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Isonitrile lipopeptides (INLPs) are known to be related to the virulence of pathogenic mycobacteria by mediating metal transport, but their biosynthesis remains obscure. In this work, we use in vitro biochemical assays, site-directed mutagenesis, chemical synthesis, and spectroscopy techniques to scrutinize the activity of core enzymes required for INLP biosynthesis in mycobacteria. Compared to environmental Streptomyces, pathogenic Mycobacterium employ a similar chemical logic and enzymatic machinery in INLP biosynthesis, differing mainly in the fatty-acyl chain length, which is controlled by multiple enzymes in the pathway. Our in-depth study on the non-heme iron(II) and α-ketoglutarate-dependent dioxygenase for isonitrile generation, including Rv0097 from Mycobacterium tuberculosis (Mtb), demonstrates that it recognizes a free-standing small molecule substrate, different from the recent hypothesis that a carrier protein is required for Rv0097 in Mtb. A key residue in Rv0097 is further identified to dictate the varied fatty-acyl chain length specificity between Streptomyces and Mycobacterium.

Natural products are a diverse class of biologically produced compounds that participate in fundamental biological processes such as cell signaling, nutrient acquisition, and interference competition. Unique triple-bond functionalities like isonitriles and alkynes often drive bioactivity and may serve as indicators of novel chemical logic and enzymatic machinery. Yet, the biosynthetic underpinnings of these groups remain only partially understood, constraining the opportunity to rationally engineer biomolecules with these functionalities for applications in pharmaceuticals, bioorthogonal chemistry, and other value-added chemical processes. Here, we focus our review on characterized biosynthetic pathways for isonitrile and alkyne functionalities, their bioorthogonal transformations, and prospects for engineering their biosynthetic machinery for biotechnological applications.

The isonitrile moiety is an electron-rich functionality that decorates various bioactive natural products isolated from diverse kingdoms of life. Isonitrile biosynthesis was restricted for over a decade to isonitrile synthases, a family of enzymes catalyzing a condensation reaction between l-Trp/l-Tyr and ribulose-5-phosphate. The discovery of ScoE, a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase, demonstrated an alternative pathway employed by nature for isonitrile installation. Biochemical, crystallographic, and computational investigations of ScoE have previously been reported, yet the isonitrile formation mechanism remains obscure. In the present work, we employed in vitro biochemistry, chemical synthesis, spectroscopy techniques, and computational simulations that enabled us to propose a plausible molecular mechanism for isonitrile formation. Our findings demonstrate that the ScoE reaction initiates with C5 hydroxylation of (R)-3-((carboxymethyl)amino)butanoic acid to generate 1, which undergoes dehydration, presumably mediated by Tyr96 to synthesize 2 in a trans configuration. (R)-3-isocyanobutanoic acid is finally generated through radical-based decarboxylation of 2, instead of the common hydroxylation pathway employed by this enzyme superfamily.

Chlorophylls are ubiquitous pigments responsible for the green color in plants. Changes in the chlorophyll content have a significant impact on photosynthesis, plant growth and development. In this study, we used a yellow stigma mutant (ys) generated from a green stigma tomato WT by using ethylmethylsulfone (EMS)-induced mutagenesis. Compared with WT, the stigma of ys shows low chlorophyll content and impaired chloroplast ultrastructure. Through map-based cloning, the ys gene is localized to a 100 kb region on chromosome 4 between dCAPS596 and dCAPS606. Gene expression analysis and nonsynonymous SNP determination identified the Solyc04g015750 , as the potential candidate gene, which encodes a magnesium chelatase H subunit (CHLH). In ys mutant, a single base C to T substitution in the SlCHLH gene results in the conversion of Serine into Leucine (Ser92Leu) at the N-terminal region. The functional complementation test shows that the SlCHLH from WT can rescue the green stigma phenotype of ys. In contrast, knockdown of SlCHLH in green stigma tomato AC, observed the yellow stigma phenotype at the stigma development stage. Overexpression of the mutant gene Slys in green stigma tomato AC results in the light green stigma. These results indicate that the mutation of the N-terminal S92 to Leu in SlCHLH is the main reason for the formation of the yellow stigma phenotype. Characterization of the ys mutant enriches the current knowledge of the tomato chlorophyll mutant library and provides a novel and effective tool for understanding the function of CHLH in tomato. • SlCHLH controlling the stigma color formation and the S92 of N-terminal affects magnesium chelatase activity. [ABSTRACT FROM AUTHOR]

Azides are energy-rich compounds with diverse representation in a broad range of scientific disciplines, including material science, synthetic chemistry, pharmaceutical science and chemical biology. Despite ubiquitous usage of the azido group, the underlying biosynthetic pathways for its formation remain largely unknown. Here we report the characterization of an enzymatic route for de novo azide construction. We demonstrate that Tri17, a promiscuous ATP- and nitrite-dependent enzyme, catalyses organic azide synthesis through sequential N-nitrosation and dehydration of aryl hydrazines. Through biochemical, structural and computational analyses, we further propose a plausible molecular mechanism for azide synthesis that sets the stage for future biocatalytic applications and biosynthetic pathway engineering., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)