Therapeutic enzymes present excellent opportunities for the treatment of human disease, modulation of metabolic pathways and system detoxification. However, current use of enzyme therapy in the ...clinic is limited as naturally occurring enzymes are seldom optimal for such applications and require substantial improvement by protein engineering. Engineering strategies such as design and directed evolution that have been successfully implemented for industrial biocatalysis can significantly advance the field of therapeutic enzymes, leading to biocatalysts with new‐to‐nature therapeutic activities, high selectivity, and suitability for medical applications. This minireview highlights case studies of how state‐of‐the‐art and emerging methods in protein engineering are explored for the generation of therapeutic enzymes and discusses gaps and future opportunities in the field of enzyme therapy.
Therapeutic enzymes have immense potential for treating human diseases, but their clinical application is hindered by the need for substantial improvement through protein engineering. Advanced techniques like design and directed evolution can enhance therapeutic enzymes, leading to biocatalysts with new therapeutic activities and high selectivity.
Fluorinated nucleoside analogues have attracted much attention as anticancer and antiviral agents and as probes for enzymatic function. However, the lack of direct synthetic methods, especially for ...2',3'-dideoxy-2',3'-difluoro nucleosides, hamper their practical utility. In order to design more efficient synthetic methods, a better understanding of the conformation and mechanism of formation of these molecules is important. Herein, we report the synthesis and conformational analysis of a 2',3'-dideoxy-2',3'-difluoro and a 2'-deoxy-2'-fluoro uridine derivative and provide an insight into the reaction mechanism. We suggest that the transformation most likely diverges from the S
1 or S
2 pathway, but instead operates via a neighbouring-group participation mechanism.
Enzyme-mediated methylation is a very important reaction in nature, yielding a wide range of modified natural products, diversifying small molecules and fine-tuning the activity of biomacromolecules. ...The field has attracted much attention over the recent years and interesting applications of the dedicated enzymes in biocatalysis and biomolecular labelling have emerged. In this review article, we summarise the concepts and recent advances in developing (chemo)-enzymatic cascades for selective methylation, alkylation and photocaging as tools to study biological methylation and as biotransformations to generate site-specifically alkylated products.
This review summarizes the recent findings and developments in the emerging area of (chemo)-enzymatic cascades using MTases and AdoMet-generating enzymes.
Therapeutic enzymes present excellent opportunities for the treatment of human disease, modulation of metabolic pathways and system detoxification. However, current use of enzyme therapy in the ...clinic is limited as naturally occurring enzymes are seldom optimal for such applications and require substantial improvement by protein engineering. Engineering strategies such as design and directed evolution that have been successfully implemented for industrial biocatalysis can significantly advance the field of therapeutic enzymes, leading to biocatalysts with new‐to‐nature therapeutic activities, high selectivity, and suitability for medical applications. This minireview highlights case studies of how state‐of‐the‐art and emerging methods in protein engineering are explored for the generation of therapeutic enzymes and discusses gaps and future opportunities in the field of enzyme therapy.
Therapeutic enzymes have immense potential for treating human diseases, but their clinical application is hindered by the need for substantial improvement through protein engineering. Advanced techniques like design and directed evolution can enhance therapeutic enzymes, leading to biocatalysts with new therapeutic activities and high selectivity.
Given the worldwide popularity of hair dyeing, there is an urgent need to understand the toxicities and risks associated with exposure to chemicals found in hair dye formulations. Hair dyes are ...categorized as oxidative and nonoxidative in terms of their chemical composition and ingredients. For several decades, the expert panel’s Cosmetic Ingredient Review (CIR) has assessed the safety of many of the chemicals used in hair dyes; however, a comprehensive review of hair dye ingredients and the risk of exposure to hair dyeing has not been documented. Herein, we review the safety of the various chemicals in oxidative and nonoxidative hair dyes, toxicities associated with hair dyeing, and the carcinogenic risks related to hair dyeing. While many compounds are considered safe for users at the concentrations in hair dyes, there are conflicting data about a large number of hair dye formulations. The CIR expert panel has ratified a number of coloring ingredients for hair dyes and banned a series of chemicals as carcinogenic to animals and unsafe for this application. The use of these chemicals as raw materials for producing hair dyes may result in the synthesis of other contaminants with potential toxicities and increased risk of carcinogenesis. It is an open question whether personal or occupational hair dyeing increases the risk of cancer; however, in specific subpopulations, a positive association between hair dye use and cancer occurrence has been reported. To address this question, a better understanding of the chemical and mechanistic basis of the reported toxicities of hair dye mixtures and individual hair dye ingredients is needed. It is anticipated that in-depth chemical and systems toxicology studies harnessing modern and emerging techniques can shed light on this public health concern in the future.
•Biotechnology-based fragrances could improve the sustainability of fragrances.•Study extents insights from prior study on naturalness and fragrances.•Does not support positive effect of natural ...ingredients claim.•Participants were open to biotechnology-based fragrances.
