Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases

Heavy enzyme isotope effects occur in proteins substituted with ²H-, ¹³C-, and ¹⁵N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme–reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization.

Enzymes use slow (millisecond to second) motions to generate reactant-enzyme geometries near the transition state and are proposed to use fast catalytic site motions (femtosecond to picosecond) to find distinct transition-state conformations along the reaction coordinate. Chemical steps occur on the timescale of bond vibrations after the fast dynamic search achieves the transition-state conformation (1, 2). The isotopic labeling of enzymes with ¹³C, ¹⁵N, and nonexchangeable ²H is an experimental strategy for probing the contributions of fast protein motions in the crossing of transition-state barriers. These “heavy” enzymes have mass-altered vibrational modes but remain electrostatically unaltered from their natural counterparts due to principles of the Born-Oppenheimer approximation (1). The decrease in bond vibrational frequencies due to the increased molecular mass results in a slowed search for the transition state and, in most cases, a decreased rate for the chemical step (3, 4). Slower chemical steps have been observed in heavy enzyme studies of purine nucleoside phosphorylase (PNP), HIV-1 protease, alanine racemase, several dihydrofolate reductases (DHFR), pentaerythritol tetranitrate reductase (PETNR), lactate dehydrogenase, and in catalytic site mutations of some of these enzymes (4, 5).Inverse enzyme isotope effects are rare cases where the chemical reaction step of the heavy enzyme is faster than its light counterpart, only reported under conditions of engineered catalytic site mutations or specific catalytic site elements being isotopically labeled. In one case, the chemical step rates of heavy and light forms of a catalytic site mutant PNP (F159Y) yielded an inverse isotope effect (k_light/k_heavy for the chemical step) of 0.75 for the phosphorolysis of guanosine (6). In other examples, PNPs engineered with asparagines labeled differently from the rest of the protein gave inverse isotope effects of 0.71 and 0.78 (7). PETNR was reported to have an enzyme isotope effect of 0.89 for deuteride ion transfer involving NADP²H with labeled enzyme and the cofactor flavin mononucleotide (FMN) in its naturally abundant form. Another inverse enzyme isotope effect of 0.92 was reported for PETNR using NADP²H and heavy FMN (8). However, when PNP and PETNR were assayed in their fully labeled native and heavy forms with substrates containing a naturally abundant isotope composition, the reported enzyme kinetic isotope effects (KIEs) were normal (9). Here, we present two unusual cases of native enzymes with natural inverse isotope effects.Methylthioadenosine nucleosidases (MTANs) are dimeric microbial nucleoside hydrolases that catalyze the hydrolysis of the N-ribosidic bond of 5′-methylthioadenosine (MTA) to 5-methylthioribose and adenine. A broad specificity for substituents at the 5′-position of adenosine permits diverse metabolic roles in bacterial genera, including functions in purine salvage, quorum sensing, S-adenosylmethionine recycling, polyamine synthesis, and the futalosine-based menaquinone biosynthetic pathway. These roles of MTANs in bacterial metabolism have made them potential drug targets. For example, interfering with bacterial quorum sensing, but not growth, avoids the selective pressure of antibiotics, and prevents the development of resistance, while inhibiting the production of virulence factors (10). Blocking menaquinone synthesis in microorganisms using the futalosine-specific biosynthetic pathway selectively targets pathogenic bacteria, including Helicobacter and Campylobacter genera without affecting the broader gut microbiome (11–13).Transition-state structures of MTANs have been solved for various bacterial species by analysis of substrate KIEs. Early transition states, with significant bond order remaining to the adenine leaving group, have been assigned to the MTANs from Neisseria meningitidis and Helicobacter pylori. However, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, and Klebsiella pneumoniae MTANs possess near-fully dissociated transition states (14). Mutational studies have highlighted the important roles that specific catalytic residues play in substrate binding and transition-state formation (15). Insight into the dynamic roles the catalytic site residues play in transition-state formation is a focus of this study.We selected MTANs from early (H. pylori MTAN; HpMTAN) and late (E. coli MTAN; EcMTAN) transition-state classes to determine the effects of heavy isotope-labeled protein. The enzymes showed normal heavy isotope effects on steady-state kinetic rates (k_cat values) and variable effects on substrate interactions (K_m values). But contrary to most enzyme systems, both MTANs revealed natural, intrinsic inverse isotope effects on the chemical step. Here we report the heavy enzyme MTAN isotope effects through kinetic experiments under steady-state and presteady-state conditions. Transition-path sampling is used to obtain mechanistic insight into how the coordination of dynamic motions at the catalytic sites confers this previously rare phenomenon. The discovery of natural inverse heavy enzyme isotope effects adds a new dimension to the usual observation that increased enzyme mass causes a decrease in the rate of the chemical step due to less-efficient barrier crossing. These findings also explain the mechanism of inverse effects and add to our understanding of rapid protein dynamic contributions to enzyme catalytic function.     

