Extremolytes – unique compatible solutes produced by extremophiles - protect biological structures like membranes, proteins, and DNA under extreme conditions, including extremes of temperature and osmotic stress. These compounds hold significant potential for applications in pharmaceuticals, healthcare, cosmetics, and life sciences. However, despite their considerable potential, only a limited number of extremolytes – most notably ectoine and hydroxyectoine – have achieved commercial relevance, primarily due to the absence of efficient production strategies for the majority of other extremolytes. Cyclic 2,3-diphosphoglycerate (cDPG), a unique metabolite found in certain hyperthermophilic methanogenic Archaea, plays a key role in thermoprotection and is synthesized from 2-phosphoglycerate (2PG) through a two-step enzymatic process involving 2-phosphoglycerate kinase (2PGK) and cyclic-2,3-diphosphoglycerate synthetase (cDPGS). In this study, we present the development of an efficient in vitro enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from Methanothermus fervidus (MfcDPGS). We optimized the heterologous production of MfcDPGS in Escherichia coli by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of MfcDPGS for both usage and storage by incorporating KCl, Mg2+, reducing agents and omission of an affinity tag. The recombinant MfcDPGS showed a Vmax of 38.2 U mg−1, with KM values of 1.52 mM for 2,3DPG and 0.55 mM for ATP. The enzyme efficiently catalyzed the complete conversion of 2,3DPG to cDPG. Remarkably, even at a scale of 100 mM, it achieved full conversion of 37.6 mg of 2,3DPG to cDPG within 180 min, using just 0.5 U of recombinant MfcDPGS at 55°C. These results highlight that MfcDPGS can be easily produced, rapidly purified, and sufficiently stabilized while delivering excellent conversion efficiency for cDPG synthesis as value added product. Additionally, a kinetic model for MfcDPGS activity was developed, providing a crucial tool to simulate and scale up cDPG production for industrial applications. This streamlined process offers significant advantages for the scalable synthesis of cDPG, paving the way for further biochemical and industrial applications of this extremolyte.

The trematode liver fluke Fasciola hepatica causes the neglected tropical disease fascioliasis in humans and is associated with significant losses in agricultural industry due to reduced animal productivity. Triosephosphate isomerase (TPI) is a glycolytic enzyme that has been researched as a drug target for various parasites, including F. hepatica. The high-resolution crystal structure of F. hepatica TPI (FhTPI) has been solved at 1.51 Å resolution in its monoclinic form. The structure has been used to perform molecular-docking studies with the most successful fasciolocide triclabendazole (TCBZ), which has recently been suggested to target FhTPI. Two FhTPI residues, Lys50 and Asp51, are located at the dimer interface and are found in close proximity to the docked TCBZ. These residues are not conserved in mammalian hosts.

A (S)-selective amine transaminase from a Streptomyces strain, Sbv333-ATA, is a biocatalyst showing both high thermostability with a melting temperature of 85 °C and broad substrate specificity for the amino acceptor. This enzyme was further characterized both biochemically and structurally. The Sbv333-ATA is stable in the presence of up to 20

Vanadium haloperoxidases have been studied to understand their mechanism and halide specificity. Crystal structures of vanadium bromoperoxidase enzyme complexes from Corallina piluifera, with vanadate and bromide and with phosphate and chloride, show significant displacement of loop residues 336-338 upon halogen binding. This shows a "closed position" of Leu337 locking the bromide ion in a hydrophobic environment favoring the vanadium peroxide reaction with the halogen by retaining the resulting hypobromite in the enzyme active site. The bound cofactor exists as a mixture of free vanadate and histidine553-vanadate adduct. A mutant enzyme Arg397Trp also has been crystallized with bound phosphate and its structure determined with and without the bound bromide ion. The precise positions of the bromine have been determined using its anomalous signal. The bromide binding site in the mutant enzyme is displaced by 2.5 & Aring; resulting in a mixed population of the "open" and the "closed" forms of Leu337. This allows additional chloroperoxidase activity due to re-positioning of the halogen ion 0.6 & Aring; closer to the vanadate ion. These studies support the application of vanadium haloperoxidase enzymes for selective halogenation of important drug molecules.

