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1.
A cascade reaction for the synthesis of d-fagomine precursor revisited: Kinetic insight and understanding of the system.
Sudar, M, Česnik, M, Clapés, P, Pohl, M, Vasić-Rački, Đ, Findrik Blažević, Z
New biotechnology. 2021;:19-28
Abstract
The synthesis of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of the interesting dietary supplement, iminosugar d-fagomine, was studied in a cascade reaction with three enzymes starting from Cbz-N-3-aminopropanol. This system was studied previously using a statistical optimization method which enabled a 79 % yield of the aldol adduct with a 10 % yield of the undesired amino acid by-product. Here, a kinetic model of the cascade, including enzyme operational stability decay rate and the undesired overoxidation of the intermediate product, was developed. The validated model was instrumental in the optimization of the cascade reaction in the batch reactor. Simulations were carried out to determine the variables with the most significant impact on substrate conversion and product yield. As a result, process conditions were found that provided the aldol adduct in 92 % yield with only 0.7 % yield of the amino acid in a one-pot one-step reaction. Additionally, compared to previous work, this improved process outcome was achieved at lower concentrations of two enzymes used in the reaction. With this study the advantages are demonstrated of a modelling approach in developing complex biocatalytical processes. Mathematical models enable better understanding of the interactions of variables in the investigated system, reduce cost, experimental efforts in the lab and time necessary to obtain results since the simulations are carried out in silico.
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2.
HSD3B1 variant and androgen-deprivation therapy outcome in prostate cancer.
Han, FF, Ren, LL, Xuan, LL, Lv, YL, Liu, H, Gong, LL, An, ZL, Liu, LH
Cancer chemotherapy and pharmacology. 2021;(1):103-112
Abstract
OBJECTIVE Rate-limiting enzyme 3b-hydroxysteroid dehydrogenase type 1 (3βHSD1) encoded by HSD3B1 catalyzes the transition of dehydroepiandrosterone (DHEA) to dihydrotestosterone (DHT). The HSD3B1 (1245C) variant renders 3bHSD1 of resistant to ubiquitination and degradation, leading to a large amount of protein accumulation in the cell. Multiple clinical studies have shown that this mutation was correlated with resistance to androgen-deprivation therapy in prostate cancer. However, the results were not consistent depending on different treatment strategy and in some researches, the number of observed cases was relatively small. METHODS To determine the effects of HSD3B1 (1245C) variant on resistance to androgen-deprivation therapy in prostate cancer, we performed a meta-analysis of the available literature. Electronic database searches identified appropriately designed studies that detected HSD3B1 in prostate cancer. We conducted a systematic search of studies in the following databases: PubMed, and EMBASE published until August 10, 2020 using the following search terms: (HSD3B1 AND ((((prostate cancer) OR prostatic neoplasm) OR prostatic carcinoma) OR prostatic cancer). RESULTS Eight researches were included in this research. The result validated that the HSD3B1 (1245C) variant allele was associated with a shorter PFS (HR, 1.97; 95% CI, 1.39-2.79; P = 0.0001) (homozygous wild-type group) in men with prostate cancer when treated with ADT, however, a higher PFS (HR, 0.68; 95% CI, 0.48-0.96; P = 0.03) when treated with ADT and CYP17A1 inhibitor. CONCLUSION The HSD3B1 (1245C) variant is a predictor of ADT plus CYP17A1 inhibitor response in prostate cancer.
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3.
Discovery and investigation of natural Diels-Alderases.
