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Modulation of OvoAMtht's dual activities: sulfoxide synthase and thiol oxygenase.

The diversity of the reactions catalyzed by radical S-adenosyl-l-methionine (SAM) enzymes is achieved at least in part through the variety of mechanisms to quench their radical intermediates. In the SPASM-twitch family, the largest family of radical SAM enzymes, the radical quenching step is thought to involve an electron transfer to or from an auxiliary 4Fe-4S cluster in or adjacent to the active site. However, experimental demonstration of such functions remains limited. As a representative member of this family, MoaA has one radical SAM cluster ([4Fe-4S](RS)) and one auxiliary cluster ([4Fe-4S](AUX)), and catalyzes a unique 3′,8-cyclization of GTP into 3′,8-cyclo-7,8-dihydro-GTP (3′,8-cH(2)GTP) in the molybdenum cofactor (Moco) biosynthesis. Here, we report a mechanistic investigation of the radical quenching step in MoaA, a chemically challenging reduction of 3′,8-cyclo-GTP-N7 aminyl radical. We first determined the reduction potentials of [4Fe-4S](RS) and [4Fe-4S](AUX) as −510 mV and −455 mV, respectively, using a combination of protein film voltammogram (PFV) and electron paramagnetic resonance (EPR) spectroscopy. Subsequent Q-band EPR characterization of 5′-deoxyadenosine C4′ radical (5′-dA-C4′●) trapped in the active site revealed isotropic exchange interaction (~260 MHz) between 5′-dA-C4′● and [4Fe-4S](AUX)(1+), suggesting that [4Fe-4S](AUX) is in the reduced (1+) state during the catalysis. Together with density functional theory (DFT) calculation, we propose that the aminyl radical reduction proceeds through a proton-coupled electron transfer (PCET), where [4Fe-4S](AUX) serves as an electron donor and R17 residue acts as a proton donor. These results provide detailed mechanistic insights into the radical quenching step of radical SAM enzyme catalysis.

Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.

Tuning the electron-transfer (ET) steps in redox reactions has proven to be an effective strategy to modulate enzyme catalysis. Here, we use 2-oxoacid:ferredoxin oxidoreductase (OFOR) as a model sy...

STUDY DESIGN Prospective, multi-centered, observational. OBJECTIVES To characterize the relationship between psychosocial aspects of health-related quality of life (HRQoL) and patient-reported bladder outcomes. SETTING Multi-institutional sites in the United States, cohort drawn from North America. METHODS We performed a cross-sectional analysis of data collected as part of the multicenter, prospective Neurogenic Bladder Research Group Spinal Cord Injury (SCI) Registry. Outcomes were: Neurogenic Bladder Symptom Score (NBSS), Neurogenic Bladder Symptom Score Satisfaction (NBSS-Satisfaction), and SCI-QoL Bladder Management Difficulties (SCI-QoL Difficulties). Adjusted multiple linear regression models were used with variables including demographic, injury characteristics, and the following psychosocial HRQoL measures; SCI-QoL Pain Interference (Pain), SCI-QoL Independence, and SCI-QoL Positive Affect and Well-being (Positive Affect). Psychosocial variables were sub-divided by tertiles for the analysis. RESULTS There were 1479 participants, 57% had paraplegia, 60% were men, and 51% managed their bladder with clean intermittent catheterization. On multivariate analysis, higher tertiles of SCI-QoL Pain were associated with worse bladder symptoms, satisfaction, and bladder management difficulties; upper tertile SCI-QoL Pain (NBSS 3.8, p

