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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Potent Inhibition of E. coli DXP Synthase by a gem -Diaryl Bisubstrate Analog

New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy- D -xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate ( D -GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens. Thus, DXPS is a promising antimicrobial target. Toward this goal, our lab has demonstrated selective inhibition of Escherichia coli DXPS by alkyl acetylphosphonate (alkylAP)-based bisubstrate analogs that exploit the requirement for ternary complex formation in the DXPS mechanism. Here, we present the first DXPS structure with a bisubstrate analog bound in the active site. Insights gained from this cocrystal structure guided structure–activity relationship studies of the bisubstrate scaffold. A low nanomolar inhibitor (compound 8) bearing a gem-dibenzyl glycine moiety conjugated to the acetylphosphonate pyruvate mimic via a triazole-based linker emerged from this study. Compound 8 was found to exhibit slow, tight-binding inhibition, with contacts to E. coli DXPS residues R99 and R478 demonstrated to be important for this behavior. This work has discovered the most potent DXPS inhibitor to date and highlights a new role of R99 that can be exploited in future inhibitor designs toward the development of a novel class of antimicrobial agents.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2006

The Influence of Microbiology on Spacecraft Design and Controls: A Historical Perspective of the Shuttle and International Space Station Programs

For over 40 years, NASA has been putting humans safely into space in part by minimizing microbial risks to crew members. Success of the program to minimize such risks has resulted from a combination of engineering and design controls as well as active monitoring of the crew, food, water, hardware, and spacecraft interior. The evolution of engineering and design controls is exemplified by the implementation of HEPA filters for air treatment, antimicrobial surface materials, and the disinfection regimen currently used on board the International Space Station. Data from spaceflight missions confirm the effectiveness of current measures; however, fluctuations in microbial concentrations and trends in contamination events suggest the need for continued diligence in monitoring and evaluation as well as further improvements in engineering systems. The knowledge of microbial controls and monitoring from assessments of past missions will be critical in driving the design of future spacecraft.

Castro, Victoria A.↗
DOE OSTI2022

Editorial: Phenylpropanoid Systems Biology and Biotechnology

Phenylpropanoids are specialized metabolites involved in several aspects of plant growth and development and in the responses of plants to environmental stimuli. These compounds are synthesized from key intermediates of the shikimate pathway, which are structurally modified by the combined activities of lyases, transferases, ligases, reductases and oxygenases, resulting in the organ- and developmental-specific synthesis and accumulation of diverse metabolites (Vogt, 2010). The phenylpropanoid pathway provides the building blocks for lignin, suberin, and condensed tannins that play a role in structural support and mechanical strength. Lignin is a major contributor to feedstock recalcitrance and negatively affects the conversion of plant biomass into downstream products in biorefineries (Liu et al., 2021). Further, this pathway is key for the production of anthocyanins for organ pigmentation, flavonols and flavones for UV protection, various flavonoids and isoflavonoids for plant-microbe interactions, and antimicrobial phytoalexins for protection against pathogens (Deng and Lu, 2017). In addition to their biological functions in planta, phenylpropanoids are economically important metabolites. They constitute important components in the human diet, acting as nutraceutical compounds with antioxidant, chemopreventive, antimitotic, neuroprotective, cardioprotective, and anti-inflammatory activities. Several phenylpropanoids are considered high-value biochemicals employed in the production of fragrances, pharmaceuticals and biopolymers (Lin and Eudes, 2020).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cellulose nanofibrils-based packaging films synergistically reinforced by lignin and tea polyphenols for strawberry preservation

Developing biomass-based food packaging materials could alleviate resource scarcity and environmental pollution. Here, this study successfully fabricated cellulose nanofibrils-based packaging films synergistically reinforced by lignin and tea polyphenols for fruit preservation. Thanks to the abundant carboxyl and hydroxyl groups in modified lignin and tea polyphenol, the composite film delivered the highest tensile strength and Young’s modulus of 230.7 MPa and 11.9 GPa, respectively. The water vapor permeability (WVP) and oxygen permeability (OP) of composite film were as low as 2.53 × 10 –11 gm –1 s –1 Pa –1 and 1.69 × 10 –16 cm m –2 s–1 Pa –1 , respectively, demonstrating excellent barrier performance. Furthermore, the composite film possessed remarkable antibacterial (100 % antibacterial activity against two types of bacteria), antioxidant (DPPH free radical scavenging activity of 90 %), and UV shielding properties (impressive UV shielding rate of 99.0 %). Strawberry preservation experiments showed that these composite films could significantly extend shelf life, owing to the excellent barrier and antimicrobial properties. Our research indicated that these composite films could be promising food packaging material compared with traditional plastic and showed great application potential in fruit preservation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1975

