Bryan Ferlez (Kerfeld lab)
Date & Location: November 19, 2019, at 12p; Room 168 Plant Biology Building
Subject: Glycyl radical enzyme-associated microcompartments: redox-replete bacterial organelles
Abstract: A growing number of ecologically diverse microbes are being identified that organize segments of their metabolism within self-assembling proteinaceous organelles known as bacterial microcompartments (BMCs). The basic BMC architecture of enzymes encapsulated in a selectively permeable protein shell is also being adapted for compartmentalizing reactions in the context of bioengineering. One of the most abundant, yet less well characterized, BMC types encapsulate glycyl radical enzymes (GREs) that require an activating enzyme (AE), S-adenosylmethionine, and an external source of electrons for activation. However, it remains unclear how the prerequisite reduction of the AE takes place if insulated from cytosolic electron transfer proteins within the BMC. One possibility is that metal centers bound by the shell mediate the transfer of electrons into and out of the lumen. While electron transfer activity has been engineered into a shell protein, little is known about how native shell proteins coordinate redox active metals and transfer electrons. In addition, although the protein shell of the carboxysome has been shown to limit CO2 diffusion, the permeability of BMC shells to O2 remains uncharacterized, and may prevent O2-mediated inactivation of encapsulated GREs.
To address these knowledge gaps, we aim to characterize two widespread iron-sulfur cluster containing shell proteins, GrpU and PduT, as well as a putative NADH-oxidizing flavoenzyme (PduS) that may serve as a luminal source of electrons. In parallel, we are using the native BMC from the phototrophic bacterium Rhodopseudomonas palustris BisB18 and a synthetic shell system developed in the Kerfeld lab to study electron transfer and O2 permeability in the context of intact compartments. To this end, we have successfully isolated BMCs from R. palustris and observed encapsulation of the predicted AE. Moreover, the shell appears to contain GrpU, which binds an oxygen labile [4Fe4S] cluster that, based on data from another GrpU homolog, has a midpoint potential of ~ –342 mV +/- 4 mV (v. SHE). Ultimately, insights from these studies will inform the design and installation of synthetic BMCs as programmable metabolic sinks that use energy or electrons generated by photochemical reaction centers to produce biotechnologically relevant products in diverse photosynthetic hosts.
Speaker Lab: Dr. Cheryl Kerfeld