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DOE OSTI2024

Preparation of coenzyme F430 biosynthetic enzymes and intermediates

Methyl-coenzyme M reductase (MCR) is the key enzyme in pathways for the formation and anaerobic oxidation of methane. As methane is a potent greenhouse gas and biofuel, investigations of MCR catalysis and maturation are of interest for the development of both methanogenesis inhibitors and natural gas conversion strategies. Here, the activity of MCR is dependent on a unique, nickel-containing coenzyme F430, the most highly reduced tetrapyrrole found in nature. Coenzyme F430 is biosynthesized from sirohydrochlorin in four steps catalyzed by the CfbABCDE enzymes. Here, methods for the expression and purification of the coenzyme F430 biosynthesis enzymes are described along with conditions for the synthesis and purification of biosynthetic intermediates on the milligram scale from commercially available porphobilinogen.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Direct root contact among neighboring plants influences activity of soil extracellular enzymes

Composition and diversity of vegetation systems can influence soil microbial activity and extracellular enzyme (EE) dynamics, which are crucial for soil carbon (C) accrual and nutrient cycling. Yet, the impact of plant interactions and competition on EE activities remains a notable knowledge gap. This study examines how direct root contact and neighboring plant identity affect the activity and spatial distribution of four key soil EEs: β-glucosidase (BGlu), chitinase, acid phosphatase (AcidP), and alkaline phosphatase (AlkP). Using three-compartment rhizoboxes with switchgrass (Panicum virgatum L.) grown alongside bush clover (Lespedeza capitata Michx.), and black-eyed Susan (Rudbeckia hirta L.), we assessed enzyme activities using zymography under conditions that either allowed or restricted direct root contact by root barriers. Results show that root proliferation and species interactions significantly influenced EE activity. While BGlu and AcidP activities were strongly correlated with root biomass, AlkP activity was consistently higher in the absence of root barriers, indicating a pronounced microbial response to plant interactions via direct/close root contacts. Additionally, soil phosphorus availability modulated enzyme activity, with higher phosphatase activities in low-P soils. Furthermore, these findings highlight the importance of root-root interactions and plant species composition in shaping soil biochemical processes.

enyzme activity↗

DOE OSTI2026

Discovery and engineering of enzymes for new-to-nature photobiocatalysis

Photobiocatalysis integrates enzymatic catalysis with photochemistry, enabling challenging radical transformations with high selectivity under mild conditions. Early developments in this field were largely driven by the discovery that enzyme-bound cofactors can form photoactive charge–transfer complexes with substrates, thereby initiating radical chemistry upon light irradiation. Recent advances, however, have substantially expanded the mechanistic landscape of photobiocatalysis through diverse mechanisms. This review summarizes major developments in photobiocatalysis reported since 2024. Rather than cataloging individual reactions, we focus on the fundamental mechanisms of radical generation and interception within enzyme active sites, and discuss how these mechanistic principles guide the discovery, engineering, and design of enzymes for new-to-nature photobiocatalysis.

Bai, Zibo [University of Illinois Urbana-Champaign↗

DOE OSTI2024

Biochemical and structural characterization of enzymes in the 4-hydroxybenzoate catabolic pathway of lignin-degrading white-rot fungi

White-rot fungi (WRF) are the most efficient lignin-degrading organisms in nature. However, their capacity to use lignin-related aromatic compounds, such as 4-hydroxybenzoate, as carbon sources has only been described recently. Previously, the hydroxyquinol pathway was proposed for the bioconversion of these compounds in fungi, but gene- and structure-function relationships of the full enzymatic pathway remain uncharacterized in any single fungal species. Here, we characterize seven enzymes from two WRF, Trametes versicolor and Gelatoporia subvermispora, which constitute a four-enzyme cascade from 4-hydroxybenzoate to β-ketoadipate via the hydroxyquinol pathway. Furthermore, we solve the crystal structure of four of these enzymes and identify mechanistic differences with the closest bacterial and fungal structural homologs. Overall, this research expands our understanding of aromatic catabolism by WRF and establishes an alternative strategy for the conversion of lignin-related compounds to the valuable molecule β-ketoadipate, contributing to the development of biological processes for lignin valorization.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Comparative transcriptomics of CAZy enzymes in white- and brown-rot agaricomycetes: Evolutionary insights into lignocellulose degradation and the relevance of GH16 glycoside hydrolase functional divergence