Watanabe, K
Journal of natural medicines. 2021;(3):434-447
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Abstract
It has been proposed that biosyntheses of many natural products involve pericyclic reactions, including Diels-Alder (DA) reaction. However, only a small set of enzymes have been proposed to catalyze pericyclic reactions. Most surprisingly, there has been no formal identification of natural enzymes that can be defined to catalyze DA reactions (DAases), despite the wide application of the reaction in chemical syntheses of complex organic compounds. However, recent studies began to accumulate a growing body of evidence that supports the notion that enzymes that formally catalyze DA reactions, in fact exist. In this review, I will begin by describing a short history behind the discovery and characterization of macrophomate synthase, one of the earliest enzymes that was proposed to catalyze an intermolecular DA reaction during the biosynthesis of a substituted benzoic acid in a phytopathogenic fungus Macrophoma commelinae. Then, I will discuss representative enzymes that have been chemically authenticated to catalyze DA reactions, with emphasis on more recent discoveries of DAases involved mainly in fungal secondary metabolite biosynthesis except for one example from a marine streptomycete. The current success in identification of a series of DAases and enzymes that catalyze other pericyclic reactions owes to the combined efforts from both the experimental and theoretical approaches in discovering natural products. Such efforts typically involve identifying the chemical features derived from cycloaddition reactions, isolating the biosynthetic genes that encode enzymes that generate such chemical features and deciphering the reaction mechanisms for the enzyme-catalyzed pericyclic reactions.
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Germline variants and response to systemic therapy in advanced prostate cancer.
Johnson, E, Nussenzveig, R, Agarwal, N, Swami, U
Pharmacogenomics. 2020;(1):75-81
Abstract
Our current understanding of prostate cancer pharmacogenomics is growing at a rapid pace. Apart from evaluating relevant biomarkers and genomic alterations in tumor tissues, an increasing focus is being placed on decoding the impact of germline alterations on prostate cancer and its treatment. Herein we summarize various germline variants that have shown to associate with response to systemic therapy in men with advanced prostate cancer. Covered biomarkers include HSD3B1, SLCO2B1, SULT1E1, TRMT11, CYP17A1, CYP1B1, genes involved in homologous recombination and DNA mismatch repair.
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HSD3B1 Genotype and Clinical Outcomes in Metastatic Castration-Sensitive Prostate Cancer.
Hearn, JWD, Sweeney, CJ, Almassi, N, Reichard, CA, Reddy, CA, Li, H, Hobbs, B, Jarrard, DF, Chen, YH, Dreicer, R, et al
JAMA oncology. 2020;(4):e196496
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Abstract
IMPORTANCE The adrenal-restrictive HSD3B1(1245A) allele limits extragonadal dihydrotestosterone synthesis, whereas the adrenal-permissive HSD3B1(1245C) allele augments extragonadal dihydrotestosterone synthesis. Retrospective studies have suggested an association between the adrenal-permissive allele, the frequency of which is highest in white men, and early development of castration-resistant prostate cancer (CRPC). OBJECTIVE To examine the association between the adrenal-permissive HSD3B1(1245C) allele and early development of CRPC using prospective data. DESIGN, SETTING, AND PARTICIPANTS The E3805 Chemohormonal Therapy vs Androgen Ablation Randomized Trial for Extensive Disease in Prostate Cancer (CHAARTED) was a large, multicenter, phase 3 trial of castration with or without docetaxel treatment in men with newly diagnosed metastatic prostate cancer. From July 28, 2006, through December 31, 2012, 790 patients underwent randomization, of whom 527 had available DNA samples. In this study, the HSD3B1 germline genotype was retrospectively determined in 475 white men treated in E3805 CHAARTED, and clinical outcomes were analyzed by genotype. Data analysis was performed from July 28, 2006, to October 17, 2018. INTERVENTIONS Men were randomized to castration plus docetaxel, 75 mg/m2, every 3 weeks for 6 cycles or castration alone. MAIN OUTCOMES AND MEASURES Two-year freedom from CRPC and 5-year overall survival, with results stratified by disease volume. Patients were combined across study arms according to genotype to assess the overall outcome associated with genotype. Secondary analyses by treatment arm evaluated whether the docetaxel outcome varied with genotype. RESULTS Of 475 white men with DNA samples, 270 patients (56.8%) inherited the adrenal-permissive genotype (≥1 HSD3B1[1245C] allele). Mean (SD) age was 63 (8.7) years. Freedom from CRPC at 2 years was diminished in men with low-volume disease with the adrenal-permissive vs adrenal-restrictive genotype: 51.0% (95% CI, 40.9%-61.2%) vs 70.5% (95% CI, 60.0%-80.9%) (P = .01). Overall survival at 5 years was also worse in men with low-volume disease with the adrenal-permissive genotype: 57.5% (95% CI, 47.4%-67.7%) vs 70.8% (95% CI, 60.3%-81.3%) (P = .03). Hazard ratios were 1.89 (95% CI, 1.13-3.14; P = .02) for CRPC and 1.74 (95% CI, 1.01-3.00; P = .045) for death. There was no association between genotype and outcomes in men with high-volume disease. There was no interaction between genotype and benefit from docetaxel. CONCLUSIONS AND RELEVANCE Inheritance of the adrenal-permissive HSD3B1 genotype is associated with earlier castration resistance and shorter overall survival in men with low-volume metastatic prostate cancer and may help identify men more likely to benefit from escalated androgen receptor axis inhibition beyond gonadal testosterone suppression.