Motor chronic inflammatory demyelinating polyneuropathy (CIDP) in 17 patients: Clinical characteristics,electrophysiological study,and response to treatment

Motor chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare and poorly described subtype of CIDP. We aimed to study their clinical and electrophysiological characteristics and response to treatment. From a prospective database of CIDP patients, we included patients with definite or probable CIDP with motor signs and without sensory signs/symptoms at diagnosis. Patients were considered to have pure motor CIDP (PM‐CIDP) if sensory conductions were normal or to have motor predominant CIDP (MPred‐CIDP) if ≥2 sensory nerve action potential amplitudes were abnormal. Among the 700 patients with CIDP, 17 (2%) were included (PM‐CIDP n = 7, MPred‐CIDP n = 10); 71% were male, median age at onset was 48 years (range: 13‐76 years), 47% had an associated inflammatory or infectious disease or neoplasia. At the more severe disease stage, 94% of patients had upper and lower limb weakness, with distal and proximal weakness in 4 limbs for 56% of them. Three‐quarters (75%) responded to intravenous immunoglobulins (IVIg) and four of five patients to corticosteroids including three of three patients with MPred‐CIDP. The most frequent conduction abnormalities were conduction blocks (CB, 82%) and F‐wave abnormalities (88%). During follow up, 4 of 10 MPred‐CIDP patients developed mild sensory symptoms; none with PM‐CIDP did so. Patients with PM‐CIDP had poorer outcome (median ONLS: 4; range: 22‐5) compared to MPred‐CIDP (2, range: 0‐4; P = .03) at last follow up. This study found a progressive clinical course in the majority of patients with motor CIDP as well as frequent associated diseases, CB, and F‐wave abnormalities. Corticosteroids might be considered as a therapeutic option in resistant IVIg patients with MPred‐CIDP.     

Disordered directional brain network interactions during learning dynamics in schizophrenia revealed by multivariate autoregressive models

Directional network interactions underpin normative brain function in key domains including associative learning. Schizophrenia (SCZ) is characterized by altered learning dynamics, yet dysfunctional directional functional connectivity (dFC) evoked during learning is rarely assessed. Here, nonlinear learning dynamics were induced using a paradigm alternating between conditions (Encoding and Retrieval). Evoked fMRI time series data were modeled using multivariate autoregressive (MVAR) models, to discover dysfunctional direction interactions between brain network constituents during learning stages (Early vs. Late), and conditions. A functionally derived subnetwork of coactivated (healthy controls [HC] ∩ SCZ] nodes was identified. MVAR models quantified directional interactions between pairs of nodes, and coefficients were evaluated for intergroup differences (HC ≠ SCZ). In exploratory analyses, we quantified statistical effects of neuroleptic dosage on performance and MVAR measures. During Early Encoding, SCZ showed reduced dFC within a frontal–hippocampal–fusiform network, though during Late Encoding reduced dFC was associated with pathways toward the dorsolateral prefrontal cortex (dlPFC). During Early Retrieval, SCZ showed increased dFC in pathways to and from the dorsal anterior cingulate cortex, though during Late Retrieval, patients showed increased dFC in pathways toward the dlPFC, but decreased dFC in pathways from the dlPFC. These discoveries constitute novel extensions of our understanding of task‐evoked dysconnection in schizophrenia and motivate understanding of the directional aspect of the dysconnection in schizophrenia. Disordered directionality should be investigated using computational psychiatric approaches that complement the MVAR method used in our work.     

Clinical and financial impacts of flexible intramedullary nailing in pediatric diaphyseal forearm fractures: A case–control study

Purpose:Flexible intramedullary nailing is regularly applied for pediatric displaced unstable forearm fractures. When compared to closed reduction and casting (orthopedic treatment), flexible intramedullary nailing decreases malalignment, shortens immobilization time, and should decrease follow-up controls. Comparing flexible intramedullary nailing and orthopedic treatment in the clinical, radiological, and financial managements of these fractures was performed.Methods:Retrospective 5 years study of pediatric cases in two pediatric orthopedic university departments. Treatment method, post-operative course, and radiological follow-up were reviewed. Number of radiographs, follow-up controls, type and duration of immobilization, final bone angulation, and reported complications were compared. Extensive financial analysis was completed.Results:Of 73 girls and 168 boys included in the study, 150 were treated by flexible intramedullary nailing and 91 by orthopedic treatment. No difference was noted with regard to total number of radiographs (7.3 vs 7.2, respectively). Total number of follow-ups was 6.4 and 5.5, respectively. Malalignment occurred in two flexible intramedullary nailing and sixteen orthopedic treatments. The least expensive cost was ambulatory orthopedic treatment.Conclusion:Flexible intramedullary nailing treated children had similar numbers of radiographs or follow-up consultation, but less malunion when compared to orthopedic treatment. Orthopedic management was systematically cheaper than flexible intramedullary nailing. Unless post-operative management guidelines decreasing the number of radiographs and follow-ups are implemented, flexible intramedullary nailing will remain a costly procedure when compared to conventional orthopedic treatment.Level of evidence:level III case–control retrospective study.     