Objectives To describe the risks of urinary diversion performed to address adverse effects of gynecologic radiation, with the hypothesis that high grade complications would be more common in sarcopenic women and in those undergoing continent diversion (vs. non-continent) or concomitant cystectomy (vs. bladder-sparing diversion). Methods : A retrospective review was performed of patient records of women who underwent urinary diversion for urinary adverse effects of gynecologic radiotherapy during the period of 2008 - 2018 from three tertiary centers. Skeletal muscle index was calculated to assess for sarcopenia based on preoperative computed tomography scans. Outcomes include high-grade post-operative complications within 90 days of surgery, 30-day readmission rate, and long-term (>90 days) outcomes. Results : Among 34 women who met the inclusion criteria, the majority were white/Caucasian (73.5%). 26/34 (76.5%) underwent non-continent diversion. Seventy nine percent (27/34) (79.4%) of women experienced complications within 90 days; 9/34 (26.5%) experienced high-grade (Clavien grade ≥3) complications. Thirty two percent (11/34) were readmitted within 30 days. Rates of high-grade complications were not significantly impacted by diversion type (p=0.49), concurrent cystectomy (p=0.70), or sarcopenia (p=1.0). Conclusions : Urinary diversion for late adverse effects of gynecologic radiotherapy is associated with high peri-operative risk, even in skilled hands. In this series, neither sarcopenia, nor continent diversion, nor cystectomy were associated with increased complications. Patients and surgeons should consider risks and benefits when deciding to proceed with urinary diversion, however further study is needed to elucidate significant pre-operative markers which may pre-dispose patients to significant complications.

Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.

BthA is a diheme enzyme that is a member of the bacterial cytochrome c peroxidase superfamily, capable of generating a highly unusual Fe(IV)Fe(IV)=O oxidation state, known to be responsible for long-range oxidative chemistry in the enzyme MauG. Here we show that installing a canonical Met ligand in lieu of the Tyr found at the heme of MauG associated with electron transfer, results in a construct that yields an unusually stable Fe(IV)=O porphyrin at the peroxidatic heme. This state is spontaneously formed at ambient conditions using either molecular O(2) or H(2)O(2). The resulting data illustrate how a ferryl iron, with unforeseen stability, may be achieved in biology.

Mononuclear non-heme iron enzymes are a large class of enzymes catalyzing a wide-range of reactions. In this work, we report that a non-heme iron enzyme in

Introduction The aim of this study was to survey pediatric urology fellowship directors (PFD) and adult reconstruction fellowship directors (AFD) to assess who they believe has sufficient training to care for adults with congenital urologic conditions (ACUC). Material and methods An online survey was created to assess attitudes towards specific training to care for ACUC. The survey was administered to 27 PFD and 26 AFD [16 from genitourinary reconstructive surgery (GURS) and 10 from female pelvic medicine and reconstructive surgery (FPMRS)]. Both groups were asked if specific training is warranted, and if general urologists, pediatric urologists or adult reconstructive urologists were sufficiently trained to care for ACUC. Results A total of 26 (96%) PFD and 10 (39%) AFD completed the survey. All PFD were fellowship trained in pediatrics. Of the AFD, 5 were GURS trained, 4 were FPMRS trained and 1 was not fellowship trained. The majority (65% PFD, 90% AFD) believed specific training is warranted. Few believed general urologists have sufficient training (8% PFD, 20% AFD). Most PFD believed pediatric urologists have sufficient training (85%), but a minority believed those with adult reconstructive training do (40%). Conversely, a minority of AFD believed that pediatric urologists have sufficient training (40%), while those with adult reconstructive training do (FPMRS: 67%, GURS: 60%). Conclusions Both pediatric and adult reconstructive urologists believe specific training to care for adults with congenital urologic conditions is warranted. Neither group considers the other to be ideally suited to care for this complex patient group. This suggests both groups may have something to learn from each other.