Storage Stability and Improvement of Intermediate Moisture Foods, Phase 3

Methods were determined for the improvement of shelf-life stability of intermediate moisture foods (IMF). Microbial challenge studies showed that protection against molds and Staphylococcus aureus could be achieved by a combination of antimicrobial agents, humectants and food acids. Potassium sorbate and propylene glycol gave the best results. It was also confirmed that the maximum in heat resistance shown by vegetative pathogens at intermediate water activities also occurred in a solid food. Glycols and sorbitol both achieve browning inhibition because of their action as a medium for reaction and effect on viscosity of the adsorbed phase. Chemical availability results showed rapid lysine loss before visual discoloration occurred. This is being confirmed with a biological test using Tetrahymena pyriformis W. Accelerated temperature tests show that effectiveness of food antioxidants against rancidity development can be predicted; however, the protection factor changes with temperature. BHA was found to be the best antioxidant for iron catalyzed oxidation.

Labuza, T. P.↗
NASA NTRS2013

Advanced Exploration Systems (AES) Logistics Reduction and Repurposing Project: Advanced Clothing Ground Study Final Report

All human space missions require significant logistical mass and volume that will become an excessive burden for long duration missions beyond low Earth orbit. The goal of the Advanced Exploration Systems (AES) Logistics Reduction & Repurposing (LRR) project is to bring new ideas and technologies that will enable human presence in farther regions of space. The LRR project has five tasks: 1) Advanced Clothing System (ACS) to reduce clothing mass and volume, 2) Logistics to Living (L2L) to repurpose existing cargo, 3) Heat Melt Compactor (HMC) to reprocess materials in space, 4) Trash to Gas (TTG) to extract useful gases from trash, and 5) Systems Engineering and Integration (SE&I) to integrate these logistical components. The current International Space Station (ISS) crew wardrobe has already evolved not only to reduce some of the logistical burden but also to address crew preference. The ACS task is to find ways to further reduce this logistical burden while examining human response to different types of clothes. The ACS task has been broken into a series of studies on length of wear of various garments: 1) three small studies conducted through other NASA projects (MMSEV, DSH, HI-SEAS) focusing on length of wear of garments treated with an antimicrobial finish; 2) a ground study, which is the subject of this report, addressing both length of wear and subject perception of various types of garments worn during aerobic exercise; and 3) an ISS study replicating the ground study, and including every day clothing to collect information on perception in reduced gravity in which humans experience physiological changes. The goal of the ground study is first to measure how long people can wear the same exercise garment, depending on the type of fabric and the presence of antimicrobial treatment, and second to learn why. Human factors considerations included in the study consist of the Institutional Review Board approval, test protocol and participants' training, and a web-based data collection questionnaire. Cardiovascular exercise was chosen as the activity in this experiment for its profuse sweating effect and because it is considered a more severe treatment applied to the clothes than every-day usage. Study garments were exercise T-shirts and shorts purchased from various vendors. Fabric construction, fabric composition, and finishing treatment were defined as the key variables. The study was divided into three balanced experiments: a cotton-polyester-wool (CPW) T-shirts study with 61 participants, a polyester-modacrylic-polyester/cocona (PMC) T-shirts study with 40 participants, and a shorts study with 70 participants. In the CPW study, the T-shirts were made of 100% cotton, or of 100% polyester or of 100% wool, and categorized into open and tight knit constructions. In the PMC study, the T-shirts were made of 100% polyester, or of 82% modacrylic, or of 95% polyester with 5% cocona fiber, without construction distinction. The shorts were made either of 100% cotton or of 100% polyester, and were knitted or woven. Some garments were treated with Bio-Protect 500 antimicrobial finish according to the experimental design. The data collected from the questionnaire included garment identification, level of exertion, duration of exercise session, number of exercise sessions, an ordinal preference scale for nine sensory elements, and reason for retiring a used garment. From the analysis of the combined CPW and PMC shirt studies, there are statistically significant differences among the mean lifetimes of various types of shirts. The exercise shirts with the longest mean lifetimes are untreated wool (600 minutes), treated cotton (526 minutes), and untreated modacrylic (515 minutes). From the combined CPW and PMC shirt studies, the most preferred material was untreated open-knit wool, which is one of the two materials that jointly were worn the longest, untreated wool, both open-knit and tight-knit. For the CP shorts study, there were no statistically significant differences in mean lifetimes of the exercise shorts at the 5% significance level due to the treatment combinations. There was therefore no justification to examine differences among levels of main effects or interactions. The preference for shorts was in this order: untreated woven polyester, untreated knitted polyester, untreated woven cotton, and treated knitted cotton. The nine preference scales were tabulated to determine the preference responses at the end of those exercise periods which were prior to the period when a garment was retired and a new garment was started. The assumption is that an unfavorable assessment of a garment leads to its retirement. The scent scale response was predominantly unfavorable at the end of the exercise period immediately prior to the exercise period when a new garment was started.

