Bioinformatic Analysis of Sulfotransferases from an Unexplored Gut Microbe, Sutterella wadsworthensis 3_1_45B: Possible Roles towards Detoxification via Sulfonation by Members of the Human Gut Microbiome.

Sulfonation, primarily facilitated by sulfotransferases, plays a crucial role in the detoxification pathways of endogenous substances and xenobiotics, promoting metabolism and elimination. Traditionally, this bioconversion has been attributed to a family of human cytosolic sulfotransferases (hSULTs) known for their high sequence similarity and dependence on 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfo donor. However, recent studies have revealed the presence of PAPS-dependent sulfotransferases within gut commensals, indicating that the gut microbiome may harbor a diverse array of sulfotransferase enzymes and contribute to detoxification processes via sulfation. In this study, we investigated the prevalence of sulfotransferases in members of the human gut microbiome. Interestingly, we stumbled upon PAPS-independent sulfotransferases, known as aryl-sulfate sulfotransferases (ASSTs). Our bioinformatics analyses revealed that members of the gut microbial genus Sutterella harbor multiple asst genes, possibly encoding multiple ASST enzymes within its members. Fluctuations in the microbes of the genus Sutterella have been associated with various health conditions. For this reason, we characterized 17 different ASSTs from Sutterella wadsworthensis 3_1_45B. Our findings reveal that SwASSTs share similarities with E. coli ASST but also exhibit significant structural variations and sequence diversity. These differences might drive potential functional diversification and likely reflect an evolutionary divergence from their PAPS-dependent counterparts.

Use of bivalirudin for anticoagulation in pediatric extracorporeal membrane oxygenation (ECMO).

This study describes the use of bivalirudin in children on extracorporeal membrane oxygenation (ECMO). Pediatric patients receiving bivalirudin were compared to patients receiving heparin as the anticoagulant on ECMO. Data was collected for children under 18 years of age supported by ECMO from January 2016 to December 2019. Data collected included demographics, diagnosis, ECMO indication, type, and duration, indication for bivalirudin use, dose range, activated partial thromboplastin time (aPTT) levels, minor and major bleeding, hemolysis, and mortality. Forty pediatric patients received ECMO; eight received bivalirudin primarily for anticoagulation. The median age was 4 months (IQR 0.5, 92) in the heparin cohort, 0.6 months (IQR 0.0, 80.0) in the primary bivalirudin cohort. The indication for ECMO was respiratory in 5 patients (18%) in the heparin group versus 6 (75%) in the primary bivalirudin group, cardiac in 18 (67%) in heparin versus 1 (12.5%) in primary bivalirudin, and extracorporeal-cardiopulmonary resuscitation (E-CPR) in 4 (15%) in heparin versus 1 (12.5%) in primary bivalirudin. Bivalirudin was the initial anticoagulant for eight patients (66.6%) while three (25%) were switched due to concern for heparin-induced thrombocytopenia (HIT) and one (8%) for heparin resistance. The median time to achieve therapeutic aPTT was 14.5 hours compared to 12 hours in the heparin group. Sixty-five percent of aPTT values in the bivalirudin and 44% of values in the heparin group were in the therapeutic range in the first 7 days. Patients with primary bivalirudin use had significantly lower dose requirement at 12 (p = 0.003), 36 (p = 0.007), and 48 (p = 0.0002) hours compared to patients with secondary use of bivalirudin. One patient (12.5%) had major bleeding, and two patients (25%) required circuit change in the primary bivalirudin cohort. Bivalirudin may provide stable and successful anticoagulation in children. Further large, multicenter studies are needed to confirm these findings.

Comparison of a predefined dose increment nomogram with a percentage adjustment nomogram in patients receiving argatroban therapy.