White-rot and brown-rot fungi (WRF and BRF, respectively) decompose lignocellulose, the main structural component of plant biomass, through distinct mechanisms. This study examines the transcriptomic responses of three WRF species (Pleurotus ostreatus, Phanerochaete chrysosporium, and Heterobasidion irregulare) and two BRF species (Fomitopsis schrenkii and Rhodonia placenta) grown on poplar wood (W) and glucose (G) as sole carbon sources. RNA-seq analysis revealed upregulation of carbohydrate-active enzymes (CAZymes) linked to lignocellulose degradation. WRF displayed a broader enzymatic repertoire, whereas BRF employed a more selective approach. Among these responses, GH16 glycoside hydrolases were consistently upregulated across all species, including BRF. Since GH16 enzymes are involved in both plant hemicellulose modification and cell wall remodeling, their wood-induced expression may reflect multiple processes rather than a single conserved wood-decay mechanism. Structural and phylogenetic analyses revealed species-specific divergence, consistent with potential functional specialization. These findings broaden our understanding of fungal enzymatic strategies and highlight GH16 enzymes as candidates for further study. Beyond the immediate context of wood decay, this work has broader implications for fungal ecology, evolutionary biology, and biotechnological applications such as biomass conversion and sustainable bioenergy.

Fungal ecological strategies↗

DOE OSTI2024

Coupling sensor to enzyme in the voltage sensing phosphatase

Voltage-sensing phosphatases (VSPs) dephosphorylate phosphoinositide (PIP) signaling lipids in response to membrane depolarization. VSPs possess an S4-containing voltage sensor domain (VSD), resembling that of voltage-gated cation channels, and a lipid phosphatase domain (PD). The mechanism by which voltage turns on enzyme activity is unclear. Structural analysis and modeling suggest several sites of VSD-PD interaction that could couple voltage sensing to catalysis. Voltage clamp fluorometry reveals voltage-driven rearrangements in three sites implicated earlier in enzyme activation—the VSD-PD linker, gating loop and R loop—as well as the N-terminal domain, which has not yet been explored. N-terminus mutations perturb both rearrangements in the other segments and enzyme activity. Our results provide a model for a dynamic assembly by which S4 controls the catalytic site.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

A global database of soil microbial phospholipid fatty acids and enzyme activities

Abstract Soil microbes drive ecosystem function and play a critical role in how ecosystems respond to global change. Research surrounding soil microbial communities has rapidly increased in recent decades, and substantial data relating to phospholipid fatty acids (PLFAs) and potential enzyme activity have been collected and analysed. However, studies have mostly been restricted to local and regional scales, and their accuracy and usefulness are limited by the extent of accessible data. Here we aim to improve data availability by collating a global database of soil PLFA and potential enzyme activity measurements from 12,258 georeferenced samples located across all continents, 5.1% of which have not previously been published. The database contains data relating to 113 PLFAs and 26 enzyme activities, and includes metadata such as sampling date, sample depth, and soil pH, total carbon, and total nitrogen. This database will help researchers in conducting both global- and local-scale studies to better understand soil microbial biomass and function.