Byrne, Vicky↗
DOE OSTI2023

Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites

Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T . atroviride exudates. In contrast, P . simiae and P . putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δ tmk 3) gene of T . atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T . atroviride , bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δ tmk3 mutant. Unexpectedly, exudates from wild-type T . atroviride and the Δ tmk 3 mutant rescued purine auxotrophic mutants of H . seropedicae , K . michiganensis and P . simiae . Metabolomic analysis on exudates from wild-type T . atroviride and the Δ tmk 3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma -metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Rational design of antimicrobial peptides targeting Gram-negative bacteria

Membrane-targeting host antimicrobial peptides (AMPs) can kill or inhibit the growth of Gram-negative bacteria. However, the evolution of resistance among microbes poses a substantial barrier to the long-term utility of the host AMPs. Combining experiment and molecular dynamics simulations, we demonstrate that terminal carboxyl capping enhances both membrane insertion and antibacterial activity of an AMP called P1. Furthermore, we show that a bacterial strain with evolved resistance to this peptide becomes susceptible to P1 variants with either backbone capping or lysine-to-arginine substitutions. Our results suggest that cocktails of closely related AMPs may be useful in overcoming evolved resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Leisingera sp. ANG59 & Pseudoalteromonas sp. JC28 Genomes Overview

The Hawaiian bobtail squid, Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A′ (compounds 1a/1b), as well as two new derivatives, BAC-D/D′ (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey’s method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola. Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host’s environment.​

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Fabrication and characterization of dextran/zein hybrid electrospun fibers with tailored properties for controlled release of curcumin

Abstract BACKGROUND In recent years, there has been considerable interest in the use of biopolymer electrospun nanofibers for various food applications due to the biocompatibility, biodegradability, and high loading capacity. Herein, we fabricated and characterized novel hybrid electrospun fibers from dextran (50%, w/v ) and zein (0–30%, w/v ) solutions, and the effects of various zein concentrations on the properties of the hybrid electrospun fibers were investigated. RESULTS When zein was added at low concentrations (5% and 10%), dextran and zein showed poor miscibility, as reflected by significantly decreased viscosity of the solutions, and the poor mechanical properties of the derived fiber membranes. When zein was added at medium concentrations (15–25%), hydrogen bonds were formed between dextran and zein molecules, as indicated by the red shift of Fourier‐transform infrared bands and β‐sheet to α‐helix structural transformations. The fiber membranes electrospun from a solution with 25% zein showed the most hydrophobic surface, with a water contact angle of 116.9°. The homogenous dispersion of dextran and zein resulted in improved mechanical properties for fibers electrospun from a solution with 30% zein. Curcumin encapsulating dextran/zein electrospun fibers exhibited effective radical scavenging activity and ferric reducing power, along with the desired controlled release behavior for curcumin delivery. CONCLUSION Food grade dextran/zein hybrid electrospun fibers demonstrated tunable properties, and appear to be promising as delivery systems for bioactive and edible antimicrobial food packaging. © 2021 Society of Chemical Industry.

Luo, Shiyuan↗
DOE OSTI2024

Exploiting bacterial effector proteins to uncover evolutionarily conserved antiviral host machinery

Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Characterization of Interactions between CTX-M-15 and Clavulanic Acid, Desfuroylceftiofur, Ceftiofur, Ampicillin, and Nitrocefin

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamases (ESBLs) are commonly associated with Gram-negative, hospital-acquired infections worldwide. Several beta-lactamase inhibitors, such as clavulanate, are used to inhibit the activity of these enzymes. To understand the mechanism of CTX-M-15 activity, we have determined the crystal structures of CTX-M-15 in complex with two specific classes of beta-lactam compounds, desfuroylceftiofur (DFC) and ampicillin, and an inhibitor, clavulanic acid. The crystal structures revealed that Ser70 and five other residues (Lys73, Tyr105, Glu166, Ser130, and Ser237) participate in catalysis and binding of those compounds. Based on analysis of steady-state kinetics, thermodynamic data, and molecular docking to both wild-type and S70A mutant structures, we determined that CTX-M-15 has a similar affinity for all beta-lactam compounds (ceftiofur, nitrocefin, DFC, and ampicillin), but with lower affinity for clavulanic acid. A catalytic mechanism for tested β-lactams and two-step inhibition mechanism of clavulanic acid were proposed. CTX-M-15 showed a higher activity toward DFC and nitrocefin, but significantly lower activity toward ampicillin and ceftiofur. The interaction between CTX-M-15 and both ampicillin and ceftiofur displayed a higher entropic but lower enthalpic effect, compared with DFC and nitrocefin. DFC, a metabolite of ceftiofur, displayed lower entropy and higher enthalpy than ceftiofur. This finding suggests that compounds containing amine moiety (e.g., ampicillin) and the furfural moiety (e.g., ceftiofur) could hinder the hydrolytic activity of CTX-M-15.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Repurposed dihydroorotate dehydrogenase inhibitors with efficacy against drug-resistant Acinetobacter baumannii