OBJECTIVE: To determine the compliance with a predefined dose increment (PDI) nomogram compared with a percentage adjustment (PA) nomogram for the dose titration of argatroban therapy. DESIGN: This was a single-center, retrospective chart review. SETTING AND PARTICIPANTS: This study was conducted in a tertiary care teaching hospital. Patients were included if they received argatroban from 2013 to 2016. OUTCOME MEASURES: The primary safety outcome was the percentage of appropriate dose titrations. The secondary safety and efficacy outcomes included the median time to therapeutic activated partial thromboplastin time (aPTT), the median argatroban dose once therapeutic, and the median time in the therapeutic, subtherapeutic, and supratherapeutic aPTT ranges, as well as the bleeding and thrombotic events during hospitalization. RESULTS: Seventy-seven patients were included in the study. There was no statistically significant difference in the percentage of titrations performed appropriately (P = 0.17). The median time to goal aPTT, the dose when first therapeutic, and the time aPTT was subtherapeutic were similar in both the arms. The patients in the PDI arm were on argatroban for a median time of 55 hours compared with 110.5 hours for the patients in the PA arm (P = 0.001). The patients in the PA arm spent more time in the therapeutic range (P < 0.05) and less time in the supratherapeutic range (P < 0.005). CONCLUSION: Although there was no difference in the percentage of appropriate dose titrations, the patients in the PA arm spent more time on argatroban, had greater time in the therapeutic aPTT range, and had less time in the supratherapeutic aPTT range. Future studies that include a larger sample size, matching therapeutic aPTT ranges, and similar initial infusion rates would help evaluate further the outcomes between the PDI and PA nomograms.

Stereospecific Formation of E- and Z-Disubstituted Double Bonds by Dehydratase Domains from Modules 1 and 2 of the Fostriecin Polyketide Synthase.

The dehydratase domain FosDH1 from module 1 of the fostriecin polyketide synthase (PKS) catalyzed the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP1 (5) and (E)-2-butenoyl-FosACP1 (11), as established by a combination of direct LC-MS/MS and chiral GC-MS. FosDH1 did not act on either (3S)-3-hydroxybutyryl-FosACP1 (6) or (Z)-2-butenoyl-FosACP1 (12). FosKR2, the ketoreductase from module 2 of the fostriecin PKS that normally provides the natural substrate for FosDH2, was shown to catalyze the NADPH-dependent stereospecific reduction of 3-ketobutyryl-FosACP2 (23) to (3S)-3-hydroxybutyryl-FosACP2 (8). Consistent with this finding, FosDH2 catalyzed the interconversion of the corresponding triketide substrates (3R,4E)-3-hydroxy-4-hexenoyl-FosACP2 (18) and (2Z,4E)-2,4-hexadienoyl-FosACP2 (21). FosDH2 also catalyzed the stereospecific hydration of (Z)-2-butenoyl-FosACP2 (14) to (3S)-3-hydroxybutyryl-FosACP2 (8). Although incubation of FosDH2 with (3S)-3-hydroxybutyryl-FosACP2 (8) did not result in detectable accumulation of (Z)-2-butenoyl-FosACP2 (14), FosDH2 catalyzed the slow exchange of the 3-hydroxy group of 8 with [18O]-water. FosDH2 unexpectedly could also support the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP2 (7) and (E)-2-butenoyl-FosACP2 (13).

Ecological, beneficial, and pathogenic functions of the Type 9 Secretion System.

The recently discovered Type 9 Secretion System (T9SS) is present in bacteria of the Fibrobacteres-Bacteroidetes-Chlorobi superphylum, which are key constituents of diverse microbiomes. T9SS is instrumental in the extracellular secretion of over 270,000 proteins, including peptidases, sugar hydrolases, metal ion-binding proteins, and metalloenzymes. These proteins are essential for the interaction of bacteria with their environment. This mini-review explores the extensive array of proteins secreted by the T9SS. It highlights the diverse functions of these proteins, emphasizing their roles in pathogenesis, bacterial interactions, host colonization, and the overall health of the ecosystems inhabited by T9SS-containing bacteria.

Spatial organization of a soil cyanobacterium and its cyanosphere through GABA/Glu signaling to optimize mutualistic nitrogen fixation.