The formate dehydrogenase enzyme from Cupriavidus necator (FdsABG) carries out the two-electron oxidation of formate to CO2, but is also capable of reducing CO2 back to formate, a potential biofuel. FdsABG is a heterotrimeric enzyme that performs this transformation using nine redox-active cofactors: a bis(molybdopterin guanine dinucleotide) (bis-MGD) at the active site coupled to seven iron–sulfur clusters, and one equivalent of flavin mononucleotide (FMN). To better understand the pathway of electron flow in FdsABG, the reduction potentials of the various cofactors were examined through direct electrochemistry. Given the redundancy of cofactors, a truncated form of the FdsA subunit was developed that possesses only the bis-MGD active site and a singular [4Fe–4S] cluster. Electrochemical characterization of FdsABG compared to truncated FdsA shows that the measured reduction potentials are remarkably similar despite the truncation with two observable features at − 265 mV and − 455 mV vs SHE, indicating that the voltammetry of the truncated enzyme is representative of the reduction potentials of the intact heterotrimer. By producing truncated FdsA without the necessary maturation factors required for bis-MGD insertion, a form of the truncated FdsA that possesses only the [4Fe–4S] was produced, which gives a single voltammetric feature at − 525 mV, allowing the contributions of the molybdenum cofactor to be associated with the observed feature at − 265 mV. This method allowed for the deconvolution of reduction potentials for an enzyme with highly complex cofactor content to know more about the thermodynamic landscape of catalysis.

The goal of this research is to devise a method of differential equation based modeling of evolution that can scale up to capture complex dynamics by enabling the inclusion of many—potentially thousands—of biological characteristics. Towards that goal, a mathematical model for evolution based on the well-established Fisher-Eigen process is built with a unique and efficient structure. The Fisher-Eigen partial differential equation (PDE) describes the evolution of a probability density function representing the distribution of a population over a phenotype space. This equation depends on the choice of a fitness function representing the likelihood of reproductive success at each point in the phenotype space. The Fisher-Eigen model has been studied analytically for simple fitness functions, but in general no analytic solution is known. Furthermore, with traditional numerical methods, the equation becomes exponentially complex to simulate as the dimensionality of the problem expands to include more phenotypes. For this research, a network model is synthesized and a set of ordinary differential equations (ODEs) is extracted based on the Fisher-Eigen PDE to describe the dynamic behavior of the system. It is demonstrated that, when juxtaposed with full numerical PDE simulations, this ODE model finds well-matched transient and precise equilibrium solutions. This prototype method makes modeling of high-dimensional data possible, allowing researchers to examine and even predict complex dynamic behavior based on a snapshot of a population. KEYWORDS: Evolutionary Modeling; Mathematical Biology; Network Dynamics; Ordinary Differential Equations; Partial Differential Equations; Fisher-Eigen model; Phenotype; Fitness Function

INTRODUCTION AND OBJECTIVE:Anterior urethral stricture disease (aUSD) is often a diagnosis of exclusion in men presenting with refractory lower urinary tract symptoms (LUTS). When the stricture is ...

INTRODUCTION AND OBJECTIVE:A successful urethroplasty has been defined in many ways. This variability poses a challenge when trying to compare success rates and techniques across studies. We aim to...

INTRODUCTION AND OBJECTIVE:Prior work has established that post-operative urethroscopy may be an effective tool to predict re-operation within 1 year following urethroplasty. Here, we examine early...

Purpose: To determine how members of the Société Internationale d’Urologie (SIU) are continuing their education in the time of COVID-19. Methods: A survey was disseminated amongst SIU members worldwide by email. Results were analyzed to examine the influence of age, practice region and settings on continuing medical education (CME) of the respondents. Results: In total, 2494 respondents completed the survey. Internet searching was the most common method of CME (76%; all ps < 0.001), followed by searching journals and textbook including the online versions (62%; all ps < 0.001). Overall, 6% of the respondents reported no time/interest for CME during the pandemic. Although most urologists report using only one platform for their CME (26.6%), the majority reported using ≥ 2 platforms, with approximately 10% of the respondents using up to 5 different platforms. Urologists < 40 years old were more likely to use online literature (69%), podcasts/AV media (38%), online CME courses/webinars (40%), and social media (39%). There were regional variations in the CME modality used but no significant difference in the number of methods by region. There was no significant difference in responses between urologists in academic/public hospitals or private practice. Conclusion: During COVID-19, urologists have used web-based learning for their CME. Internet learning and literature were the top frequently cited learning methods. Younger urologists are more likely to use all forms of digital learning methods, while older urologists prefer fewer methods.

