Engineering of Rieske dioxygenase variants with improved cis-dihydroxylation activity for benzoates.

Rieske dioxygenases have a long history of being utilized as green chemical tools in the organic synthesis of high-value compounds, due to their capacity to perform the cis-dihydroxylation of a wide variety of aromatic substrates. The practical utility of these enzymes has been hampered however by steric and electronic constraints on their substrate scopes, resulting in limited reactivity with certain substrate classes. Herein, we report the engineering of a widely used member of the Rieske dioxygenase class of enzymes, toluene dioxygenase (TDO), to produce improved variants with greatly increased activity for the cis-dihydroxylation of benzoates. Through rational mutagenesis and screening, TDO variants with substantially improved activity over the wild-type enzyme were identified. Homology modeling, docking studies, molecular dynamics simulations, and substrate tunnel analysis were applied in an effort to elucidate how the identified mutations resulted in improved activity for this polar substrate class. These analyses revealed modification of the substrate tunnel as the likely cause of the improved activity observed with the best-performing enzyme variants.

Characterization of Dichloroisoeverninic Acid Biosynthesis and Chemoenzymatic Synthesis of New Orthosomycins.

The orthosomycins are highly modified oligosaccharide natural products with a broad spectrum and potent antimicrobial activities. These include everninomicins and avilamycins, which inhibit protein translation by binding a unique site on the bacterial ribosome. Notably, ribosomal bound structures reveal a network of interactions between the 50S subunit and dichloroisoeverninic acid (DCIE), the aromatic A1-ring conserved across orthosomycins, but the relationship of these interactions to their antimicrobial activity remains undetermined. Genetic functional analysis of three genes putatively associated with DCIE biosynthesis in the everninomicin producer Micromonospora carbonacea delineates the native biosynthetic pathway and provides previously unreported advanced biosynthetic intermediates. Subsequent in vitro biochemical analyses demonstrate the complete DCIE biosynthetic pathway and provide access to novel everninomicin analogs. In addition to the orsellinate synthase EvdD3 and a flavin-dependent halogenase EvdD2, our results identified a key acyltransferase, EvdD1, responsible for transferring orsellinate from the acyl carrier protein domain of EvdD3 to a heptasaccharide orthosomycin biosynthetic intermediate. We have also shown that EvdD1 is able to transfer unnatural aryl groups via their N-acyl cysteamine thioesters to the everninomicin scaffold and used this as a biocatalyst to generate a panel of unnatural aryl analogs. The impact of diverse aryl functional group substitution on both ribosome inhibition and antibacterial activities demonstrates the importance of the DCIE moiety in the pharmacology of orthosomycins, notably revealing an uncoupling between ribosomal engagement and antibiotic activity. Control of A1-ring functionality in this class of molecules provides a potential handle to explore and address pharmacological roles of the DCIE ring in this potent and unique class of antibiotics.

Development and application of a high throughput assay system for the detection of Rieske dioxygenase activity.

Herein we report the development of a new periodate-based reactive assay system for the fluorescent detection of the cis-diol metabolites produced by Rieske dioxygenases. This sensitive and diastereoselective assay system successfully evaluates the substrate scope of Rieske dioxygenases and determines the relative activity of a rationally designed Rieske dioxygenase variant library. The high throughput capacity of the assay system enables rapid and efficient substrate scope investigations and screening of large dioxygenase variant libraries.

Discovery of human cell selective effector molecules using single cell multiplexed activity metabolomics.

Discovering bioactive metabolites within a metabolome is challenging because there is generally little foreknowledge of metabolite molecular and cell-targeting activities. Here, single-cell response profiles and primary human tissue comprise a response platform used to discover novel microbial metabolites with cell-type-selective effector properties in untargeted metabolomic inventories. Metabolites display diverse effector mechanisms, including targeting protein synthesis, cell cycle status, DNA damage repair, necrosis, apoptosis, or phosphoprotein signaling. Arrayed metabolites are tested against acute myeloid leukemia patient bone marrow and molecules that specifically targeted blast cells or nonleukemic immune cell subsets within the same tissue biopsy are revealed. Cell-targeting polyketides are identified in extracts from biosynthetically prolific bacteria, including a previously unreported leukemia blast-targeting anthracycline and a polyene macrolactam that alternates between targeting blasts or nonmalignant cells by way of light-triggered photochemical isomerization. High-resolution cell profiling with mass cytometry confirms response mechanisms and is used to validate initial observations.