Soil biocrusts are characterized by the spatial self-organization of resident microbial populations at small scales. The cyanobacterium Microcoleus vaginatus, a prominent primary producer and pioneer biocrust former, relies on a mutualistic carbon (C) for nitrogen (N) exchange with its heterotrophic cyanosphere microbiome, a mutualism that may be optimized through the ability of the cyanobacterium to aggregate into bundles of trichomes. Testing both environmental populations and representative isolates, we show that the proximity of mutualistic diazotroph populations results in M. vaginatus bundle formation orchestrated through chemophobic and chemokinetic responses to gamma-aminobutyric acid (GABA) /glutamate (Glu) signals. The signaling system is characterized by: a high GABA sensitivity (nM range) and low Glu sensitivity (µM to mM), the fact that GABA and Glu are produced by the cyanobacterium as an autoinduction response to N deficiency, and by the presence of interspecific signaling by heterotrophs in response to C limitation. Further, it crucially switches from a positive to a negative feedback loop with increasing GABA concentration, thus setting maximal bundle sizes. The unprecedented use of GABA/Glu as an intra- and interspecific signal in the spatial organization of microbiomes highlights the pair as truly universal infochemicals.

Synthesis of versatile neuromodulatory molecules by a gut microbial glutamate decarboxylase.

Dysbiosis of the microbiome correlates with many neurological disorders, yet very little is known about the chemistry that controls the production of neuromodulatory molecules by gut microbes. Here, we found that an enzyme glutamate decarboxylase (BfGAD) of a gut microbe Bacteroides fragilis forms multiple neuromodulatory molecules such as γ-aminobutyric acid (GABA), hypotaurine, taurine, homotaurine, and ß-alanine. We evolved BfGAD and doubled its taurine productivity. Additionally, we increased its specificity towards the substrate L-glutamate. Here, we provide a chemical strategy via which the BfGAD activity could be fine-tuned. In future, this strategy could be used to modulate the production of neuromodulatory molecules by gut microbes.

Intermediate partitioning kinetic isotope effects for the NIH shift of 4-hydroxyphenylpyruvate dioxygenase and the hydroxylation reaction of hydroxymandelate synthase reveal mechanistic complexity.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) are similar enzymes that catalyze complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. Both enzymes decarboxylate HPP and then hydroxylate the resulting hydroxyphenylacetate (HPA). The hydroxylation reaction catalyzed by HPPD displaces the aceto substituent of HPA in a 1,2-shift to form 2,5-dihydroxyphenylacetate (homogentisate, HG), whereas the hydroxylation reaction of HMS places a hydroxyl on the benzylic carbon forming 3'-hydroxyphenylacetate (S-hydroxymandelate, HMA) without ensuing chemistry. The wild-type form of HPPD and variants of both enzymes uncouple to form both native and non-native products. We have used intermediate partitioning to probe bifurcating steps that form these products by substituting deuteriums for protiums at the benzylic position of the HPP substrate. These substitutions result in altered ratios of products that can be used to calculate kinetic isotope effects (KIE) for the formation of a specific product. For HPPD, secondary normal KIEs indicate that cleavage of the bond in the displacement reaction prior to the shift occurs by a homolytic mechanism. NMR analysis of HG derived from HPPD reacting with enantiomerically pure R-3'-deutero-HPP indicates that no rotation about the bond to the radical occurs, suggesting that collapse of the biradical intermediate is rapid. The production of HMA was observed in HMS and HPPD variant reactions. HMS hydroxylates to form exclusively S-hydroxymandelate. When HMS is reacted with R-3'-deutero-HPP, the observed kinetic isotope effect represents geometry changes in the initial transition state for the nonabstracted proton. These data show evidence of sp(3) hybridization in a HPPD variant and sp(2) hybridization in HMS variants, suggesting that HMS stabilizes a more advanced transition state in order to catalyze H-atom abstraction.

Renalase is an α-NAD(P)H oxidase/anomerase.

Renalase is a protein hormone secreted into the blood by the kidney that is reported to lower blood pressure and slow heart rate. Since its discovery in 2005, renalase has been the subject of conjecture pertaining to its catalytic function. While it has been widely reported that renalase is the third monoamine oxidase (monoamine oxidase C) that oxidizes circulating catecholamines such as epinephrine, there has been no convincing demonstration of this catalysis in vitro. Renalase is a flavoprotein whose structural topology is similar to known oxidases, lysine demethylases, and monooxygenases, but its active site bears no resemblance to that of any known flavoprotein. We have identified the catalytic activity of renalase as an α-NAD(P)H oxidase/anomerase, whereby low equilibrium concentrations of the α-anomer of NADPH and NADH initiate rapid reduction of the renalase flavin cofactor. The reduced cofactor then reacts with dioxygen to form hydrogen peroxide and releases nicotinamide dinucleotide product in the ß-form. These processes yield an apparent turnover number (0.5 s(-1) in atmospheric dioxygen) that is at least 2 orders of magnitude more rapid than any reported activity with catechol neurotransmitters. This highly novel activity is the first demonstration of a role for naturally occurring α-NAD(P)H anomers in mammalian physiology and the first report of a flavoprotein catalyzing an epimerization reaction.