Science & Technology - Other Topics↗

DOE OSTI2024

Lysosomal enzyme binding to the cation-independent mannose 6-phosphate receptor is regulated allosterically by insulin-like growth factor 2

The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. CI-MPR is multifunctional and modulates embryonic growth and fetal size by downregulating circulating levels of the peptide hormone insulin-like growth factor 2 (IGF2). The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. How a particular ligand affects the receptor’s conformation or its ability to bind other ligands remains poorly understood. To address these questions, we purified a soluble form of the receptor from newborn calf serum, carried out glycoproteomics to define the N-glycans at its 19 potential glycosylation sites, probed its ability to bind lysosomal enzymes in the presence and absence of IGF2 using surface plasmon resonance, and assessed its conformation in the presence and absence of IGF2 by negative-staining electron microscopy and hydroxyl radical protein footprinting studies. Together, our findings support the hypothesis that IGF2 acts as an allosteric inhibitor of lysosomal enzyme binding by inducing global conformational changes of CI-MPR.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Enzymatic cleavage of model lignin dimers depends on pH, enzyme, and bond type

Lignin is composed of phenylpropanoid monomers linked by ether and carbon-carbon bonds to form a complex heterogeneous structure. Bond-specific studies of lignin-modifying enzymes (LMEs; e.g., laccases and peroxidases) are limited by the polymerization of model lignin substrates and repolymerization of cleavage products. Here we present a high throughput platform to screen LME activities on four tagged model lignin compounds that represent the β-O-4’, β-β’, 5–5’, and 4-O-5’ linkages in lignin. We utilized nanostructure-initiator mass spectrometry (NIMS) and model lignin compounds with tags containing perfluorinated and cationic moieties, which effectively limit polymerization and condensation of the substrates and their degrading products. Sub-microliter sample droplets were printed on the NIMS chip with a novel robotics method. This rapid platform enabled characterization of LMEs across a range of pH 3–10 and relative quantification of modified (typically oxidized), cleaved, and polymerized products. All tested enzymes oxidized the four substrates and cleaved the β-O-4’ and β-β’ substrates to monomeric products. We discovered that the active pH range depended on both the substrate and the enzyme type. This has important applications for biomass conversion to biofuels and bioproducts, where the relative percentages of different bond types in lignin varies depending on feedstock and chemical pretreatment methods.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Impact of moisture on microbial decomposition phenotypes and enzyme dynamics

Soil organic matter decomposition is a complex process reflecting microbial composition and environmental conditions. Moisture can modulate the connectivity and interactions of microbes. Due to heterogeneity, a deeper understanding of the influence of soil moisture on the dynamics of organic matter decomposition and resultant phenotypes remains a challenge. Soils from a long-term field experiment exposed to high and low moisture treatments were incubated in the laboratory to investigate organic matter decomposition using chitin as a model substrate. By combining enzymatic assays, biomass measurements, and microbial enrichment via activity-based probes, we determined the microbial functional response to chitin amendments and field moisture treatments at both the community and cell scales. Chitinolytic activities showed significant responses to the amendment of chitin, independent of differences in field moisture treatments. However, for other measurements of carbon metabolism and cellular functions, soils from high moisture field treatments had greater potential enzyme activity than soils from low moisture field treatments. A cell tagging approach was used to enrich and quantify bacterial taxa that are actively producing chitin-degrading enzymes. By integrating organism, community, and soil core measurements we show that (i) a small subset of taxa compose the majority (>50%) of chitinase production despite broad functional redundancy, (ii) the identity of key chitin degraders varies with moisture level, and (iii) extracellular enzymes that are not cell-associated account for most potential chitinase activity measured in field soil.

activity-based probes↗

DOE OSTI2025

Sustainable production of plastic-degrading enzymes in Chlamydomonas pacifica

AbstractThe discovery of a new extremophile alga,Chlamydomonas pacifica, provides an opportunity to expand on heterologous protein expression beyond the traditionalChlamydomonas reinhardtii. C. pacificais a unicellular extremophile capable of surviving at high pH, high temperatures, and high salinity. These various growth conditions allow C. pacifica to outcompete any invading contaminants in open-air environments. Developing this novel species as a platform for recombinant protein production could significantly advance commercial microalgal recombinant protein production. We have previously shown thatC. reinhardtiican secrete a plastic-degrading enzyme: a PETase known as PHL7. This PETase is capable of cleaving ester bonds and has been used commercially for the degradation of PET plastics. However, the expression of such an enzyme has yet to be done in open raceway ponds and on a large scale. Here, we describe the culturing of PHL7 transgenic C. pacifica strain in three 80L raceway ponds and the measurements of recombinant enzymatic expression and activity found in the culture media. Our work provides proof of concept that this new organism can produce functional PHL7 enzymes in addition to producing the valuable components that inherently exist in theC. pacificaalgae biomass.Graphical Abstract

