scholarly journals Tuning of pKa values activates substrates in flavin-dependent aromatic hydroxylases

2020 ◽  
Vol 295 (12) ◽  
pp. 3965-3981 ◽  
Author(s):  
Warintra Pitsawong ◽  
Pirom Chenprakhon ◽  
Taweesak Dhammaraj ◽  
Dheeradhach Medhanavyn ◽  
Jeerus Sucharitakul ◽  
...  
Keyword(s):  
Active Sites ◽  
Single Component ◽  
Electrophilic Aromatic Substitution ◽  
Aromatic Substitution ◽  
Substrate Analogs ◽  
Two Component ◽  
Group A ◽  
Catalytic Mechanisms ◽  
Protein Variants ◽  
Group D

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.

scholarly journals Structural basis of the Cope rearrangement and C–C bond-forming cascade in hapalindole/fischerindole biogenesis

2017 ◽  
Author(s):  
Sean A. Newmister ◽  
Shasha Li ◽  
Marc Garcia-Borràs ◽  
Jacob N. Sanders ◽  
Song Yang ◽  
...  
Keyword(s):  
Active Sites ◽  
Mutational Analysis ◽  
Biological Activities ◽  
Sigmatropic Rearrangement ◽  
National Institutes Of Health ◽  
Structural Basis ◽  
Electrophilic Aromatic Substitution ◽  
Cope Rearrangement ◽  
Aromatic Substitution

STRUCTURESThe atomic coordinates and structure factors for:HpiC1 W73M/K132M SeMet (P212121) –1.7 ÅHpiC1 native (C2) –1.5 ÅHpiC1 native (P42) –2.1 ÅHpiC1 Y101F (C2) –1.4 ÅHpiC1 Y101S (C2) –1.4 ÅHpiC1 F138S (P21) –1.7 ÅHpiC1 Y101F/F138S (P21 –1.65 Å have been deposited with the Research Collaboratory for Structural Bioinformatics as Protein Data Bank entries 5WPP, 5WPR, 6AL6, 5WPR, 5WPU, 6AL7, and 6AL8 (www.rcsb.org).GRANTSThis work was supported by: The authors thank the National Science Foundation under the CCI Center for Selective C-H Functionalization (CHE-1205646), the National Institutes of Health (CA70375 to RMW and DHS), R35 GM118101, R01 GM076477 and the Hans W. Vahlteich Professorship (to DHS) for financial support. M.G-B. thanks the Ramón Areces Foundation for a postdoctoral fellowship. J.N.S. acknowledges the support of the National Institute of General Medical Sciences of the National Institutes of Health under Award Number F32GM122218. Computational resources were provided by the UCLA Institute for Digital Research and Education (IDRE) and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the NSF (OCI-1053575). The content does not necessarily represent the official views of the National Institutes of Health.ABSTRACTHapalindole alkaloids are a structurally diverse class of cyanobacterial natural products defined by their varied polycyclic ring systems and diverse biological activities. These polycyclic scaffolds are generated from a common biosynthetic intermediate by the Stig cyclases in three mechanistic steps, including a rare Cope-rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution. Here we report the structure of HpiC1, a Stig cyclase that catalyzes the formation of 12-epi-hapalindole U in vitro. The 1.5 Å structure reveals a dimeric assembly with two calcium ions per monomer and the active sites located at the distal ends of the protein dimer. Mutational analysis and computational methods uncovered key residues for an acid catalyzed [3,3]-sigmatropic rearrangement and specific determinants that control the position of terminal electrophilic aromatic substitution leading to a switch from hapalindole to fischerindole alkaloids.

Single-component and two-component para -nitrophenol monooxygenases: structural basis for their catalytic difference

2021 ◽  
Author(s):  
Yuan Guo ◽  
De-Feng Li ◽  
Jianting Zheng ◽  
Ying Xu ◽  
Ning-Yi Zhou
Keyword(s):  
Crystal Structure ◽  
Rational Design ◽  
Single Component ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Gram Positive ◽  
Two Component ◽  
Group D

