Sulfatases catalyze the cleavage of a variety of cellular sulfate esters via a novel mechanism that requires the action of a protein-derived formylglycine cofactor. Formation of the cofactor is catalyzed by an accessory protein and involves the two-electron oxidation of a specific cysteinyl or seryl residue on the relevant sulfatase. Although some sulfatases undergo maturation via mechanisms in which oxygen serves as an electron acceptor, AtsB, the maturase from Klebsiella pneumoniae, catalyzes the oxidation of Ser72 on AtsA, its cognate sulfatase, via an oxygen-independent mechanism. Moreover, it does not make use of pyridine or flavin nucleotide cofactors as direct electron acceptors. In fact, AtsB has been shown to be a member of the radical S-adenosylmethionine superfamily of proteins, suggesting that it catalyzes this oxidation via an intermediate 5′-deoxyadenosyl 5′-radical that is generated by a reductive cleavage of S-adenosyl-L-methionine. In contrast to AtsA, very little in vitro characterization of AtsB has been conducted. Herein we show that coexpression of the K. pneumoniae atsB gene with a plasmid that encodes genes that are known to be involved in ironsulfur cluster biosynthesis yields soluble protein that can be characterized in vitro. The as-isolated protein contained 8.7 ± 0.4 irons and 12.2 ± 2.6 sulfides per polypeptide, which existed almost entirely in the [4Fe-4S] 2+ configuration, as determined by Mössbauer spectroscopy, suggesting that it contained at least two of these clusters per polypeptide. Reconstitution of the as-isolated protein with additional iron and sulfide indicated the presence of 12.3 ± 0.2 irons and 9.9 ± 0.4 sulfides per polypeptide. Subsequent characterization of the reconstituted protein by Mössbauer spectroscopy indicated the presence of only [4Fe-4S] clusters, suggesting that reconstituted AtsB contains three per polypeptide. Consistent with this stoichiometry, an as-isolated AtsB triple variant containing Cys → Ala substitutions at each of the cysteines in its CX 3 CX 2 C radical SAM motif contained 7.3 ± 0.1 irons and 7.2 ± 0.2 sulfides per polypeptide while the reconstituted triple variant contained 7.7 ± 0.1 irons and 8.4 ± 0.4 sulfides per polypeptide, indicating that it was unable to incorporate an additional cluster. UV-visible and Mössbauer spectra of both samples indicated the presence of only clusters. AtsB was capable of catalyzing multiple turnovers and exhibited a V max /[E T ] of ~0.36 min −1 for an 18-amino acid peptide substrate using dithionite to supply the requisite electron and a value of ~0.039 min −1 for the same substrate using the physiologically relevant flavodoxin reducing system. Simultaneous quantification of formylglycine and 5′-deoxyadenosine as a function of time indicates an approximate 1:1 stoichiometry. Use of a peptide substrate in which the target serine is † This work was supported by NIH Grant GM-63847 (S.J.B.), the Dreyfus Foundation (Teacher Scholar Award to C.K.), and the Beckman Foundation (Young Investigator Award to...
The enzyme isopenicillin N synthase (IPNS) installs the β-lactam and thiazolidine rings of the penicillin core into the linear tripeptide, L-δ-aminoadipoyl-L-Cys-D-Val (ACV), on the pathways to a number of important antibacterial drugs. A classic set of enzymological and crystallographic studies by Baldwin and co-workers established that this overall four-electron oxidation occurs by a sequence of two oxidative cyclizations, with the β-lactam ring being installed first and the thiazolidine ring second. Each phase requires cleavage of an aliphatic C–H bond of the substrate: the pro-S-CCys,β-H bond for closure of the β-lactam ring, and the CVal,β-H bond for installation of the thiazolidine ring. IPNS uses a mononuclear non-heme-iron(II) cofactor and dioxygen as co-substrate to cleave these C–H bonds and direct the ring closures. Despite the intense scrutiny to which the enzyme has been subjected, the identities of the oxidized iron intermediates that cleave the C–H bonds have been addressed only computationally; no experimental insight into their geometric or electronic structures has been reported. In this work, we have employed a combination of transient-state-kinetic and spectroscopic methods, together with the specifically deuterium-labeled substrates, A[d2-C]V and AC[d8-V], to identify both C–H-cleaving intermediates. The results show that they are high-spin Fe(III)-superoxo and high-spin Fe(IV)-oxo complexes, respectively, in agreement with published mechanistic proposals derived computationally from Baldwin’s founding work.