Implementing a Community-Based Initiative to Improve Nutritional Intake among Home-Delivered Meal Recipients

Home-delivered meal (HDM) recipients are a highly vulnerable group of older adults at risk for malnutrition and subsequent health decline. To help HDM recipients increase their nutritional intake, HDM agencies may provide expanded meal options that allow older adults to have greater autonomy over their meal selection; however, the extent to which recipients are able to select nutritious meals that are responsive to their health complexities is unknown. This study examined the nutritional content of meals selected by HDM recipients enrolled in an expanded menu plan through a large HDM agency. Data were drawn from a retrospective chart review of 130 HDM recipients who had the option of selecting their own HDM meals and frequency of meal delivery. Findings indicate that older adults who selected their own meals chose meals that were significantly lower in protein, potassium, fat, and calories. The lack of these nutrients suggests that older adults enrolled in expanded menu plans should be referred to registered dietitian nutritionists who can provide skilled guidance in meal selection. To address this need, we also describe and provide preliminary data representing a referral program designed to connect HDM recipients to dietetic services with the goal of optimizing older adult nutrition and health-related outcomes.     

The Role of Gut-Derived,Protein-Bound Uremic Toxins in the Cardiovascular Complications of Acute Kidney Injury

Acute kidney injury (AKI) is a frequent disease encountered in the hospital, with a higher incidence in intensive care units. Despite progress in renal replacement therapy, AKI is still associated with early and late complications, especially cardiovascular events and mortality. The role of gut-derived protein-bound uremic toxins (PBUTs) in vascular and cardiac dysfunction has been extensively studied during chronic kidney disease (CKD), in particular, that of indoxyl sulfate (IS), para-cresyl sulfate (PCS), and indole-3-acetic acid (IAA), resulting in both experimental and clinical evidence. PBUTs, which accumulate when the excretory function of the kidneys is impaired, have a deleterious effect on and cause damage to cardiovascular tissues. However, the link between PBUTs and the cardiovascular complications of AKI and the pathophysiological mechanisms potentially involved are unclear. This review aims to summarize available data concerning the participation of PBUTs in the early and late cardiovascular complications of AKI.     

Sulfur and methane oxidation by a single microorganism

Natural and anthropogenic wetlands are major sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic bacteria at the oxic–anoxic interface, a zone of intense redox cycling of carbon, sulfur, and nitrogen compounds. Here, we report on the isolation of an aerobic methanotrophic bacterium, ‘Methylovirgula thiovorans'' strain HY1, which possesses metabolic capabilities never before found in any methanotroph. Most notably, strain HY1 is the first bacterium shown to aerobically oxidize both methane and reduced sulfur compounds for growth. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are responsible for methane and methanol oxidation, respectively. Various pathways for respiratory sulfur oxidation were present, including the Sox–rDsr pathway and the S₄I system. Strain HY1 employed the Calvin–Benson–Bassham cycle for CO₂ fixation during chemolithoautotrophic growth on reduced sulfur compounds. Proteomic and microrespirometry analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of the respective substrates. Methane and thiosulfate could therefore be independently or simultaneously oxidized. The discovery of this versatile bacterium demonstrates that methanotrophy and thiotrophy are compatible in a single microorganism and underpins the intimate interactions of methane and sulfur cycles in oxic–anoxic interface environments.