New antimicrobials are needed for the treatment of extensively drug-resistantAcinetobacter baumannii. The de novo pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated drug target for malaria and human autoimmune diseases. Here, we provide genetic evidence thatA. baumanniiDHODH (AbDHODH) is essential for bacterial survival in rodent infection models. We chemically validate the target by repurposing a unique library of ~450 triazolopyrimidine/imidazopyrimidine analogs developed for our malaria DHODH program to identify 21 compounds with submicromolar activity onAbDHODH. The most potent (DSM186, DHODH IC 50 28 nM) had a minimal inhibitory concentration of ≤1 µg/ml against geographically diverseA. baumanniistrains, including meropenem-resistant isolates. A structurally related analog (DSM161) with a long in vivo half-life conferred significant protection in the neutropenic mouse thigh infection model. Encouragingly, the development of resistance to these compounds was not identified in vitro or in vivo. Lastly, the X-ray structure ofAbDHODH bound to DSM186 was solved to 1.4 Å resolution. These data support the potential ofAbDHODH as a drug target for the development of antimicrobials for the treatment ofA. baumanniiand potentially other high-risk bacterial infections.

Acinetobacter baumannii↗
DOE OSTI2024

Activity of Cannabidiol on Ex Vivo Amino Acid Fermentation by Bovine Rumen Microbiota

Amino-acid-fermenting bacteria are wasteful organisms within the rumens of beef cattle that remove dietary amino nitrogen by producing ammonia, which is then excreted renally. There are currently no on-label uses for the control of this microbial guild, but off-label use of broad-spectrum antimicrobials has shown efficacy, which contributes to antimicrobial resistance. Plant-derived antimicrobials supplemented into the diets of cattle may offer worthwhile alternatives. This study sought to investigate the role of cannabidiol (CBD) as a terpenophenolic antimicrobial. Ex vivo cell suspensions were harvested from the rumen fluid of Angus × Holstein steers in non-selective media with amino acid substrates. The suspensions were treated with five concentrations of CBD (860 μg mL−1–0.086 μg mL−1) and incubated (24 h), after which ammonia production and viable number of cells per substrate and treatment were measured. The data demonstrated a ~10–15 mM reduction in ammonia produced at the highest concentration of CBD and negligible changes in the viable number of amino-acid-fermenting bacteria. CBD does not appear to be a biologically or economically viable terpenophenolic candidate for the control of amino acid fermentation in beef cattle.

Biotechnology & Applied Microbiology↗
DOE OSTI2020

Antibacterial Minerals: A geochemical approach to combating antibiotic resistance

The rapid rise of antibiotic resistant bacteria is seriously threatening our ability to treat lifethreatening infections. As a result, our approach to medicine and agriculture will require significant changes if we are to successfully maintain current levels of healthcare and food security. Certain naturally occurring clays have been shown to harbor antimicrobial properties and kill antibiotic resistant bacteria. These clays have been proposed as a new paradigm for fighting the potentially devastating effects of the post antibiotic era. However, natural samples, by their inherent properties, exhibit variable antibacterial effectiveness and the synthesis of minerals with reproducible antibacterial activity is needed. Our project focused on 3 specific aims; 1) develop synthetic versions of naturally occurring bactericidal minerals to control the chemical purity and antibacterial potency, 2) determine how effective these mineral formulations are at killing antibiotic resistant bacteria and 3) investigate the response of human skin cell cultures to these antibacterial minerals. Utilizing hydrothermal based methods our team was able to reproduce the antibacterial activity observed in natural antibacterial minerals, while eliminating unwanted elemental impurities. Importantly, we were able to create a mineral based bactericide that has reproducible reactivity that could provide a consistent dose response. These antibacterial mineral formulations are effective at killing common hospital acquired antibiotic resistant bacteria while providing minimal toxicity to human fibroblasts. Our research has optimized the antibacterial properties of antibacterial minerals to allow greater efficacy from a public health perspective.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography

Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B 6 -derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