Eric L. Hegg

Mitigating the release of the greenhouse gas nitrous oxide (N2O) is a major focus of sustainable agricultural practices. Currently, application of nitrogen-based fertilizers to the field drives significant microbial N2O production. Understanding nitrogen flux through microbial metabolic processes, such as nitrification and denitrification, under different soil conditions is key to tracking sources of N2O emissions in agricultural soils. Stable isotopes are a powerful tool for tracing sources and sinks of N2O because isotopic preferences (e.g., isotope effects) of key enzymes in nitrogen cycling pathways allow for the differentiation of microbial sources of N2O. Ascertaining source contributions of microbial N2O production can be complicated, however, by the consumption of N2O by nitrous oxide reductase (N2OR), which itself exhibits isotopic preferences that influence the isotopic composition of the remaining N2O. To help provide the isotopic parameters required to quantify and distinguish sources and sinks of N2O, we determined the kinetic isotope effects (KIEs) for N2O consumption by purified form II N2OR (resting state: CuZ*) from Stutzerimonas stutzeri. The enrichment factors (ε) were –5.4 ± 0.2‰, -10.0 ± 0.4‰, -0.6 ± 0.1‰, -9.5 ± 0.4‰, and –13.5 ± 0.5‰ for ε15Nbulk, ε15Nα, ε15Nβ, ε15NSP, and ε18O, respectively. Thus, the kinetic isotope effects were normal for bulk δ15N (1.0054 ± 0.0002), δ15Nα (1.0101 ± 0.0004), and δ15NSP (1.0095 ± 0.0004), and δ18O (1.0137 ± 0.0005). The KIE for δ15Nβ was 1.0006 ± 0.0001, indicating there is essentially no isotopic preference for this position. These data improve our understanding of the isotopic fractionation associated with enzymatic N2O consumption, which will inform modeling of nitrogen flux pathways leading to N2O emissions in soil microbial communities.

Bacterial microcompartments (BMCs) are protein-based organelles found in diverse bacteria that encapsulate a sequence of enzymes to accelerate specific metabolic pathways and limit toxicity by confining intermediate products. To facilitate catalysis in BMCs, reactant and products must transit across the protein shells, which are made up of multimeric protein tiles. Quantifying the permeability for transport of small molecules across the shell is a key engineering consideration to design novel catalytic microcompartments. We examine the permeability of reactants and products for two reaction pathways, the degradation of 2-aminophenol by AmnAB and the reduction of nitrate to ammonium by NrfA, through a computational lens to quantify permeability for these metabolites at the nanoscale. From Hamiltonian replica exchange umbrella sampling simulations, we determine that the energetic barriers to permeation for the relevant metabolites along both pathways are small, with permeability coefficients in the range of 0.1-4cm/s. The high permeabilities calculated for each metabolite through the inhomogeneous solubility diffusion model are corroborated by enzyme activity measurements in vitro that point to enzymatic activity within the shell system. We expand on these findings by quantifying the scale of transport and catalysis that can be supported by in vitro BMC systems. The results highlight the importance of substrate permeability across BMCs within a biological context, and represent steps toward generating synthetic shells or bioengineering novel nanoreactors.

Non-biodegradable synthetic plastics are accumulating in the environment and current recycling methods are limited by harsh processing conditions and inferior quality of recycled products. While biological approaches to plastic degradation offer promise, available enzymes remain inefficient at degrading synthetic plastics due to their inherent physical and chemical properties. Inspired by nature's strategies for degrading resistant natural polymers like cellulose and chitin with enzyme complexes co-localized to polymer surfaces, we quantitatively evaluated a range of previously reported plastic-binding peptides (PBPs) for their capacity to anchor soluble proteins specifically to polystyrene and polypropylene. Among the PBPs tested, LCIM3 demonstrated better specificity when compared to other peptides, binding to polystyrene and polypropylene with an apparent dissociation constant (K_D) in the nanomolar range. By analogy to natural cellulosome complexes, we further investigated the rational design of scaffolds utilizing paired dockerin–cohesin protein binding domains and LCIM3 to enable recruitment of multiple user-defined proteins to plastics. Our study demonstrates that plastic binding peptides were marginally able to increase the localization of proteins and scaffolds onto plastic substrates. However, we also identify a significant non-specific adsorption of many untargeted proteins on plastic as a potential bottleneck for approaches to improve plastic degradation via rational engineering. This proof-of-concept study highlights both the opportunities and challenges in designing bio-inspired scaffolds to improve plastic degradation, laying a foundation for future advancements in sustainable plastic recycling and upcycling.

