Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases.

Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2'-fucosyllactose (2'-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2'-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2'-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2'-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2'-FL production. Therefore, to decrease the formation of byproduct 2'-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2'-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154-171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154-171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2'-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2'-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2'-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions.

Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR2 E2 , with efficient native transaminase (kcat : 69.49±1.77 min-1 ) and artificial esterase (kcat : 3908-0.41 min-1 ) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR2 E2 (pHopt : 8.0-9.5; Topt : 60-65 °C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the ß-keto ester into the ß-keto acid at the hydrolytic site and subsequently into the ß-amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR2 E2 PluriZyme was proven with a set of ß-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

Impact of SARS-CoV-2 (COVID-19) pandemic on patients with lysosomal storage disorders and restoration of services: experience from a specialist centre.

This study aims to evaluate the impact of the COVID-19 pandemic on the lysosomal disorders unit (LSDU) at Royal Free London NHS Foundation Trust (RFL), a highly specialised national service for diagnosis and management of adults with lysosomal storage disorders (LSD). Review of home care enzyme replacement therapy (ERT) and emergency care, and COVID-19 shielding categories as per UK government guidance. New clinical pathways were developed to manage patients safely during the pandemic; staff well-being initiatives are described. LSDU staff were redeployed and/or had additional roles to support increased needs of hospitalised COVID-19 patients. During the first lockdown in March 2020, 286 of 602 LSD patients were shielding; 72 of 221 had home care ERT infusions interrupted up to 12 weeks. During the pandemic, there was a 3% reduction in home care nursing support required, with patients learning to self-cannulate or require support for cannulation only. There were no increased adverse clinical events during this period. Twenty-one contracted COVID-19 infection, with one hospitalised and no COVID-19 related deaths. In 2020, virtual clinics were increased by 88% (video and/or telephone) compared to 2019. RFL well-being initiatives supported all staff. We provide an overview of the impact of the COVID-19 pandemic on staff and patients attending a highly specialised rare disease service. As far as we are aware, this is the first detailed narrative on the challenges and subsequent rapid adaptations made, both as part of a large organisation and as a specialist centre. Lessons learnt could be translated to other rare disease services and ensure readiness for any future pandemic.

Mechanistic alternatives for peptide bond formation on the ribosome.

The peptidyl transfer reaction on the large ribosomal subunit depends on the protonation state of the amine nucleophile and exhibits a large kinetic solvent isotope effect (KSIE ∼8). In contrast, the related peptidyl-tRNA hydrolysis reaction involved in termination shows a KSIE of ∼4 and a pH-rate profile indicative of base catalysis. It is, however, unclear why these reactions should proceed with different mechanisms, as the experimental data suggests. One explanation is that two competing mechanisms may be operational in the peptidyl transferase center (PTC). Herein, we explored this possibility by re-examining the previously proposed proton shuttle mechanism and testing the feasibility of general base catalysis also for peptide bond formation. We employed a large cluster model of the active site and different reaction mechanisms were evaluated by density functional theory calculations. In these calculations, the proton shuttle and general base mechanisms both yield activation energies comparable to the experimental values. However, only the proton shuttle mechanism is found to be consistent with the experimentally observed pH-rate profile and the KSIE. This suggests that the PTC promotes the proton shuttle mechanism for peptide bond formation, while prohibiting general base catalysis, although the detailed mechanism by which general base catalysis is excluded remains unclear.

Origins of Enantiopreference of Mycobacterium smegmatis Acyl Transferase: A Computational Analysis.

Acyl transferase from Mycobacterium smegmatis (MsAcT) is a promising biocatalyst because it catalyzes an acyl transfer reaction in aqueous solution, thereby accepting many primary and secondary alcohols as substrates. MsAcT also exhibits high enantioselectivity for a selected number of secondary alcohols. To increase the applicability of this enzyme for the production of optically active compounds, a detailed understanding of the reaction mechanism and the factors that affect enantioselectivity is essential. Herein, quantum chemical calculations are employed to study the reactions of two secondary alcohols, 1-isopropyl propargyl alcohol and 2-hydroxy propanenitrile, for which the enzyme displays opposite enantiopreference, favoring the S enantiomer in the former case and R enantiomer in the latter. A model of the active site has been designed and for both substrates various binding modes are evaluated and the intermediates and transition states along the reaction path are then located. The calculated energy profiles agree with the experimental observations, and reproduce the selectivity outcome. Through a detailed analysis of the geometries of key transition states, insights into the origins of the enantiopreference are obtained.