BACKGROUND Patients diagnosed with primary brain tumors (pPBTs) exhibit high psychological distress. This study assessed how symptoms of anxiety and depression change over time in pPBTs and identified factors that may predict patients’ symptom trajectories. METHODS Ninety-nine adult pPBTs completed psychosocial assessments at routine neuro-oncology clinical appointments over 6 to 18 months, with a minimum 8-week interval between assessments. Symptoms of anxiety and depression were assessed with the PROMIS Anxiety and Depression Short-Forms. The prevalence and incidence of patients with clinically elevated anxiety and depression symptoms throughout follow-up were examined, along with the prevalence of patients that experienced clinically meaningful changes in symptoms between follow-ups. Linear mixed-effects models evaluated changes in symptoms over time at the group level and latent class growth analysis (LCGA) evaluated changes in symptoms over time at the individual level. RESULTS At baseline, 51.5% and 32.3% of patients exhibited clinically elevated levels of anxiety and depression, respectively. Of patients with any follow-up data (N = 74), 54.1% and 50% experienced clinically meaningful increases in anxiety and depression scores, respectively. The incidence of moderate to severe anxiety and depression was 15.6% and 8.3%, respectively. There was no significant change in symptoms over time at the group level, but better physical, functional, and brain-cancer well-being predicted significantly lower anxiety and depression symptoms (p< .001). Results from LCGA showcased 5-unique subgroups of patients with distinct anxiety and depression symptom trajectories. CONCLUSION pPBTs commonly experience elevated anxiety and depression. Symptoms of anxiety and depression change in clinically meaningful manners throughout the disease, even in the absence of significant group-level time effects. Routine screening for elevated symptoms is needed to capture clinically meaningful changes in symptoms and identify factors affecting symptoms to intervene.
e24131 Background: A primary brain tumor (PBT) is a distressing diagnosis that impacts the psychosocial well-being of patients and caregivers. Validated questionnaires are useful to assess PBT patient and caregiver psychosocial outcomes. However, it is unknown whether it is feasible to assess these outcomes in a routine clinical setting. This study evaluated the feasibility of assessing psychosocial outcomes for PBT patients and caregivers at the UF Health Neuro-Oncology clinic. Methods: 171 participants (100 PBT patients, 75 with malignant glioma; 71 caregivers) completed a battery of questionnaires to assess psychosocial outcomes during routine clinical appointments. At the end of the battery, a Participant Experience Form (PEF) assessed participant perceptions about the overall experience of completing the battery on a 7-point Likert Scale. Based on criteria from Bowen and colleagues (2009), feasibility and acceptability of assessment procedures were conceptualized and operationalized by assessing: Acceptability (80% of participants will have an average score ≥ 4 on the PEF); Practicality (average time of completion will be < 35 minutes); and Implementation (80% of participants will complete the entire battery). Descriptive statistics were used to assess these outcomes. Results: Patient and caregiver scores of feasibility and acceptability outcomes were as follows: Acceptability (Average PEF score: patients = 5.75 [ SD= 0.95]; caregivers = 5.96 [ SD= 0.83]); Practicality (Average time to complete (minutes): patients = 28.7 [ SD= 11.68]; caregivers = 26.0 [ SD= 12.01]); Implementation (% of participants that completed entire battery: patients = 88.0; caregivers = 84.5). Conclusions: The results indicate that assessing psychosocial outcomes in a routine clinical setting for PBT patients and caregivers was feasible, acceptable, and practical. Future research will use this battery to evaluate longitudinal psychosocial outcomes for this population in a clinical setting.
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