para -Nitrophenol (PNP) is a hydrolytic product of organophosphate insecticides, such as parathion and methylparathion, in soil. Aerobic microbial degradation of PNP has been classically shown to proceed via ‘Hydroquinone (HQ) pathway’ in Gram-negative degraders, whereas via ‘Benzenetriol (BT) pathway’ in Gram-positive ones. ‘HQ pathway’ is initiated by a single-component PNP 4-monooxygenase and ‘BT pathway’ by a two-component PNP 2-monooxygenase. Their rigio-selectivity intrigues us to investigate their catalytic difference through structural study. PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP). However, the mechanisms are unknown. Here, PnpA1 was structurally determined to be a member of group D flavin-dependent monooxygenases with an acyl-CoA dehydrogenase fold. The crystal structure and site-directed mutagenesis underlined the direct involvement of Arg100 and His293 in catalysis. The bulky side chain of Val292 was proposed to push the substrate towards FAD, hence positioning the substrate properly. A variant N450A was found with improved activity for 4NC and 2C4NP, probably because of the reduced steric hindrance. PnpA1 shows obvious difference in substrate selectivity with its close homologues TcpA and TftD, which may be determined by Thr296 and loop 449–454. Above all, our study allows the structural comparison between the two types of PNP monooxygenases. An explanation that accounts for their regio-selectivity was proposed: the different PNP binding manner determines their choice of ortho - or para -hydroxylation on PNP. IMPORTANCE Single-component PNP monoxygenases hydroxylate PNP at 4-position while two-component ones at 2-position. However, their catalytic and structural differences remain elusive. The structure of single-component PNP 4-monooxygenase has previously been determined. In this study, to illustrate their catalytic difference, we resolved the crystal structure of, PnpA1, a typical two-component PNP 2-monooxygenase. The roles of several key amino acid residues in substrate binding and catalysis were revealed and a variant with improved activities towards 4NC and 2C4NP was obtained. Moreover, through comparing the two types of PNP monooxygenases, a hypothesis was proposed to account for their catalytic difference, which gives us a better understanding of these two similar reactions at molecular level. And these results will also be of further aid in enzyme rational design in bioremediation and biosynthesis.

Survival of classic swine fever virus in hams made from the meat of pigs vaccinated with the PAV-250 strain and unvaccinated pigs

2019 ◽  
Vol 10 (3) ◽  
pp. 536-551
Author(s):  
Heidi Amezcua Hempel ◽  
María Salud Rubio Lozano ◽  
Eliseo Manuel Hernández Baumgarten ◽  
Pablo Correa Girón † ◽  
Oscar Torres Ángeles ◽  
...  
Keyword(s):  
Reference Strain ◽  
Clinical Signs ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Direct Immunofluorescence ◽  
Biometric Analysis ◽  
Treatment Groups ◽  
Group A ◽  
Group B ◽  
Group D

The study was to determine the presence of Classical Swine Fever virus (CSFv), in the meat of vaccinated pigs with the PAV-250 strain and then challenged using the same strain. Five treatment groups were established (each with four pigs). Group A: Pigs thatwere fed with processed hams from negative animals; Group B: Pigs that were fed with processed hams from commercial pigs inoculated with the ALD (reference strain) (titre of 104.0/ml); Group C: Pigs fed with processed hams from pigs infected with the virulent ALD strain (titre of 102.5/ml); Group D: Pigs fed with processed hams from pigs vaccinated with the PAV-250 strain and challenged with the ALD strain (titre of 101.1/ml); and Group E: Pigs fed with processed hams from pigs vaccinated with two doses of the PAV-250 strain and challenged with the ALD strain (negative). Blood samples were taken at d 1, 5, 10, 15 and 20 for biometric analysis. Groups B, C and D manifested clinical signs of CSFv: 40 °C temperature, anorexia, paralysis, vomiting, diarrhea, tremor, hirsute hair and cyanosis. Pigs were slaughtered and necropsies performed to identify lesions in tissues. Results of direct immunofluorescence testing of tissues were positive and the virus was recovered. Under these study conditions, it was found that CSFv resisted the cooking method at 68 °C for 40 min in hams from unvaccinated pigs, and that the virus was able to transmit the disease to healthy unvaccinated pigs, whereas the hams from the vaccinated animals did not transmit the virus.

scholarly journals The Role of Obesity in Predicting the Clinical Outcomes of COVID-19

Obesity Facts ◽  
2021 ◽  
pp. 1-9
Author(s):  
Serdar Sahin ◽  
Havva Sezer ◽  
Ebru Cicek ◽  
Yeliz Yagız Ozogul ◽  
Murat Yildirim ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Health Care Providers ◽  
Study Groups ◽  
Normal Weight ◽  
Overweight And Obesity ◽  
Care Providers ◽  
Significant Difference ◽  
Group A ◽  
Group B ◽  
Group D