The prevalence of motility within the human oral microbiota.

The human oral and nasal microbiota contains approximately 770 cultivable bacterial species. More than 2000 genome sequences of these bacteria can be found in the expanded Human Oral Microbiome Database (eHOMD). We developed HOMDscrape, a freely available Python software tool to programmatically retrieve and process amino acid sequences and sequence identifiers from BLAST results acquired from the eHOMD website. Using the data obtained through HOMDscrape, the phylogeny of proteins involved in bacterial flagellar motility, Type 4 pilus driven twitching motility, and Type 9 Secretion system (T9SS) driven gliding motility was constructed. A comprehensive phylogenetic analysis was conducted for all components of the rotary T9SS, a machinery responsible for secreting various enzymes, virulence factors, and enabling bacterial gliding motility. Results revealed that the T9SS outer membrane ß-barrel protein SprA of human oral microbes underwent horizontal evolution. Overall, we catalog motile microbes that inhabit the human oral microbiota and document their evolutionary connections. These results will serve as a guide for further studies exploring the impact of motility on shaping of the human oral microbiota.

Evidence for the mechanism of hydroxylation by 4-hydroxyphenylpyruvate dioxygenase and hydroxymandelate synthase from intermediate partitioning in active site variants.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) each catalyze similar complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. The reactions differ in that HPPD hydroxylates at the ring C1 and HMS at the benzylic position. The HPPD reaction is more complex in that hydroxylation at C1 instigates a 1,2-shift of an aceto substituent. Despite that multiple intermediates have been observed to accumulate in single turnover reactions of both enzymes, neither enzyme exhibits significant accumulation of the hydroxylating intermediate. In this study we employ a product analysis method based on the extents of intermediate partitioning with HPP deuterium substitutions to measure the kinetic isotope effects for hydroxylation. These data suggest that, when forming the native product homogentisate, the wild-type form of HPPD produces a ring epoxide as the immediate product of hydroxylation but that the variant HPPDs tended to also show the intermediacy of a benzylic cation for this step. Similarly, the kinetic isotope effects for the other major product observed, quinolacetic acid, showed that either pathway is possible. HMS variants show small normal kinetic isotope effects that indicate displacement of the deuteron in the hydroxylation step. The relatively small magnitude of this value argues best for a hydrogen atom abstraction/rebound mechanism. These data are the first definitive evidence for the nature of the hydroxylation reactions of HPPD and HMS.

Osmolality of Commonly Used Oral Medications in the Neonatal Intensive Care Unit.

OBJECTIVE: The administration of hyperosmolar oral products in neonates has been associated with gastrointestinal complications. The American Academy of Pediatrics recommends a maximum osmolality of 450 mOsm/kg for formulas and enteral nutrition for term infants, and recent studies reported intolerance to enteral nutrition with osmolality above 500 mOsm/kg in low birthweight infants. The osmolality of medications administered to neonates is often not available in the literature or from manufacturers. The purpose of this study was to determine the osmolality of oral medications commonly administered to neonates in the NICU. METHODS: Fifty-two oral medications were chosen for this study, including solutions, suspensions, syrups, elixirs, and intravenous solutions administered orally. The osmolality of each medication was measured in triplicate by using freezing point depression. RESULTS: Thirty-seven of the 43 medications with measurable values (86.1%) had an osmolality greater than 500 mOsm/kg, and 6 medications (14%) had an osmolality less than 500 mOsm/kg. Nine medications did not result in a value. CONCLUSIONS: Our study provides osmolality data on oral medications commonly used in neonates with most oral medications having an osmolality greater than 500 mOsm/kg.