Enzyme catalysis by entropy without Circe effect.

Entropic effects have often been invoked to explain the extraordinary catalytic power of enzymes. In particular, the hypothesis that enzymes can use part of the substrate-binding free energy to reduce the entropic penalty associated with the subsequent chemical transformation has been very influential. The enzymatic reaction of cytidine deaminase appears to be a distinct example. Here, substrate binding is associated with a significant entropy loss that closely matches the activation entropy penalty for the uncatalyzed reaction in water, whereas the activation entropy for the rate-limiting catalytic step in the enzyme is close to zero. Herein, we report extensive computer simulations of the cytidine deaminase reaction and its temperature dependence. The energetics of the catalytic reaction is first evaluated by density functional theory calculations. These results are then used to parametrize an empirical valence bond description of the reaction, which allows efficient sampling by molecular dynamics simulations and computation of Arrhenius plots. The thermodynamic activation parameters calculated by this approach are in excellent agreement with experimental data and indeed show an activation entropy close to zero for the rate-limiting transition state. However, the origin of this effect is a change of reaction mechanism compared the uncatalyzed reaction. The enzyme operates by hydroxide ion attack, which is intrinsically associated with a favorable activation entropy. Hence, this has little to do with utilization of binding free energy to pay the entropic penalty but rather reflects how a preorganized active site can stabilize a reaction path that is not operational in solution.

Catalytic Adaptation of Psychrophilic Elastase.

The class I pancreatic elastase from Atlantic salmon is considered to be a cold-adapted enzyme in view of the cold habitat, the reduced thermostability of the enzyme, and the fact that it is faster than its mesophilic porcine counterpart at room temperature. However, no experimental characterization of its catalytic properties at lower temperatures has actually been reported. Here we use extensive computer simulations of its catalytic reaction, at different temperatures and with different peptide substrates, to compare its characteristics with those of porcine pancreatic elastase, with which it shares 67% sequence identity. We find that both enzymes have a preference for smaller aliphatic residues at the P1 position, while the reaction rate with phenylalanine at P1 is predicted to be substantially lower. With the former class of substrates, the calculated reaction rates for salmon enzyme are consistently higher than those of the porcine ortholog at all temperatures examined, and the difference is most pronounced at the lowest temperature. As observed for other cold-adapted enzymes, this is caused by redistribution of the activation free energy in terms of enthalpy and entropy and can be linked to differences in the mobility of surface-exposed loops in the two enzymes. Such mobility changes are found to be reflected by characteristic sequence conservation patterns in psychrophilic and mesophilic species. Hence, calculations of mutations in a single surface loop show that the temperature dependence of the catalytic reaction is altered in a predictable way.

Entropy and Enzyme Catalysis.