<b><i>Introduction:</i></b> The aim of this was to describe the predictors of mortality related to COVID-19 infection and to evaluate the association between overweight, obesity, and clinical outcomes of COVID-19. <b><i>Methods:</i></b> We included the patients &#x3e;18 years of age, with at least one positive SARS-CoV-2 reverse transcriptase-polymerase chain reaction. Patients were grouped according to body mass index values as normal weight &#x3c;25 kg/m<sup>2</sup> (Group A), overweight from 25 to &#x3c;30 kg/m<sup>2</sup> (Group B), Class I obesity 30 to &#x3c;35 kg/m<sup>2</sup> (Group C), and ≥35 kg/m<sup>2</sup> (Group D). Mortality, clinical outcomes, laboratory parameters, and comorbidities were compared among 4 groups. <b><i>Results:</i></b> There was no significant difference among study groups in terms of mortality. Noninvasive mechanical ventilation requirement was higher in group B and D than group A, while it was higher in Group D than Group C (Group B vs. Group A [<i>p</i> = 0.017], Group D vs. Group A [<i>p</i> = 0.001], and Group D vs. Group C [<i>p</i> = 0.016]). Lung involvement was less common in Group A, and presence of hypoxia was more common in Group D (Group B vs. Group A [<i>p</i> = 0.025], Group D vs. Group A [<i>p</i> &#x3c; 0.001], Group D vs. Group B [<i>p</i> = 0.006], and Group D vs. Group C [<i>p</i> = 0.014]). The hospitalization rate was lower in Group A than in the other groups; in addition, patients in Group D have the highest rate of hospitalization (Group B vs. Group A [<i>p</i> &#x3c; 0.001], Group C vs. Group A [<i>p</i> &#x3c; 0.001], Group D vs. Group A [<i>p</i> &#x3c; 0.001], Group D vs. Group B [<i>p</i> &#x3c; 0.001], and Group D vs. Group C [<i>p</i> = 0.010]). <b><i>Conclusion:</i></b> COVID-19 patients with overweight and obesity presented with more severe clinical findings. Health-care providers should take into account that people living with overweight and obesity are at higher risk for COVID-19 and its complications.

scholarly journals AB0422 LEFT VENTRICULAR ABNORMALITIES IN SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS FOLLOWED BY SEQUENTIAL ECHOCARDIOGRAPHY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1510.1-1511
Author(s):  
T. Kuga ◽  
M. Matsushita ◽  
K. Tada ◽  
K. Yamaji ◽  
N. Tamura
Keyword(s):  
Lupus Erythematosus ◽  
Systolic Dysfunction ◽  
Left Ventricular ◽  
Monocyte Count ◽  
Lv Dysfunction ◽  
Group A ◽  
Group B ◽  
Lv Diastolic Dysfunction ◽  
Group D