Nitrous oxide (N₂O) is a serious concern due to its role in global warming and ozone destruction. Agricultural practices account for ∼80% of all anthropogenic N₂O produced in the US, due in large part to the stimulation of microbial denitrification. Stable isotopes are uniquely suited to examine both microbial N₂O sources and the mechanism of N₂O biosynthesis through the use of Site Preference (δ¹⁵N^SP; the difference in δ¹⁵N between the central and outer N atoms in N₂O) and kinetic isotope effects (KIEs), respectively. Using trace gas isotope ratio mass spectrometry (TG-IRMS), we determined the δ¹⁵N, δ¹⁵N^α, δ¹⁵N^β, and δ¹⁸O of N₂O produced by a purified cytochrome c nitric oxide reductase (cNOR) from Paracoccus denitrificans. We also calculated δ¹⁵N^SP, the KIEs, and associated isotopic enrichment factors (ε) for N^bulk, N^α, and N^β. A normal isotope effect was observed for bulk ¹⁵N, with a KIE value of 1.0086 ± 0.0009 (ε = -8.6 ± 0.9‰). The isotope effects for both ¹⁵N^α and ¹⁵N^β were also normal, with position-specific KIEs of 1.0072 ± 0.0010 (ε = -7.2 ± 1.0‰) and 1.0100 ± 0.0010 (ε = -9.9 ± 1.0‰), respectively, and δ¹⁵N^SP values ranged from 0.5 to 8.7‰ with no significant trend as the reaction proceeded. Values of δ¹⁸O increased with N₂O production (slope of δ¹⁸O against [-f ln f/(1 - f)] = -19.9 ± 1.9‰). We present implications for the mechanism of N₂O production from cNOR based on our data.

Bacterial microcompartments (BMCs) are large protein-based organelles found in many bacteria that encapsulate a sequence of enzymes to accelerate specific metabolic pathways and limit toxicity by confining intermediate products. In BMCs to facilitate catalysis, reactant and products must transit across the protein shells, which are made up of multimeric protein tiles. Quantifying the permeability for transport of small molecules across the shell is a key engineering consideration to design novel catalytic microcompartments. We examine the permeability for reactants and products for two reaction pathways, the degradation of 2-aminophenol by AmnAB, and the reduction of nitrate to ammonium by NrfA, through a computational lens to quantify permeability for these metabolites at the nanoscale. From Hamiltonian replica exchange umbrella sampling simulations, we determine that the energetic barriers to permeation for the relevant metabolites along both pathways are small, with permeability coefficients in the range of 0.1-4 cm/s. The high permeabilities calculated for each metabolite through the inhomogeneous solubility diffusion model are corroborated by enzyme activity measures \emph{in vitro} that point to enzymatic activity within the shell system. We expand on these findings by quantifying the scale of transport and catalysis that can be supported in realistic BMC systems. The results highlight the importance of substrate permeability across BMCs within a biological context, and represent steps toward generating synthetic shells or bioengineering novel nanoreactors.

The dissimilatory nitrate reduction to ammonium pathway converts nitrate to ammonium, a vital reaction in the global nitrogen cycle. The second step of the pathway is performed by cytochrome c nitrite reductase (NrfA), a soluble, periplasmic cytochrome responsible for the reduction of nitrite to ammonium. The pentaheme NrfA catalyzes this six-electron and eight-proton reduction of nitrite at a single active site with the help of its quinol oxidase partners. In this review, we discuss our current understanding of (i) the structure, homology, and evolution of both NrfA and its redox partners, (ii) the regulation of the nrf operon, and (iii) the maturation of NrfA proteins via unique cytochrome maturation pathways.

Bacterial microcompartments (BMCs) are proteinaceous organelles that self-assemble into selectively permeable shells that encapsulate enzymatic cargo. BMCs enhance catalytic pathways by reducing crosstalk among metabolites, preventing harmful intermediates from leaking into the cytosol and increasing reaction efficiency via enzyme colocalization. The intrinsic properties of BMCs make them attractive for biotechnological engineering. However, in vivo expression methods for shell synthesis have significant drawbacks that limit the potential design space for these nanocompartments. Here, we describe the development of an efficient and rapid method for the in vitro assembly of BMC shells from their protein building blocks. Our method enables large-scale construction of BMC shells by utilizing urea as a chaotropic agent to control self-assembly and provides an approach for encapsulation of both biotic and abiotic cargo under a broad range of reaction conditions. We demonstrate an enhanced level of control over the assembly of BMC shells in vitro and expand the design parameter space for engineering BMC systems with specialized and enhanced catalytic properties.