The role played by entropy for the enormous rate enhancement achieved by enzymes has been debated for many decades. There are, for example, several confirmed cases where the activation free energy is reduced by around 10 kcal/mol due to entropic effects, corresponding to a rate enhancement of ∼107 compared to the uncatalyzed reaction. However, despite substantial efforts from both the experimental and theoretical side, no real consensus has been reached regarding the origin of such large entropic contributions to enzyme catalysis. Another remarkable instance of entropic effects is found in enzymes that are adapted by evolution to work at low temperatures, near the freezing point of water. These cold-adapted enzymes invariably show a more negative entropy and a lower enthalpy of activation than their mesophilic orthologs, which counteracts the exponential damping of reaction rates at lower temperature. The structural origin of this universal phenomenon has, however, remained elusive. The basic problem with connecting macroscopic thermodynamic quantities, such as activation entropy and enthalpy derived from Arrhenius plots, to the 3D protein structure is that the underlying detailed (microscopic) energetics is essentially inaccessible to experiment. Moreover, attempts to calculate entropy contributions by computer simulations have mostly focused only on substrate entropies, which do not provide the full picture. We have recently devised a new approach for accessing thermodynamic activation parameters of both enzyme and solution reactions from computer simulations, which turns out to be very successful. This method is analogous to the experimental Arrhenius plots and directly evaluates the temperature dependence of calculated reaction free energy profiles. Hence, by extensive molecular dynamics simulations and calculations of up to thousands of independent free energy profiles, we are able to extract activation parameters with sufficient precision for making direct comparisons to experiment. We show here that the agreement with the measured quantities, for both enzyme catalyzed and spontaneous solution reactions, is quite remarkable. Importantly, we can now address some of the most spectacular entropy effects in enzymes and clarify their detailed microscopic origin. Herein, we discuss as examples the conversion of cytidine to uridine catalyzed by cytidine deaminase and reactions taking place on the ribosome, namely, peptide bond formation and GTP hydrolysis by elongation factor Tu. It turns out that the large entropy contributions to catalysis in these cases can now be rationalized by our computational approach. Finally, we address the problem of cold adaptation of enzyme reaction rates and prove by computational experiments that the universal activation enthalpy-entropy phenomenon originates from mechanical properties of the outer protein surface.

Origin of the Enigmatic Stepwise Tight-Binding Inhibition of Cyclooxygenase-1.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain, fever, inflammation, and some types of cancers. Their mechanism of action is the inhibition of isoforms 1 and 2 of the enzyme cyclooxygenase (COX-1 and COX-2, respectively). However, both nonselective and selective NSAIDs may have side effects that include gastric intestinal bleeding, peptic ulcer formation, kidney problems, and occurrences of myocardial infarction. The search for selective high-affinity COX inhibitors resulted in a number of compounds characterized by a slow, tight-binding inhibition that occurs in a two-step manner. It has been suggested that the final, only very slowly reversible, tight-binding event is the result of conformational changes in the enzyme. However, the nature of these conformational changes has remained elusive. Here we explore the structural determinants of the tight-binding phenomenon in COX-1 with molecular dynamics and free energy simulations. The calculations reveal how different classes of inhibitors affect the equilibrium between two conformational substates of the enzyme in distinctly different ways. The class of tight-binding inhibitors is found to exclusively stabilize an otherwise unfavorable enzyme conformation and bind significantly stronger to this state than to that normally observed in crystal structures. By also computing free energies of binding to the two enzyme conformations for 16 different NSAIDs, we identify an induced-fit mechanism and the key structural features associated with high-affinity tight binding. These results may facilitate the rational development of new COX inhibitors with improved selectivity profiles.

AsiteDesign: a Semirational Algorithm for an Automated Enzyme Design.

With advances in protein structure predictions, the number of available high-quality structures has increased dramatically. In light of these advances, structure-based enzyme engineering is expected to become increasingly important for optimizing biocatalysts for industrial processes. Here, we present AsiteDesign, a Monte Carlo-based protocol for structure-based engineering of active sites. AsiteDesign provides a framework for introducing new catalytic residues in a given binding pocket to either create a new catalytic activity or alter the existing one. AsiteDesign is implemented using pyRosetta and incorporates enhanced sampling techniques to efficiently explore the search space. The protocol was tested by designing an alternative catalytic triad in the active site of Pseudomonas fluorescens esterase (PFE). The designed variant was experimentally verified to be active, demonstrating that AsiteDesign can find alternative catalytic triads. Additionally, the AsiteDesign protocol was employed to enhance the hydrolysis of a bulky chiral substrate (1-phenyl-2-pentyl acetate) by PFE. The experimental verification of the designed variants demonstrated that F158L/F198A and F125A/F158L mutations increased the hydrolysis of 1-phenyl-2-pentyl acetate from 8.9 to 66.7 and 23.4%, respectively, and reversed the enantioselectivity of the enzyme from (R) to (S)-enantiopreference, with 32 and 55% enantiomeric excess (ee), respectively.

Structural-Based Modeling in Protein Engineering. A Must Do.