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Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle.
Klein, WP, Thomsen, RP, Turner, KB, Walper, SA, Vranish, J, Kjems, J, Ancona, MG, Medintz, IL
ACS nano. 2019;(12):13677-13689
Abstract
Developing reliable methods of constructing cell-free multienzyme biocatalytic systems is a milestone goal of synthetic biology. It would enable overcoming the limitations of current cell-based systems, which suffer from the presence of competing pathways, toxicity, and inefficient access to extracellular reactants and removal of products. DNA nanostructures have been suggested as ideal scaffolds for assembling sequential enzymatic cascades in close enough proximity to potentially allow for exploiting of channeling effects; however, initial demonstrations have provided somewhat contradictory results toward confirming this phenomenon. In this work, a three-enzyme sequential cascade was realized by site-specifically immobilizing DNA-conjugated amylase, maltase, and glucokinase on a self-assembled DNA origami triangle. The kinetics of seven different enzyme configurations were evaluated experimentally and compared to simulations of optimized activity. A 30-fold increase in the pathway's kinetic activity was observed for enzymes assembled to the DNA. Detailed kinetic analysis suggests that this catalytic enhancement originated from increased enzyme stability and a localized DNA surface affinity or hydration layer effect and not from a directed enzyme-to-enzyme channeling mechanism. Nevertheless, the approach used to construct this pathway still shows promise toward improving other more elaborate multienzymatic cascades and could potentially allow for the custom synthesis of complex (bio)molecules that cannot be realized with conventional organic chemistry approaches.
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Why is glycine cleavage system segmentally expressed in radial glia?
Sato, K
Journal of theoretical biology. 2019;:17-19
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Steered molecular dynamics simulations reveal the role of Ca2+ in regulating mechanostability of cellulose-binding proteins.
Gunnoo, M, Cazade, PA, Orlowski, A, Chwastyk, M, Liu, H, Ta, DT, Cieplak, M, Nash, M, Thompson, D
Physical chemistry chemical physics : PCCP. 2018;(35):22674-22680
Abstract
The conversion of cellulosic biomass into biofuels requires degradation of the biomass into fermentable sugars. The most efficient natural cellulase system for carrying out this conversion is an extracellular multi-enzymatic complex named the cellulosome. In addition to temperature and pH stability, mechanical stability is important for functioning of cellulosome domains, and experimental techniques such as Single Molecule Force Spectroscopy (SMFS) have been used to measure the mechanical strength of several cellulosomal proteins. Molecular dynamics computer simulations provide complementary atomic-resolution quantitative maps of domain mechanical stability for identification of experimental leads for protein stabilization. In this study, we used multi-scale steered molecular dynamics computer simulations, benchmarked against new SMFS measurements, to measure the intermolecular contacts that confer high mechanical stability to a family 3 Carbohydrate Binding Module protein (CBM3) derived from the archetypal Clostridium thermocellum cellulosome. Our data predicts that electrostatic interactions in the calcium binding pocket modulate the mechanostability of the cellulose-binding module, which provides an additional design rule for the rational re-engineering of designer cellulosomes for biotechnology. Our data offers new molecular insights into the origins of mechanostability in cellulose binding domains and gives leads for synthesis of more robust cellulose-binding protein modules. On the other hand, simulations predict that insertion of a flexible strand can promote alternative unfolding pathways and dramatically reduce the mechanostability of the carbohydrate binding module, which gives routes to rational design of tailormade fingerprint complexes for force spectroscopy experiments.