The aim of this article is to understand the extreme variability in estimates of the reproduction ratio R-0 observed in practice. For expository purposes, we consider a discrete-time, stochastic version of the susceptible-infected-recovered model and introduce different approximate maximum likelihood estimators of R-0. We carefully discuss the properties of these estimators and illustrate, by a Monte Carlo study, the widths of confidence intervals for R-0.

The BthA protein from the microorganism Burkholderia thailandensis contains two hemes with axial His/OH(2) and His/Tyr coordinations separated by the closest interheme distance of 14 Å. BthA has a similar structure and belongs to the same family of multiheme cytochrome c peroxidases as MauG, which performs long-range oxidation of the partner protein methylamine dehydrogenase. Magnetic Mössbauer spectroscopy of the diferric state of BthA corroborates previous structural work identifying a high-spin (His/OH(2)) peroxidatic heme and a low-spin (His/Tyr) electron transfer heme. Unlike MauG, addition of H(2)O(2) fully converts the diferric form of BthA to a stable 2e(−) oxidized state, allowing a new assessment of this state. The peroxidatic heme is found to be oxidized to a canonical compound II, S = 1 oxoiron(IV) heme. In contrast, the electronic properties of the oxidized His/Tyr heme are puzzling. The isomer shift of the His/Tyr heme (0.17 mm/s) is close to that of the precursor S = 1/2 Fe(3+) heme (0.21 mm/s) which suggests oxidation of the Tyr. However, the spin-dipolar hyperfine coupling constants are found here to be the same as those for the ferryl peroxidatic heme, indicating that the His/Tyr heme is also a compound II, S = 1 Fe(4+) heme and ruling out oxidation of the Tyr. DFT calculations indicate that the unusually high isomer shift is not attributable to the rare axial His/Tyr heme coordination. The calculations are only compatible with spectroscopy for an unusually long Fe(4+)–O(Tyr) distance, which is presumably under the influence of the protein environment of the His/Tyr heme moiety in the H(2)O(2) oxidized state of the protein. The results offer new insights into how high valence intermediates can be tuned by the protein environment for performing long-range oxidation.

2-oxoglutarate:ferredoxin oxidoreductase (OGOR) is a thiamine pyrophosphate (TPP) and [4Fe-4S] cluster-dependent enzyme from the reductive tricarboxylic acid (rTCA) cycle that fixes CO 2 to succinyl-CoA, forming 2-oxoglutarate and CoA. Here we report an OGOR from the rTCA cycle of Magnetococcus marinus MC-1, along with all three potential ferredoxin (Fd) redox partners. We demonstrate that MmOGOR operates bidirectionally (both CO 2 -fixing and 2-oxoglutarate-oxidizing) and that only one Fd (MmFd1) supports efficient catalysis. Our 1.94-Å- and 2.80-Å-resolution crystal structures of native and substrate-bound forms of MmOGOR reveal the determinants of substrate specificity and CoA-binding in an OGOR and illuminate the [4Fe-4S] cluster environment, portraying the electronic conduit through which MmFd1 is wired to the bound TPP. Structural and biochemical data further identify Glu45α as a mobile residue that impacts catalytic bias toward CO 2 fixation, although it makes no direct contact with TPP-bound intermediates, indicating that reaction directionality can be tuned by second-layer interactions. The transformation of CO 2 into chemicals or fuels is a tantalizing chemistry at the heart of efforts to achieve sustainable carbon usage. The search continues for scalable catalysts that can be used to capture CO 2 in waste gas streams. Remarkably, microbial organisms contain enzymes that engage in CO 2 transformations routinely at ambient temperature and pressure, and often, such enzymes are also incredibly efficient in terms of minimal thermodynamic waste spent while engaging in catalytic chemistry. Further industrial promise is held in the implementation of engineered micro-organisms that contain CO 2 -reducing enzymes. Here, we investigate one of the reversible catalysts found widely in nature that can engage in the transformation of CO 2 into 2-oxoglutarate. The lessons from our study may impact how industrial microbiologists re-wire carbon pathways in scalable microbial bioreactors where CO 2 can be transformed into fuels or high-value molecules. We describe an oxoglutarate:ferredoxin oxidoreductase (OGOR) enzyme that is found natively in the reverse TCA cycle and demonstrate that it participates in the two-electron inter-conversion of CO 2 to 2-oxoglutarate while possessing only a single iron-sulfur cluster. X-ray crystal structures reveal how substrate specificity is generated, and determining the identity of the catalytically relevant redox partner yields insight into how electron-transfer steps may be key to guiding CO 2 reduction., National Institutes of Health (U.S.) (Grant R35 GM126982), National Institutes of Health (U.S.) (Grant P41 GM103403), National Institutes of Health (U.S.). Office of Research Infrastructure Programs. High-End Instrumentation (HEI) Grant Program (S10 RR029205), United States. Department of Energy. Office of Science (Contract DE-AC02-06CH11357)

