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Results for “substrate promiscuity”

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PubMed2026

Exploring the substrate promiscuity and functional residues of UGT73 family enzymes in Entada phaseoloides.

Flavonoid glycosides and triterpenoid saponins are bioactive plant metabolites with broad applications in food, medicine, and agriculture. These compounds are typically synthesized through glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferases (UGTs). In this study, phylogenetic analysis across multiple species revealed a lineage-specific expansion of the UGT73 family in legumes such as Entada phaseoloides and Glycine max. The genome of the medicinal legume E. phaseoloides was re-annotated using integrated Oxford Nanopore Technologies and Illumina transcriptomic data to identify target genes. Four expanded UGT73 family genes were selected and functionally characterized. UGT73AA6 specifically glycosylates flavonoids, while UGT73CG48 and UGT73CG49 catalyze glycosylation of both flavonoids and pentacyclic triterpenoids. UGT73CG49 exhibits higher catalytic activity for the glucosylation of flavonoids and pentacyclic triterpenes compared to its xylosylation activity. Structural modeling and molecular docking identified key active sites, and site-directed mutagenesis revealed Gly194 as a critical residue enhancing catalytic activity in UGT73CG49. This study provides new insights into the functional evolution and metabolic versatility of the UGT73 family in legumes. The identification and engineering of UGT73 genes from E. phaseoloides lay a foundation for future applications in biosynthetic pathway engineering and the industrial production of high-value glycosides.

Substrate Specificity↗

PubMed2024

Multisubstrate specificity shaped the complex evolution of the aminotransferase family across the tree of life.

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

Substrate Specificity↗

PubMed2026

Iterative Enoyl Reduction by a FabV-Family Enzyme Expands the Chemical Landscape of Discrete Polyketide Synthases.

Polyketides are a structurally diverse class of natural products with immense therapeutic potential. However, the biosynthetic output of discrete polyketide synthases (PKSs) has been constrained by a fundamental functional limitation: unlike modular Type I systems, discrete PKS systems typically lack integrated enoyl reductase (ER) activity. This constraint restricts their chemical repertoire primarily to unsaturated polyenes or aromatic scaffolds. Here, we characterize PbrC16, a FabV-family ER from a manumycin-type biosynthetic gene cluster (BGC) in Peterkaempfera bronchialis. This enzyme represents the first experimentally validated ER capable of functioning within discrete PKS architectures. In vitro biochemical reconstitution demonstrates that PbrC16 along with its homologue ScFabV catalyze iterative enoyl reductions in both β-ketoacyl-acyl carrier protein synthase III (KAS III)-dependent and highly reducing (HR) Type II PKS contexts, enabling the complete saturation of long-chain polyketide intermediates. Structural and computational analyses reveal the molecular basis for its exceptional substrate promiscuity and versatile acyl carrier protein (ACP) recognition. These findings resolve a long-standing "reductive gap" in discrete PKS biology and provide a "plug-and-play" module for the rational engineering of saturated polyketide scaffolds.

Polyketide Synthases↗

PubMed2025

Engineering of xylose metabolic pathways in Rhodotorula toruloides for sustainable biomanufacturing.

The oleaginous yeast Rhodotorula toruloides is a promising microbial cell factory for the sustainable production of biofuels and value-added chemicals from renewable carbon sources. Unlike the conventional yeast Saccharomyces cerevisiae, R. toruloides can naturally metabolize xylose, the second most abundant sugar in lignocellulosic hydrolysates. However, its native xylose metabolism is inefficient, characterized by slow xylose uptake and accumulation of D-arabitol. Moreover, despite its phenotype, research on the enzymes involved in xylose metabolism has yet to reach a consensus. Therefore, this review provides a comprehensive analysis of the non-canonical xylose metabolism in R. toruloides, focusing on the properties of key enzymes involved in xylose metabolism. Native xylose reductase and xylitol dehydrogenase exhibit broad substrate promiscuity compared to their counterparts in the xylose-fermenting Scheffersomyces stipitis. Additionally, the absence of xylulokinase expression under xylose-utilizing conditions redirects metabolism toward D-arabitol accumulation. Consequently, D-arabitol dehydrogenases and ribulokinase play essential roles in the xylose metabolism of R. toruloides. These findings highlight the fundamental differences between R. toruloides xylose metabolism and the oxidoreductase pathways observed in other xylose-fermenting yeast, providing insights for metabolic engineering strategies to improve xylose utilization and enhance bioconversion of cellulosic hydrolysates to different bioproducts by R. toruloides.

Xylose↗

PubMed2026

In-Silico and Functional Characterization of EcdLp, an ABC Transporter of Aspergillus nidulans NRRL11440.

Echinocandin B (ECB) biosynthesis in Aspergillus nidulans is primarily governed by multiple genes located within the biosynthetic echinocandin (ecd) gene cluster. The contributory functions of many genes, including transcription factors and tailoring enzymes of the ecd gene cluster, have been previously studied. The present study focused on determining the role of transporter proteins, EcdLp, EcdCp, and EcdDp, in ECB efflux using in silico and biochemical approaches. The molecular docking analysis revealed that ECB relatively showed higher binding affinity for EcdLp than the other co-clustered MFS transporters EcdCp and EcdDp, suggesting a preferred substrate of EcdLp. These results were further confirmed by heterologous integration of the ecdL gene in the ABC transporters-deficient Saccharomyces cerevisiae AD1-8u⁻, confirming active efflux. However, the binding of ECB in EcdLp is distinct from the R6G binding, overlapping the promiscuous site of farnesol, resulting in inhibition of R6G efflux in a dose-dependent manner. In conclusion, these results decipher the ECB binding and efflux mechanism and unveil the evolutionarily specialized architecture of EcdLp that permits targeted metabolite export in addition to environmental responsiveness, and lay the groundwork for optimizing ECB production via transporter engineering.

Aspergillus nidulans↗