Biotechnological solutions will be a key aspect in our immediate future society, where optimized enzymatic processes through enzyme engineering might be an important solution for waste transformation, clean energy production, biodegradable materials, and green chemistry, for example. Here we advocate the importance of structural-based bioinformatics and molecular modeling tools in such developments. We summarize our recent experiences indicating a great prediction/success ratio, and we suggest that an early in silico phase should be performed in enzyme engineering studies. Moreover, we demonstrate the potential of a new technique combining Rosetta and PELE, which could provide a faster and more automated procedure, an essential aspect for a broader use.

The relationship between Helicobacter pylori infection and gastric adenocarcinoma in Northern Iran.

Colonization of the human stomach with Helicobacter pylori induces chronic gastritis and is associated with the development of gastric and duodenal ulcers, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Infection with an H. pylori strain containing the cytotoxin-associated (cagA) gene (a marker for a pathogenicity island) may increase the risk of atrophic gastritis and gastric cancer. The exact role of H. pylori in gastric carcinogenesis is still being investigated. Hence, we assessed whether H. pylori infection is associated with the development of gastric adenocarcinoma in northern Iran. Gastric biopsy specimens from 168 patients suffering from gastric adenocarcinoma, gastric ulcer, and non-ulcer dyspepsia were analyzed by means of the polymerase chain reaction. H. pylori was detected in the gastric mucosa of 34 (75.5%) gastric adenocarcinoma, 56 (88.8%) gastric ulcer, and 36 (60%) non-ulcer dyspepsia. In patients with gastric adenocarcinoma, the cagA was less commonly found than those in noncancer patients (4/34 vs. 58/92, p < 0.05). Our work suggests that although H. pylori infection is significantly associated with gastric adenocarcinoma in northern Iran, the cagA is not the dominant virulence in development of gastric adenocarcinoma.

Transoral Laser-Assisted Total Laryngectomy: Expanding the TLM's World.

INTRODUCTION: The introduction of laryngeal transoral procedures has created a shift in the treatment of laryngeal cancers towards the primary surgical management of patients. In this study, we aimed to evaluate the safety, efficacy, and feasibility of the transoral laser-assisted total laryngectomy (TLM-TL) in advanced laryngeal cancer. Case presentation. In this case report, we describe a case of a 50-year-old male patient presented to the otorhinolaryngology clinic with a history of hoarseness and odynophagia since 6 months. Based on the pathological and imaging findings, the diagnosis of stage IVa laryngeal squamous cell carcinoma with the involvement of the base, tongue, and left palatine tonsil was made for the patient, and transoral total laryngectomy with partial glossectomy via the TLM technique was planned. RESULT: The tumor was successfully resected by TLM-TL with clear surgical margins. No complication was observed after the surgery. Good functional recovery was obtained regarding swallowing and speech. The patient's oncologic and functional outcomes were evaluated for 2 years. Everything was satisfactory with good long-term cosmetic and laryngopharyngeal functional outcome and no sign of tumor recurrence. CONCLUSIONS: TLM-TL is a minimally invasive and cost-benefit endoscopic surgical procedure feasible in advanced laryngeal cancer with good long-term oncological and functional outcome. It could limit postoperative complications, mainly the incidence of pharyngocutaneous fistulae. It is also associated with better satisfaction after TL due to cosmetic benefits.

Mechanism of Biocatalytic Friedel-Crafts Acylation by Acyltransferase from Pseudomonas protegens.

Acyltransferases isolated from Pseudomonas protegens (PpATase) and Pseudomonas fluorescens (PfATase) have recently been reported to catalyze the Friedel-Crafts acylation, providing a biological version of this classical organic reaction. These enzymes catalyze the cofactor-independent acylation of monoacetylphloroglucinol (MAPG) to diacetylphloroglucinol (DAPG) and phloroglucinol (PG) and have been demonstrated to have a wide substrate scope, making them valuable for potential applications in biocatalysis. Herein, we present a detailed reaction mechanism of PpATase on the basis of quantum chemical calculations, employing a large model of the active site. The proposed mechanism is consistent with available kinetics, mutagenesis, and structural data. The roles of various active site residues are analyzed. Very importantly, the Asp137 residue, located more than 10 Å from the substrate, is predicted to be the proton source for the protonation of the substrate in the rate-determining step. This key prediction is corroborated by site-directed mutagenesis experiments. Based on the current calculations, the regioselectivity of PpATase and its specificity toward non-natural substrates can be rationalized.