Background:Cardiovascular disease (CVD) is detected in up to 50% of systemic lupus erythematosus (SLE) patients1and major cause of death2. Even clinically silent SLE patients can develop left ventricular (LV) diastolic dysfunction3. Proper echocardiographic follow up of SLE patients is required.Objectives:To clarify how the prevalence of LV abnormalities changes over follow-up period and identify the associated clinical factors, useful in suspecting LV abnormalities.Methods:29 SLE patients (24 females and 5 men, mean age 52.8±16.3 years, mean disease duration 17.6±14.5 years) were enrolled. All of them underwent echocardiography as the baseline examination and reexamined over more than a year of follow-up period(mean 1075±480 days) from Jan 2014 to Sep 2019. Patients complicated with pulmonary artery hypertension, deep venous thrombosis or pulmonary embolism and underwent cardiac surgery during the follow-up period were excluded. Left ventricular(LV) systolic dysfunction was defined as ejection fraction (EF) < 50%. LV diastolic dysfunction was defined according to ASE/EACVI guideline4. LV dysfunction (LVD) includes one or both of LV systolic dysfunction and LV diastolic function. Monocyte to HDL ratio (MHR) was calculated by dividing monocyte count with HDL-C level.Prevalence of left ventricular abnormalities was analysed at baseline and follow-up examination. Clinical characteristics and laboratory data were compared among patient groups as follows; patients with LV dysfunction (Group A) and without LV dysfunction (Group B) at the follow-up echocardiography, patients with LV asynergy at any point of examination (Group C) and patients free of LV abnormalities during the follow-up period (Group D).Results:At the baseline examination, LV dysfunction (5/29 cases, 13.8%), LV asynergy (6/29 cases, 21.7%) were detected. Pericarditis was detected in 7 patients (24.1%, LVD in 3 patients, LV asynergy in 2 patients) and 2 of them with subacute onset had progressive LV dysfunction, while 5 patients were normal in echocardiography after remission induction therapy for SLE. At the follow-up examination, LV dysfunction (9/29 cases, 31.0%, 5 new-onset and 1 improved case), LV asynergy (6/29 cases, 21.7%, 2 new-onset and 2 improved cases) were detected. Though any significant differences were observed between Group A and Group B at the baseline, platelet count (156.0 vs 207.0, p=0.049) were significantly lower in LV dysfunction group (Group A) at the follow-up examination. Group C patients had significantly higher uric acid (p=0.004), monocyte count (p=0.009), and MHR (p=0.003) than Group D(results in table).Conclusion:LV dysfunction is progressive in most of patients and requires regular follow-up once they developed. Uric acid, monocyte count and MHR are elevated in SLE patients with LV asynergy. Since MHR elevation was reported as useful marker of endothelial dysfunction5, our future goal is to analyse involvement of monocyte activation and endothelial dysfunction in LV asynergy of SLE patients.References:[1]Doria A et al. Lupus. 2005;14(9):683-6.[2]Manger K et al. Ann Rheum Dis. 2002 Dec;61(12):1065-70.[3]Leone P et al. Clin Exp Med. 2019 Dec 17.[4]Nagueh SF et al. J Am Soc Echocardiogr. 2016 Apr;29(4):277-314.[5]Acikgoz N et al. Angiology. 2018 Jan;69(1):65-70.Numbers are median (interquartile range), Mann-Whitney u test were performed, p value less than 0.05 was considered statistically significant.Disclosure of Interests: :None declared

scholarly journals Prophylactic temporary abdominal aortic balloon occlusion for patients with pernicious placenta previa: a retrospective study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fei Huo ◽  
Hansheng Liang ◽  
Yi Feng
Keyword(s):  
Placenta Previa ◽  
Balloon Occlusion ◽  
Artery Embolism ◽  
Abdominal Aortic ◽  
Significant Difference ◽  
Group A ◽  
Group B ◽  
Clinical Records ◽  
Group D ◽  
Central Placenta Previa

Abstract Background Pernicious placenta previa (PPP) can increase the risk of perioperative complications. During caesarean section in patients with adherent placenta, intraoperative blood loss, hysterectomy rate and transfusion could be reduced by interventional methods. Our study aimed to investigate the influence of maternal hemodynamics control and neonatal outcomes of prophylactic temporary abdominal aortic balloon (PTAAB) occlusion for patients with pernicious placenta previa. Methods This was a retrospective study using data from the Peking University People’s Hospital from January 2014 through January 2020. Clinical records of pregnant women undergoing cesarean section were collected. Patients were divided into two groups: treatment with PTAAB placement (group A) and no balloon placement (group B). Group A was further broken down into two groups: prophylactic placement (Group C) and balloon occlusion (group D). Results Clinical records of 33 cases from 5205 pregnant women underwent cesarean section were collected. The number of groups A, B, C, and D were 17, 16, 5 and 12.We found that a significant difference in the post-operative uterine artery embolism rates between group A and group B (0% vs.31.3%, p = 0.018). There was a significant difference in the Apgar scores at first minute between group A and group B (8.94 ± 1.43 vs 9.81 ± 0.75,p = 0.037),and the same significant difference between two groups in the pre-operative central placenta previa (29.4% vs. 0%,p = 0.044), complete placenta previa (58.8% vs 18.8%, p = 0.032),placenta implantation (76.5% vs 31.3%, p = 0.015). We could also observe the significant difference in the amount of blood cell (2.80 ± 2.68vs.10.66 ± 11.97, p = 0.038) and blood plasma transfusion (280.00 ± 268.32 vs. 1033.33 ± 1098.20, p = 0.044) between group C and group D. The significant differences in the preoperative vaginal bleeding conditions (0% vs 75%, p = 0.009), the intraoperative application rates of vasopressors (0% vs. 58.3%, p = 0.044) and the postoperative ICU (intensive care unit) admission rates (0% vs. 58.3%, p = 0.044) were also kept. Conclusions PTAAB occlusion could be useful in reducing the rate of post-operative uterine artery embolism and the amount of transfusion, and be useful in coping with patients with preoperative vaginal bleeding conditions, so as to reduce the rate of intraoperative applications of vasopressors and the postoperative ICU (intensive care unit) admission. In PPP patients with placenta implantation, central placenta previa and complete placenta previa, we advocate the utilization of prophylactic temporary abdominal aortic balloon placement.