Diaz, Crisandra Jade (ORCID:0000000173429041)↗

NASA NTRS1966

Microorganisms detected by enzyme-catalyzed reaction

Enzymes detect the presence of microorganisms in soils. The enzyme lysozymi is used to release the enzyme catalase from the microorganisms in a soil sample. The catalase catalyzes the decomposition of added hydrogen peroxide to produce oxygen which is detected manometrically. The partial pressure of the oxygen serves as an index of the samples bacteria content.

Vango, S. P.↗

NASA NTRS1972

Enzyme activity in terrestrial soil in relation to exploration of the Martian surface

Urease activity in soil is persistent for long periods under low water, low temperature, and sterile regimes, and it was suggested that some form of enzyme-protective mechanism exists in soil. Dublin soil was extracted by sonication in water followed by adding a mixture of salts. Urease activity is associated with the organo-mineral complex thus obtained and is resistant to the activities of proteolytic enzymes. Clay free soil organic matter prepared subsequently by filtration also exhibits urease activity which is resistant to proteolysis. Models consisting of enzymes with bentonite and lignin were found to mimic this resistance to proteolysis. A model system is presented which suggests both the origin and location of soil ureases and a reason for their persistence in nature.

Ardakani, M. S.↗

NASA NTRS1972

The synthesis of starch from carbon dioxide using isolubilized stabilized enzymes

Systems for artificial manufacture of starch and for delineation of technological areas, and the rationale for studying them are considered. A discussion of the enzyme-catalyzed routes of synthesis available and a choice as to the most promising route are presented. A discussion of the enzymes involved, of enzyme insolubilization technology, and of possible engineering approaches, with examples in the form of model calculations for both reactors and separators, are also presented.

Bassham, J. A.↗

NASA NTRS1972

Solvent effects on enzymes - Implications for extraterrestrial life.

Review of several studies on the alterations taking place in the structure, catalytic activity, specificity, and stability of an enzyme when some or all of the water in the medium is replaced by another solvent. These studies show the utility of solvents as a tool for probing enzyme function. They also suggest that solvents other than water should be investigated as media for controlling and directing enzyme reactions.

Heinrich, M. R.↗

NASA NTRS1970

Subcellular localization of pituitary enzymes

A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

Smith, R. E.↗

NASA NTRS1981

The effects of space flight on some rat liver enzymes regulating carbohydrate and lipid metabolism

The effects of space flight conditions on the activities of certain enzymes regulating carbohydrate and lipid metabolism in rat liver are investigated in an attempt to account for the losses in body weight observed during space flight despite preflight caloric consumption. Liver samples were analyzed for the activities of 32 cytosolic and microsomal enzymes as well as hepatic glycogen and individual fatty acid levels for ground control rats and rats flown on board the Cosmos 936 biosatellite under normal space flight conditions and in centrifuges which were sacrificed upon recovery or 25 days after recovery. Significant decreases in the activities of glycogen phosphorylase, alpha-glycerol phosphate acyl transferase, diglyceride acyl transferase, aconitase and 6-phosphogluconate dehydrogenase and an increase in palmitoyl CoA desaturase are found in the flight stationary relative to the flight contrifuged rats upon recovery, with all enzymes showing alterations returning to normal values 25 days postflight. The flight stationary group is also observed to be characterized by more than twice the amount of liver glycogen of the flight centrifuged group as well as a significant increase in the ratio of palmitic to palmitoleic acid. Results thus indicate metabolic changes which may be involved in the mechanism of weight loss during weightlessness, and demonstrate the equivalence of centrifugation during space flight to terrestrial gravity.

Abraham, S.↗