Choi, C. J. | bioRxiv Science

bioRxiv2025

CURE-ating the substrate scope and functional residues of nonheme iron(II) α-ketoglutarate dependent hydroxylase BesE

Nonheme iron (II) -ketoglutarate-dependent dioxygenases (Fe/KGs) play important roles in functionalizing biological substrates from individual amino acids up to macromolecules. BesE, a unique homolog from the actinobacterial {beta}-ethynylserine biosynthetic pathway, catalyzes a highly selective hydroxylation on dipeptide substrate {gamma}-L-glutamyl-L-propargylglycine (1). Inspired by this transformation, our year-long Course-based Undergraduate Research Experience (CURE) laboratory interrogated BesE catalysis using an interdisciplinary approach. After establishing a modular chemical synthesis of 1, we rationally designed fourteen non-native analogs with key alterations to specific substrate moieties putatively involved in enzymatic recognition. Following in vitro enzymology and the application of chemical derivatization techniques compatible with all substrate analogs and putative products, we determined that the terminal alkyne moiety is not essential while reinforcing the significance of the {gamma}-glutamyl moiety for hydroxylation activity. Thirteen rationally designed BesE mutants established the importance of polar active site residues for substrate recognition and catalysis. This work establishes a baseline of BesE recognition from both a chemical and biochemical perspective and contributes to the growing understanding about Fe/KG recognition on biomedically relevant targets. Moreover, this contributes to the growing examples of using natural products and their biosynthetic enzymology as a vibrant platform for the interdisciplinary training of early career biomedical researchers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=53 SRC="FIGDIR/small/681649v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1d6f5aaorg.highwire.dtl.DTLVardef@19aa649org.highwire.dtl.DTLVardef@1b2f8dorg.highwire.dtl.DTLVardef@cf0867_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

3D-intrusions transport active surface microbial assemblages to the dark ocean

Subtropical oceans contribute significantly to global primary production, but the fate of the picophytoplankton that dominate in these low nutrient regions is poorly understood. Working in the subtropical Mediterranean, we demonstrate that subduction of water at ocean fronts generates 3D intrusions with uncharacteristically high carbon, chlorophyll, and oxygen that extend below the sunlit photic-zone into the dark ocean. These contain "fresh" picophytoplankton assemblages that resemble the photic-zone regions where the water originated. Intrusions propagate depth-dependent seasonal variations in microbial assemblages into the ocean interior. Strikingly, the intrusions included dominant biomass contributions from non-photosynthetic bacteria and enrichment of enigmatic heterotrophic bacterial lineages. Thus, the intrusions not only deliver material that differs in composition and nutritional character from sinking detrital particles, but also drive shifts in bacterial community composition, organic matter processing, and interactions between surface and deep communities. Modeling efforts paired with global observations demonstrate that subduction can flux similar magnitudes of particulate organic carbon as sinking export, but is not accounted for in current export estimates and carbon cycle models. Intrusions formed by subduction are a particularly important mechanism for enhancing connectivity between surface and upper mesopelagic ecosystems in stratified subtropical ocean environments that are expanding due to the warming climate.

ecology↗