scholarly journals Biomechanical study of Tile C3 pelvic fracture fixation using an anterior internal system combined with sacroiliac screws

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Lin Liu ◽  
Donggui Zeng ◽  
Shicai Fan ◽  
Yongxing Peng ◽  
Hui Song ◽  
...  
Keyword(s):  
Minimally Invasive ◽  
Steel Plate ◽  
Standing Position ◽  
Pelvic Fractures ◽  
Internal Fixator ◽  
Sacroiliac Joints ◽  
Sacroiliac Screws ◽  
Group A ◽  
Group B ◽  
Group D

Abstract Background How to perform minimally-invasive surgery on Tile C pelvic fractures is very difficult, and it is also a hot topic in orthopedic trauma research. We applied minimally-invasive treatment using an anterior internal fixator combined with sacroiliac screws. Objectives To compare the biomechanical properties of different fixation models in pelvic facture specimens, using an internal fixation system or a steel plate combined with sacroiliac screws. Methods Sixteen fresh adult cadaver pelvic specimens were randomly separated into four groups named A, B, C, and D. The four groups were respectively stabilized using a two-screwed, three-screwed, or four-screwed anterior internal fixator or a steel plate with sacroiliac screws. All models were tested in both standing and sitting positions. Vertical loads of 600 N were applied increasingly. Shifts of bilateral sacroiliac joints and pubis rupture were measured. Results The shifts in sacroiliac joints and pubis rupture in the standing position were all less than 3.5 mm, and the shifts in the sitting position were all less than 1 mm. In the standing position, the results of shifts in the sacroiliac joints were group C < group D < group B < group A. For comparisons between A:B and C:D, P > 0.05. For comparisons between A, B:C, and D, P < 0.05. The results of shifts in pubis ruptures were group D < group C < group B < group A. In the comparison between C:D, P > 0.05; for comparisons between A:B, A:C, A:D, B:C, and B:D, P < 0.05. In the sitting posture, the results of shifts in the sacroiliac joints were group C < group D < group B < group A, and the shifts in the pubis ruptures were group D < group C < roup B < group A. For comparison between C:D, P > 0.05. For comparisons between A:B, A:C, A:D, B:C, and B:D, P < 0.05. Conclusion Use of an anterior internal fixator combined with sacroiliac screws effectively stabilized Tile C3 pelvic fractures. The stability of specimens increased as the number of screws in the internal fixator increased.

scholarly journals Hematological Features in Sheep with IgG and IgM Antibodies against Borrelia burgdorferi sensu lato

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 164
Author(s):  
Labrini V. Athanasiou ◽  
Victoria M. Spanou ◽  
Eleni G. Katsogiannou ◽  
Panagiotis D. Katsoulos
Keyword(s):  
Borrelia Burgdorferi ◽  
Rural Areas ◽  
Blood Analysis ◽  
Borrelia Burgdorferi Sensu Lato ◽  
Igm Antibodies ◽  
Group A ◽  
Group B ◽  
Group D ◽  
Immunofluorescence Antibody

Exposure of sheep to Borreliaburgdorferi sensulato (s.I.) complex, the causative agent of Lyme borreliosis (LB), has been reported in tick-abundant areas worldwide, while no data have been reported in Greece. The aim of the study was to identify the hematological alterations in sheep with seropositivity against Borrelia burgdorferi (s.I.). Blood samples were obtained from 318 tick infested sheep for blood analysis and serological determination of IgG and IgM antibodies against B. burgdorferi by indirect immunofluorescence antibody (IFA) assay after exclusion of endo-ectoparasites and other tick-borne infections. A total number of 162 sheep met the inclusion criteria, allocated in four groups based on the presence or absence of IgG and/or IgM; sheep found negative for IgM and IgG (Group A), positive for IgM (Group B), positive for both IgM and IgG (Group C) and positive for IgG (Group D). Anemia, thrombocytopenia and normal or decreased leukocyte count, mainly due to lymphopenia were the main hematological features observed in seropositive sheep. The presence of these features raises the suspicion of Borrelia infection in tick infested sheep. The seropositivity of 23.58% in sheep raises concerns of Borrelia circulation, especially in rural areas and potential risk of transmission to humans.

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