we identified CsUGT91A1 (CsGT3) from tea plants, which localizes to the cytoplasm and nucleus. Recombinant CsGT3 converted (Z)-3-hexenyl glucoside into vicianoside and primeveroside, with optimal activity at 30 °C and pH 7.5. Kinetic and docking studies indicated higher catalytic efficiency for UDParabinose than UDPxylose. Transient overexpression in tobacco and tea plants confirmed CsGT3 catalyzes this conversion. Moreover, infestation by tea green leafhoppers, tea gray geometrids, or tea aphids consistently increased the accumulation of (Z)-3-hexenyl glycosides, associated with the increased precursor (Z)-3-hexenol. These findings elucidate the role of CsGT3 in the biosynthesis of (Z)-3-hexenyl disaccharides and enhance the understanding of volatile glycoside metabolism in tea plants.
Y. Liao, Zeyuan Zou, Xiaochen Zhou et al.· Journal of Agricultural and...· 0 citations
Geranylgeranoic acid (GGA) is an endogenous acyclic diterpenoid metabolite of the mevalonate pathway that has been implicated in programmed cell death in hepatoma cells and may contribute to the elimination of premalignant hepatocytes. Recent metabolomic studies have identified 2,3-dihydrogeranylgeranoic acid (2,3-diGGA), an α-saturated derivative of GGA with reduced cell-death-inducing activity compared with GGA. This concise review examines the hypothesis that diversion of bioactive GGA toward 2,3-diGGA formation represents a metabolic shift that may attenuate hepatic tumor surveillance and influence hepatocellular carcinoma susceptibility. The review summarizes current evidence for endogenous GGA biosynthesis through the mevalonate pathway, MAO-B- and CYP3A4-associated GGA formation, and the tissue- and age-associated distribution of GGA and 2,3-diGGA. It also discusses the proposed 2,3-diGGA-forming activity, whose molecular identity remains unknown, and evaluates the potential utility of the GGA/2,3-diGGA ratio as a candidate biomarker. Finally, future experimental strategies are outlined to identify the responsible enzyme, clarify causality in cellular and animal models, and validate this metabolic framework in human liver tissues, chronic liver disease cohorts, and HCC-associated settings.
Findings provide insights into the potential multifaceted insecticidal action of benzothiazole in T. castaneum at the transcriptomic level and support its further development as a target-specific grain fumigant.
Kaidi Cui, Xueran Hu, Weifeng Cheng et al.· Pest Management Science· 0 citations
Glycosylation is a crucial modification in plant metabolism and is primarily catalyzed by UDP-glycosyltransferases (UGTs). Among these, the UGT84 subfamily is known to catalyze not only O-glycosylation of hydroxyl groups but also glucose esterification of carboxylic acids, but the underlying mechanism remains unclear. In this study, we identified the MaUGT84R1 gene from Morus alba and heterologously expressed it in both Escherichia coli and Nicotiana benthamiana. In vitro enzymatic assays confirmed its dual catalytic activity. Structural comparisons combined with phylogenetic analysis revealed, for the first time, a unique His-Asn-Ser catalytic triad within the UGT84 subfamily. Molecular docking elucidated the sugar acceptor binding pocket of MaUGT84R1, and site-directed mutagenesis validated the functional roles of key residues, including Phe17, His22, Phe87, Tyr90, Asn122, Phe199, and Val279. On the basis of these findings, we propose a molecular mechanism for the pH-dependent dual catalytic function of MaUGT84R1. In addition, we determined the cytosolic localization of MaUGT84R1 in mulberry protoplasts. Collectively, our study provides novel mechanistic and evolutionary insights into the dual catalytic activity of the UGT84 subfamily, and lays a foundation for future protein engineering efforts.
Jiahui Gong, Wan Zeng, Zerong Wu et al.· Plant physiology and biochem...· 0 citations
Myxoglucamides, natural products recently isolated from Cystobacterineae sp., are featured by an unprecedented vinyl-substituted α-keto-γ-amino acid that is linked to a glycosylated 14-methyl-pentadecanoic acid. Thus, they unite elements from three biomolecular classes in a compact glycolipopeptide. To elucidate the biological relevance of this arrangement, we searched for molecular targets by activity-based protein profiling (ABPP)-although a phenotypic bioactivity has not been reported. An access to the compound class was established through the first total synthesis of myxoglucamide A in 11 steps. A proteome-wide ABPP study led to the identification of aldo-keto reductase 1C3 (AKR1C3) as the primary target of myxoglucamides in human cells. AKR1C3 is an oncogenic factor involved in prostaglandin and steroid synthesis, promoting the growth, proliferation, and metastasis of carcinoma cells. The functional inhibition of AKR1C3 by a competitive mechanism (IC50 = 1.61 µm) was validated in vitro, and 20 analogs provided structure-activity relationships and more potent analogs (IC50 = 181 nm). Biophysical interactions were quantified by thermal shift assays, and essential molecular protein-ligand interactions were characterized by X-ray crystallography at 2.0 Å resolution. The study implies that the search for targets of natural products is rewarding even in the absence of an initial phenotypic activity.
Thomas Siemon, Vivek K. Mishra, Mingming Zhao et al.· Angewandte Chemie· 0 citations