INTRODUCTION AND OBJECTIVE:Post-void dribbling (PVD) and ejaculatory dysfunction (EjD) are common after urethroplasty (UP). Although the etiology remains unknown, they are thought to share a simila...

INTRODUCTION AND OBJECTIVE:The Urethral Stricture Symptom and Impact Measure (USSIM) is a condition-specific outcome measure for men with urethral stricture. Concept elicitation, cognitive, and pri...

INTRODUCTION AND OBJECTIVE:Urinary tract infections (UTIs) are a major source of morbidity after spinal cord injury (SCI). People with SCI attribute a variety of symptoms to UTI; but up to 60% of t...

INTRODUCTION AND OBJECTIVE:Nephrectomy can be avoided in most patients even in cases of high-grade renal trauma (HGRT). The reasons for nephrectomy after trauma are complex but it is often done in ...

INTRODUCTION AND OBJECTIVE:We report 2-year outcomes of ROBUST I, a multi-center, single-arm, open-label study of the safety and efficacy of a paclitaxel drug-coated balloon (DCB) for treatment of ...

Most organisms contain multiple soluble protein-based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron-sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the "radical SAM," or S-adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron-sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe4 S4 ]1+ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side-by-side fashion for the first time. While a single [Fe4 S4 ]-cluster bearing Fd (TM0927) has a potential of -420 mV, the other four 2x[Fe4 S4 ]-bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).

MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6 -isopentenyl (i6 A) group found at position 37 in several tRNAs to afford 2-methylthio-N6 -(isopentenyl)adenosine (ms2 i6 A). MiaB uses a reduced [4Fe-4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5'-deoxyadenosyl 5'-radical (5'-dA•)-a required intermediate in its reaction-as well as an additional [4Fe-4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe-4S]2+ cluster to the [4Fe-4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5'-dA• and ms2 i6 A production, indicating that only one hydrogen atom abstraction is required for the reaction.

Cytochrome c peroxidases (bCcPs) are diheme enzymes required for the reduction of H2O2 to water in bacteria. There are two classes of bCcPs: one is active in the diferric form (constitutively active), and the other requires the reduction of the high-potential heme (H-heme) before catalysis commences (reductively activated) at the low-potential heme (L-heme). To improve our understanding of the mechanisms and heme electronic structures of these different bCcPs, a constitutively active bCcP from Nitrosomonas europaea ( NeCcP) and a reductively activated bCcP from Shewanella oneidensis ( SoCcP) were characterized in both the diferric and semireduced states by electron paramagnetic resonance (EPR), resonance Raman (rRaman), and magnetic circular dichroism (MCD) spectroscopy. In contrast to some previous crystallographic studies, EPR and rRaman spectra do not indicate the presence of significant amounts of a five-coordinate, high-spin ferric heme in NeCcP or SoCcP in either the diferric or semireduced state in solution. This observation points toward a mechanism of activation in which the active site L-heme is not in a static, five-coordinate state but where the activation is more subtle and likely involves formation of a six-coordinate hydroxo complex, which could then react with hydrogen peroxide in an acid-base-type reaction to create Compound 0, the ferric hydroperoxo complex. This mechanism lies in stark contrast to the diheme enzyme MauG that exhibits a static, five-coordinate open heme site at the peroxidatic heme and that forms a more stable FeIV═O intermediate.