Escherichia coli C-glycosyltransferase IroB catalyzes the formation of a CC bond between enterobactin and the glucose moiety of UDP-glucose, resulting in the production of mono-, di- and ...tri-glucosylated enterobactin (MGE, DGE, TGE). To identify catalytic residues, we generated a homology model of IroB from aligned structures of two similar C-glycosyltransferases as templates. Superposition of our homology model onto the structure of a TDP-bound orthologue revealed residue W264 as a possible stabilizer of UDP-glucose. D304 in our model was located near the predicted site of the glucose moiety of UDP-glucose. A loop containing possible catalytic residues (H65, H66, E67) was found at the predicted enterobactin-binding site. We generated IroB variants at positions 65–67, 264, and 304 and investigated variant protein conformations and enzymatic activities. Variants were found to have Tm values similar to wild-type IroB. Fluorescence emission spectra of H65A/H66A, E67A, and D304N were superimposable with wild-type IroB. However, the emission spectrum of W264L was blue-shifted, suggesting solvent exposure of W264. While H65A/H66A retained activity (92% conversion of enterobactin, with MGE as a major product), all other IroB variants were impaired in their abilities to glucosylate enterobactin: E67A catalyzed partial (29%) conversion of enterobactin to MGE; W264L converted 55% of enterobactin to MGE; D304N was completely inactive. Activity-impaired variants were found to bind enterobactin with affinities within 2.5-fold of wild-type IroB. Given our outcomes, we propose that IroB W264 and D304 are required for binding and orienting UDP-glucose, while E67, possibly supported by H65/H66, participates in enterobactin/MGE/DGE deprotonation.
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•Using C-glycosyltransferase structures, we generated an E. coli IroB homology model.•Five potentially catalytic IroB residues were identified in our model.•Mutations of these residues did not affect overall protein fold.•Variant D304N was found to inactive. W264L, E67A and H65A/H66A were partially active.•Proposed roles: IroB W264 & D304 - donor binding; IroB E67 - acceptor deprotonation.
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•Benchmarking the E. coli S30 lysate core proteome.•Classification of proteome subsets relevant for cell-free expression.•Tuning S30 cell-free lysate production for improved protein ...quality.•Quantitative proteome analysis of SOS response induced S30 lysates.
Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a “black box”. Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures.
This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.
Bacterial iron acquisition by the means of enterobactin (ENT) is constrained in mammalian hosts due to ENT-binding proteins such as siderocalin and serum albumin. To evade sequestration by these ...proteins, ENT can be modified by the C glycosyltransferase IroB, which is located in the iroA locus of Salmonella and certain extraintestinal E. coli strains such as uropathogenic E. coli CFT073. The glycosylation of ENT has been reported to be a bacterial evasion mechanism to restore the iron scavenging ability of ENT in the presence of mammalian ENT-binding proteins by the installation of a steric impediment.
The C glycosyltransferase IroB catalyses the transfer of a glucose moiety to the DHB subunit of ENT under formation of a C-C bond between the anomeric C1 of the glucose moiety and the C5 of the 2,3-DHB subunit of ENT. The formation of mono-, di- and triglycosylated Ent (MGE/DGE/TGE) products where observed in vitro. The formation of a C-C bond is remarkable because of its chemical stability and resilience against enzymatic degradation.
In this M.Sc. thesis, we initially identified the iroB gene product in the iroA harbouring E. coli strain Nissle 1917 on transcriptional and translational level and expressed and purified IroB recombinant. Then, we investigated the mechanism of the C-C bond formation catalysed by IroB in vitro. Based on the hypothesis that deprotonation of the catechol 2 hydroxyl renders the catechol C5 para to the 2-hydroxyl nucleophilic, the C-C bond would then be formed in a general SN2 reaction between the attacking nucleophile and the anomeric carbon of glucose, which is further facilitated by the excellent phosphate leaving group of the UDP-glucose donor. By the means of homology modelling and superposition strategies, we were able to identify the binding sites of the glycosyl donor UDP-glucose and the glycosyl acceptor ENT and to locate residues that could potentially act as base catalysts to increase the phenolate anionic character of the 2,3-DHB subunit during catalysis.
We established an activity assay for IroB, separated products arising from IroB activity by reversed phase chromatography and compared so the activity of wild-type IroB and several variants. Additionally, all variants were characterized biophysically, mainly to confirm that the structural integrity was not impaired by mutations. Ultimately, our results enable us to propose a mechanism for C-glycosylation of IroB that is consistent with other glycosyltransferases found in nature.
Systemic therapy for metastatic melanoma has evolved rapidly during the last decade, and patient treatment has become more complex.
To evaluate the survival benefit achieved through surgical ...resection of melanoma metastatic to the abdominal viscera in patients treated in the modern treatment environment.
This retrospective review of the institutional melanoma database from the John Wayne Cancer Institute at Providence St Johns Health Center, a tertiary-level melanoma referral center, included 1623 patients with melanoma diagnosed as having potentially resectable abdominal metastases before (1969-2003) and after (2004-2014) advances in systemic therapy.
Overall survival (OS).