A New Approach to Survival Analysis of Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Squamous cell carcinoma is the most common histological subtype of head and neck cancers. METHODS: In a retrospective longitudinal study, we assessed the risk of local or metastatic recurrence and death in 140 patients with head and neck squamous cell carcinoma (HNSCC). Multivariate and shared frailty models were used for survival analysis with sex, primary tumor site, grade and stage of the tumor, and treatment modalities as contributing factors. RESULTS: The most frequent site for HNSCC was the oral cavity (30%), followed by the tongue (26.4%). For most primary sites, men were at nearly 2-fold higher risk of local recurrence than women, but there was no difference by sex in the risk of metastatic recurrence. Undifferentiated HNSCC was associated with a higher risk of local recurrence (nearly 4-fold) and metastasis (6-15-fold based on the primary site) than well-differentiated tumors. In early months after surgical resection alone, the risk of local recurrence was higher compared to other treatment modalities. There was a strong dependency between the risk of local and metastatic recurrence. CONCLUSION: In conclusion, men diagnosed with HNSCC, those with higher grade or advanced state tumor, and those treated by surgery alone are at higher risk of unfavorable outcomes than others and may need more frequent follow-up visits.

Chemical reaction mechanisms in solution from brute force computational Arrhenius plots.

Decomposition of activation free energies of chemical reactions, into enthalpic and entropic components, can provide invaluable signatures of mechanistic pathways both in solution and in enzymes. Owing to the large number of degrees of freedom involved in such condensed-phase reactions, the extensive configurational sampling needed for reliable entropy estimates is still beyond the scope of quantum chemical calculations. Here we show, for the hydrolytic deamination of cytidine and dihydrocytidine in water, how direct computer simulations of the temperature dependence of free energy profiles can be used to extract very accurate thermodynamic activation parameters. The simulations are based on empirical valence bond models, and we demonstrate that the energetics obtained is insensitive to whether these are calibrated by quantum mechanical calculations or experimental data. The thermodynamic activation parameters are in remarkable agreement with experiment results and allow discrimination among alternative mechanisms, as well as rationalization of their different activation enthalpies and entropies.

Antimalarial Effects of Iranian Flora Artemisia sieberi on Plasmodium berghei In Vivo in Mice and Phytochemistry Analysis of Its Herbal Extracts.

The aim of this study is pharmacochemistry of Iranian flora Artemisia sieberi and its antimalarial effects on Plasmodium berghei in vivo. This is the first application of A. sieberi for treatment of murine malaria. A. sieberi were collected at flowering stage from the Khorassan and Semnan provinces of Iran; the aerial parts were air-dried at room temperature and then powdered. The powder was macerated in methanol, filtered with Bokhner hopper and solvent was separated in rotary evaporator. Total herbal extract was subsequently processed for ether and chloroform extracts preparation. The toxicity of herbal extract was assessed on naive NMRI mice with high, average and low doses; then pathophysiological signs were assessed. Finally, the antimalarial efficacy was investigated on two groups of Plasmodium berghei infected mice. Percentage of parasitaemia and pathophysiology were also evaluated. The results of this assessment showed no toxicity even by high concentration of herbal extract. A significant reduction in percentage of parasitaemia was observed; no alterations of hepatosplenomegaly and body weight were indicated in study group. A. sieberi extracts showed antimalarial effects against murine malaria with some efficacies on reducing pathophysiology. However, there is requirement to find the major component of this herbal extract by further studies.

Composition and antimicrobial activity of the essential oil of Artemisia kulbadica from Iran.

The hydrodistilled volatile oil from the aerial parts of Artemisia kulbadica Boiss. & Buhse was investigated by a combination of GC and GC/MS. Twenty-seven compounds were identified, representing 92.9% of the total oil. Sabinene (25.1%), trans-thujone (18.7%) and gamma-cadinene (16.0%) were the main components. The antimicrobial activity was determined against six bacterial strains and one fungal. The oil was active against all the tested strains.