Atmospheric methane (CH₄) is a potent greenhouse gas responsible for about 15% of the total greenhouse effect (1). The amount of CH₄ in Earth’s atmosphere is gradually increasing (2). The world’s largest single CH₄ source is natural wetlands, one-third of which are temperate and boreal northern wetlands (3, 4). Methane produced by the degradation of organic matter in anoxic sediments reaches the atmosphere via diffusion, transport through aerenchymous roots, or ebullition. Much of the diffusive flux of CH₄ is oxidized by aerobic methanotrophic bacteria at oxic–anoxic interfaces in wetlands, thereby limiting CH₄ emission (5–8). Methane formation is also prevented by the activity of microorganisms that redirect the flow of electrons and carbon away from methanogenic archaea, such as certain sulfur-cycling microorganisms. Microorganisms that respire sulfate (SO₄²⁻) or other oxidized sulfur compounds can contribute considerably to the anaerobic degradation of organic carbon in wetlands and outcompete methanogenic archaea (9, 10).Aerobic methanotrophic bacteria were long assumed to have a limited substrate spectrum, including methane, methanol, and occasionally other C1 compounds, but no other substrates (11). This assumption was overturned when it was discovered that methanotrophs of the genus Methylocella (family Beijerinckiaceae) use some simple organic acids, alcohols, and short-chain alkanes as alternative substrates to methane (11, 12). A few other alphaproteobacterial methanotrophs belonging to the Methylocystaceae or Beijerinckiaceae families, while not as versatile as Methylocella, have also been shown to metabolize acetate and/or ethanol (13–15). Two cultured Beijerinckiaceae methanotrophs even possess the genetic capacity for aerobic CO oxidation (16, 17), and the growth of one of them, ‘Methylocapsa gorgona'' MG08, was supported by CO in the presence of methane (18). In addition, thermophilic and mesophilic verrucomicrobial methanotrophs of the proposed genera ‘Methylacidiphilum'' and ‘Methylacidimicrobium'' grow autotrophically on CO₂ with H₂ as an electron donor (19–23). In fact, genes encoding NiFe hydrogenase are widespread in all major taxonomic families of methanotrophs, suggesting that H₂ may be a common supplemental energy source for these bacteria in nature. Recently, members of the genus Methylacidiphilum were also found to grow heterotrophically on various C3 compounds (24). Clearly, some methanotrophs can take advantage of other small-molecule substrates besides methane and methanol, which may enhance their survival and/or growth in natural habitats where CH₄ concentrations are low and/or variable (13).The oxidation of H₂ to two protons and the oxidation of CO to CO₂ both yield considerably lower standard free-energy changes, ΔG°′ = −237 kJ⋅mol⁻¹ H₂ and ΔG°′ = −249 kJ⋅mol⁻¹ CO, respectively, than the complete oxidation of CH₄ to CO₂ (ΔG°′ = −818 kJ⋅mol⁻¹ CH₄). In comparison, the standard free-energy changes for the oxidation of H₂S to SO₄²⁻ (ΔG°′ = −797 kJ⋅mol⁻¹ H₂S) and S₂O₃²⁻ to SO₄²⁻ (ΔG°′ = −818 kJ⋅mol⁻¹ S₂O₃²⁻) are similar to that for CH₄ oxidation. Based on these considerations, reduced sulfur compounds would be well-suited alternative substrates for methanotrophs.To date, there has been a clear distinction between thiotrophic and methanotrophic microorganisms. The growth of methanotrophs using reduced sulfur compounds as electron donors has never been reported. Recently, a member of the genus Methylacidiphilum was found to degrade methanethiol and sulfide for detoxification, but no growth benefit was observed from their oxidation (25). The common occurrence of sqr (encoding sulfide:quinone oxidoreductase) (Fig. 1) and mtoX (encoding methanethiol oxidase) (25) in methanotroph genomes suggests that detoxification mechanisms are common. However, genomes of some methanotrophs also harbor a complete Sox system (Dataset S1 and Fig. 1), and a recent study unveiled the co-occurrence of genes encoding sulfur (Sox and reverse dissimilatory sulfite reductase [rDsr]) and CH₄ (methane monooxygenase) oxidation systems in a metagenome-assembled genome recovered from a permafrost thaw wetland (26). These findings provide hints for a possible combination of thiotrophy and methanotrophy in particular bacteria. Here, we experimentally confirmed this hypothesis and isolated a facultative methanotroph (strain HY1) that harbors a complete repertoire of sulfur oxidation genes encoding the Sox–rDsr system (without soxCD).Open in a separate windowFig. 1.Phylogenomic tree and distribution of distinctive metabolic traits in methane- and sulfur-oxidizing bacteria in the classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Chlorobia. The tree includes 37 genomes and 2 metagenome-assembled genomes. Representative genomes of Sox-containing Alphaproteobacterial and Gammaproteobacterial methanotrophs were included. The tree was constructed based on 27 concatenated ribosomal proteins with FastTree implemented within Anvi’o phylogenomics workflow (details are in Materials and Methods). Black circles indicate 70% bootstrap support for nodes along the tree. A homology-based search for functional genes was performed by using BLAST (124), OrthoFinder (125), and manual examination (details are in Materials and Methods). Solid and open squares indicate the presence and absence of the genes, respectively.