With growing global environmental concerns, consumer acceptance of new technologies or innovation-derived products can be tied strongly with how natural the latter are perceived to be. A prior study found that fragrances were evaluated more positively if participants were informed that the fragrances were of natural origin. This led the authors to conclude that the perceived naturalness of a fragrance activates a positive effect, similar as for other consumer products. However, obtaining fragrant molecules from natural sources can pose a strain on the environment and biotechnology-based fragrances could potentially improve the sustainability of the perfumery industry. The primary goal of this study was to extent the findings of this initial study with a focus on biocatalysis-based fragrances. For this, a laboratory experiment was conducted with a 3x2x2 mixed design: participants smelled either three fragrances of natural origin or three biocatalysis-based fragrances and were either informed or not informed about the origin of the fragrances. In contrast to expectations, no consistent positive effect of natural origin or negative effect of biocatalysis-based origin of fragrances was found in our study. The results suggest that the participants were open to novel production methods in the fragrance industry. The discussion comprises a methodological comparison to the prior study, alternative explanations for these findings, and discusses insights for the fragrance industry and future research. Further research is needed to fully understand the complex factors that shape consumer behaviour in the fragrance industry.
Methyltransferases (MTases) modify a wide range of biomolecules using
S
-adenosyl-
l
-methionine (AdoMet) as the cosubstrate. Synthetic AdoMet analogues are powerful tools to site-specifically ...introduce a variety of functional groups and exhibit potential to be converted only by distinct MTases. Extending the size of the substituent at the sulfur/selenium atom provides selectivity among MTases but is insufficient to discriminate between promiscuous MTases. We present a panel of AdoMet analogues differing in the nucleoside moiety (NM-AdoMets). These NM-AdoMets were efficiently produced by a previously uncharacterized methionine adenosyltransferase (MAT) from methionine and ATP analogues, such as ITP and
N
6
-propargyl-ATP. The
N
6
-modification changed the relative activity of three representative MTases up to 13-fold resulting in discrimination of substrates for the methyl transfer and could also be combined with transfer of allyl and propargyl groups.
Methyltransferases modify a wide range of biomolecules using
S
-adenosyl-
l
-methionine (AdoMet) as cosubstrate. Enzymatic generation of nucleoside-modified AdoMet analogues and conversion by different methyltransferases is shown.
Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site‐specific ...inhibition and timed removal. S‐Adenosyl‐L‐methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC‐ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC‐ChMAT at 1.87 Å revealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC‐MATs were compatible with DNA‐ and RNA‐MTases, enabling sequence‐specific modification (“writing”) of plasmid DNA and light‐triggered removal (“erasing”).
S‐Adenosyl‐L‐methionine (AdoMet) analogs provide a way to study and control methyltransferase target sites. Their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered and crystallized the first SAM synthetases (PC‐MATs) able to generate AdoMet analogs with photocaging groups. In combination with DNA‐MTases, sequence‐specific modification (“writing”) of plasmid DNA and light‐triggered removal (“erasing”) is demonstrated.
Sequence‐specific modification (“writing”) and light‐triggered removal (“erasing”) of DNA is reported for the first time in an enzymatic cascade. In their Research Article on page 480, Andrea ...Rentmeister and co‐workers engineered SAM synthetases suitable for converting methionine analogs with photo‐cleavable groups and show their compatibility with methyltransferases to provide DNA with a light‐sensitive “cage”. Crystal structure analysis revealed how substitutions in the active site led to accommodation of the photo‐cleavable group.
Nucleic acids are ubiquitous in nature and modified nucleosides are present in a wide range of anti-viral, anti-cancer drugs and antibiotics. Although a variety of naturally occurring nucleoside ...analogues exist, few include modifications to the ribose or deoxyribose ring. Intriguingly, the uridyl peptide antibiotics (UPAs), such as pacidamycin, contain a biosynthetically unique 3'-deoxyuridine which resembles synthetic anti-retrovirals. Elucidation of the biosynthesis of this structuraly unique nucleoside motif suggests a degree of substrate flexibility, making it a highly attractive prospect for biosynthetic approaches to nucleoside modification. In order to fully exploit the biotransformative potential, a detailed mechanistic understanding of the individual enzymes involved in the biosynthesis of the nucleoside moiety, and especially the enzyme employed at the installation of the 3'-deoxy modification, is required. Chapter 1, the introduction the thesis, discusses the importance of nucleosides for Chemistry and Biology. The section describes the biosynthesis of the nucleoside antibiotics and reviews the recent advances relating to the synthesis and biosynthesis of 3'-deoxy-nucleosides. The Chapter proceeds to describes the biosynthesis of deoxy-sugars, deoxy-nucleosides and nucleotides, reviewing the most common dehydratase mechanisms in addition to examining unusual dehydratases involved in carbohydrate metabolism. Chapter 2, the study of Pac13, the uridine-5'-aldehyde dehydratase of the pacidamyicin nucleoside cluster, is reported. Through detailed functional, structural and kinetic analysis of the wild-type enzyme as well a series of mutants, Chapter 2 provides insight into the mechanism emplyed by this unusual enzyme. Chapter 3 describes the structural and functional analysis of Pac11, the flavin-dependent oxidoreductase of the nucleoside biosynthetic cluster, while Chapter 4 revolves around Pac5, the PLP-dependent aminotransferase. In Chapter 5, the chemical synthesis of fluorinated nucleosides, as probes for exploring the enzymes' mechanism is investigated. Chapter 7 reports the experimental procedures for the research described in this document. The work described in this thesis broadens the understanding of the biosynthesis of deoxy-nucleosides and constitutes the first structural and mechanistic study of the biosynthesis of the biosynthesis of the valuable yet, synthetically challenging 3'-deoxy nucleosides.
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