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HSD3B1 and Response to a Nonsteroidal CYP17A1 Inhibitor in Castration-Resistant Prostate Cancer.
Almassi, N, Reichard, C, Li, J, Russell, C, Perry, J, Ryan, CJ, Friedlander, T, Sharifi, N
JAMA oncology. 2018;(4):554-557
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Abstract
IMPORTANCE The HSD3B1 (1245C) germline variant encodes for a gain-of-function missense in 3β-hydroxysteroid dehydrogenase isoenzyme 1 (3βHSD1) that results in increased dihydrotestosterone synthesis from extragonadal precursors and is predictive of more rapid progression to castration-resistant prostate cancer (CRPC). OBJECTIVE To determine whether the HSD3B1 (1245C) genotype is predictive of clinical response to extragonadal androgen ablation with nonsteroidal 17α-hydroxylase/17,20-lyase (CYP17A1) inhibition in men with metastatic CRPC. DESIGN, SETTING, AND PARTICIPANTS An observational study of men with metastatic CRPC treated with ketoconazole between June 1998 and December 2012 was conducted at the University of California, San Francisco. EXPOSURES Extragonadal androgen ablation with the nonsteroidal CYP17A1 inhibitor ketoconazole among men with metastatic CRPC. MAIN OUTCOMES AND MEASURES The primary end points of analysis were duration of ketoconazole therapy and time to disease progression stratified by HSD3B1 genotype. Disease progression was defined as either biochemical or radiographic progression, using the Prostate Cancer Working Group 3 and Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 definitions, respectively. Kaplan-Meier analysis was used to estimate time on therapy and time to disease progression. A log-rank test for trend was used to compare outcomes by HSD3B1 genotype. RESULTS A total of 90 men (median [interquartile range] age, 61.5 [55.3-67.0] years) with metastatic CRPC were included in the analysis, with sufficient data to determine duration of ketoconazole therapy and time to disease progression in 88 and 81 patients, respectively. The median duration of therapy increased with the number of inherited HSD3B1 (1245C) variant alleles: 5.0 months (95% CI, 3.4-10.4) for 0 variant alleles; 7.5 months (95% CI, 4.9-19.2) for 1; and 12.3 months (95% CI, 1.8-not reached) for 2 (overall comparison for trend, P = .01). Median progression-free survival also increased with number of HSD3B1 (1245C) variant alleles inherited: 5.4 months (95% CI, 3.7-7.5) for 0 variant alleles; 9.7 months (95% CI, 5.6-32.9) for 1; and 15.2 months (95% CI, 7.8-not reached) for 2 (overall comparison for trend, P = .03). CONCLUSIONS AND RELEVANCE Inheritance of the HSD3B1 (1245C) variant allele, which is a predictive biomarker of resistance to castration, is also a predictive biomarker of sensitivity to extragonadal androgen ablation with a nonsteroidal CYP17A1 inhibitor. These findings signal a possible pathway of treatment stratification for patients with prostate cancer.
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Widespread distribution of encapsulin nanocompartments reveals functional diversity.
Giessen, TW, Silver, PA
Nature microbiology. 2017;:17029
Abstract
Cells organize and regulate their metabolism via membrane- or protein-bound organelles. In this way, incompatible processes can be spatially separated and controlled. In prokaryotes, protein-based compartments are used to sequester harmful reactions and store useful compounds. These protein compartments play key roles in various metabolic and ecological processes, ranging from iron homeostasis to carbon fixation. One of the newest types of protein organelle are encapsulin nanocompartments. They are able to encapsulate specific protein cargo and are proposed to be involved in redox-related processes. We identified more than 900 putative encapsulin systems in bacterial and archaeal genomes. Encapsulins can be found in fifteen bacterial and two archaeal phyla. Our analysis reveals one new capsid type and nine previously unknown cargo proteins targeted to the interior of encapsulins. We experimentally characterize three newly identified encapsulin systems and illustrate their probable involvement in iron mineralization, oxidative and nitrosative stress resistance and anaerobic ammonium oxidation, a process responsible for 30% of the nitrogen lost from the oceans.