Of the 1623 patients identified in the database with abdominal melanoma metastases, 1097 were men (67.6%), and the mean (SD) age was 54.6 (14.6) years. Of the patients with metastatic melanoma, 1623 (320 19.7% in the 2004-2014 period) had abdominal metastases, including 336 (20.7%) with metastases in the gastrointestinal tract, 697 (42.9%) in the liver, 138 (8.5%) in the adrenal glands, 38 (2.3%) in the pancreas, 109 (6.7%) in the spleen, and 305 (18.8%) with multiple sites. Median OS was superior in surgical (n = 392; 18.0 months) vs nonsurgical (n = 1231; 7.0 months) patients (P < .001). The most favorable 1-year and 2-year OS was seen after surgery for gastrointestinal tract (52% and 41%) and liver (51% and 38%) metastases, respectively. Multivariable analysis found increasing age (hazard ratio HR, 1.01; 95% CI, 1.00-1.01; P = .02) and the presence of ulceration (HR, 1.21; 95% CI, 1.01-1.45; P = .04) were associated with a worse OS. Alternatively, treatment with metastasectomy (HR, 0.59; 95% CI, 0.46-0.74; P < .001) and metastases involving the gastrointestinal tract (HR, 0.65; 95% CI, 0.48-0.87; P = .004) were associated with a better OS. The systemic treatment era did not significantly affect outcomes (HR, 0.82; 95% CI, 0.67-1.02; P = .15). Overall, patients with gastrointestinal tract metastases undergoing complete, curative resection derived the greatest benefit, with a median OS of 64 months.
To our knowledge, this series is the largest single-institution experience with abdominal melanoma metastases, demonstrating that surgical resection remains an important treatment consideration even in the systemic treatment era.
Background
Recommended treatment for patients with sentinel lymph node (SLN)-positive melanoma has recently changed. Randomized trials demonstrated equivalent survival with close observation versus ...completion lymph node dissection (CLND), but increased regional node recurrence. We evaluated factors related to in-basin nodal recurrence after lymphadenectomy (LND) for SLN-positive or macroscopic nodal metastases.
Methods
An institutional database and the first Multicenter Selective Lymphadenectomy Trial (MSLT-I) were analyzed independently. Exclusions were multiple primaries, multi-basin involvement, or in-transit metastases. Patient demographics, primary tumor thickness and ulceration, lymph nodes retrieved, and use of adjuvant radiotherapy were analyzed. Multivariate analyses were performed to determine factors predicting in-basin nodal recurrence (significance
p
≤ 0.05).
Results
The retrospective cohort (577 patients) showed an in-basin failure rate of 6.6% after CLND for a positive SLN and 13.1% after LND for palpable disease (
p
= 0.001). This recurrence risk persisted after adjustment for patient, tumor, and LND factors hazard ratio (HR) 2.32;
p
= 0.004. In the MSLT-I cohort (326 patients), the failure rate after CLND following SLNB was 6.2%, but 10.1% after LND for palpable recurrence in observation patients. After adjustment for other factors, macroscopic disease was associated with an increased risk of recurrence after LND (HR 2.24;
p
= 0.05).
Conclusion
After LND for melanoma, in-basin recurrence is infrequent, but a clinically significant fraction will fail. Failure is less likely if dissection is performed for clinically occult disease. Further research is warranted to evaluate the long-term regional control and quality of life associated with nodal basin observation, which has now become standard practice.
Mucosal melanoma represents a distinct minority of disease sites and portends a worse outcome. The ideal treatment and role of adjuvant therapy remains unknown at this time. We hypothesized that a ...combination of neoadjuvant and adjuvant therapies would improve survival in these aggressive melanomas. Our large, prospectively maintained melanoma database was queried for all patients diagnosed with mucosal melanoma. Over the past five decades, 227 patients were treated for mucosal melanoma. There were 82 patients with anorectal, 75 with sinonasal, and 70 with urogenital melanoma. Five-year overall survival and melanoma-specific survival for the entire cohort were 32.8 and 37.5 per cent, respectively, with median overall survival of 38.7 months. One hundred forty-two patients (63.8%) underwent adjuvant therapy and 15 were treated neo-adjuvantly (6.6%). There was no survival difference by therapy type or timing, disease site, or decade of diagnosis. There was improved survival in patients undergoing multiple surgeries (Hazard Ratio HR 0.55, P = 0.0005). Patients receiving neoadjuvant therapy had significantly worse survival outcomes (HR 2.49, P = 0.013). Over the past five decades, improvements have not been seen in outcomes for mucosal melanoma. Although multiple surgical interventions portend a better outcome in patients with mucosal melanoma, adjuvant treatment decisions must be individualized.
Surgical resection of metastases to the adrenal gland can improve overall survival of patients with stage IV melanoma, but its relative value with respect to current nonsurgical therapies is unknown. ...We hypothesized that surgery remains an optimal first-line treatment approach for resectable adrenal metastases. A search of our institution's prospectively collected melanoma database identified stage IV patients treated for adrenal metastases between January 1, 2000, and August 11, 2014. The 91 study patients had a mean age of 60.3 years at diagnosis of adrenal metastasis and 24 had undergone adrenalectomy. Improved survival was associated with an unknown primary lesion, surgical resection, and nonsurgical therapies. Median overall survival from diagnosis of adrenal metastases was 29.2 months with adrenalectomy versus 9.4 months with nonoperative treatment. Adrenalectomy, either as complete metastasectomy or targeted to lesions resistant to systemic therapy, is associated with improved long-term survival in metastatic melanoma.
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