The enzyme cyclic di-phosphoglycerate synthetase that is involved in the production of the osmolyte cyclic 2,3-diphosphoglycerate has been studied both biochemically and structurally. Cyclic 2,3-diphosphoglycerate is found exclusively in the hyperthermophilic archaeal methanogens, such as Methanothermus fervidus, Methanopyrus kandleri, and Methanothermobacter thermoautotrophicus. Its presence increases the thermostability of archaeal proteins and protects the DNA against oxidative damage caused by hydroxyl radicals. The cyclic 2,3-diphosphoglycerate synthetase enzyme has been crystallized and its structure solved to 1.7 Å resolution by experimental phasing. It has also been crystallized in complex with its substrate 2,3 diphosphoglycerate and the co-factor ADP and this structure has been solved to 2.2 Å resolution. The enzyme structure has two domains, the core domain shares some structural similarity with other NTP-dependent enzymes. A significant proportion of the structure, including a 127 amino acid N-terminal domain, has no structural similarity to other known enzyme structures. The structure of the complex shows a large conformational change that occurs in the enzyme during catalytic turnover. The reaction involves the transfer of the γ-phosphate group from ATP to the substrate 2,3 -diphosphoglycerate and the subsequent SN2 attack to form a phosphoanhydride. This results in the production of the unusual extremolyte cyclic 2,3 -diphosphoglycerate which has important industrial applications.

A branched chain aminotransferase from Thermoproteus tenax has been identified, cloned, over-expressed and biochemically characterised. A molecular modelling approach has been used to predict the 3D structure allowing its comparison with other related enzymes. This enzyme has high similarity to a previously characterised aminotransferase from Thermoproteus uzoniensis however its substrate specificity shows key differences towards the substrate α-ketoglutarate. Examination of the active sites of the two related enzymes reveals a single amino acid substitution of a glycine residue to a serine residue which could be responsible for this difference. When Gly104 in T. tenax was mutated to a serine residue and the resultant enzyme characterised, this single amino acid change resulted in a dramatic reduction in activity towards α-ketoglutarate with an 18-fold reduction in Vmax and a 20-fold Km increase, resulting in a 370-fold lower catalytic efficiency. Structural comparisons between the two related Thermoproteus enzymes and another branched chain aminotransferase from Geoglobus acetivorans has revealed that the serine residue affects the flexibility of a key loop involved in catalysis. This subtle difference has provided further insight into our understanding of the substrate specificity of these industrially important enzymes.

The role and power of biocatalysis in sustainable chemistry has been continuously brought forward step by step to its present outstanding position. The problem-solving capabilities of biocatalysis have been realized by numerous substantial achievements in biology, chemistry and engineering. Advances and breakthroughs in the life sciences and interdisciplinary cooperation with chemistry have clearly accelerated the implementation of biocatalytic synthesis in modern chemistry. Resource-efficient biocatalytic manufacturing processes have already provided numerous benefits to sustainable chemistry as well as customer-centric value creation in the pharmaceutical, food, flavor, fragrance, vitamin, agrochemical, polymer, specialty, and fine chemical industries. Biocatalysis can make significant contributions not only to manufacturing processes, but also to the design of completely new value-creation chains. Biocatalysis can now be considered as a key enabling technology to implement sustainable chemistry.

The biosynthesis of metabolites from available starting materials is becoming an ever important area due to the increasing demands within the life science research area. Access to metabolites is making essential contributions to analytical, diagnostic, therapeutic and different industrial applications. These molecules can be synthesized by the enzymes of biological systems under sustainable process conditions. The facile synthetic access to the metabolite and metabolite-like molecular space is of fundamental importance. The increasing knowledge within molecular biology, enzyme discovery and production together with their biochemical and structural properties offers excellent opportunities for using modular cell-free biocatalytic systems. This reduces the complexity of synthesizing metabolites using biological whole-cell approaches or by classical chemical synthesis. A systems biocatalysis approach can provide a wealth of optimized enzymes for the biosynthesis of already identified and new metabolite molecules.

The Front Cover shows the treasure box of biocatalysis, which can now be considered as a key enabling technology for the development of sustainable chemistry. The implementation of biocatalytic syntheses in modern chemistry for different industrial sectors has been accelerated by advances and breakthroughs in understanding structure, function, discovery, design, development, and application of enzymes as Nature′s inherently chiral catalysts. Therefore, a “Golden Age of Biocatalysis” can be envisioned. More information can be found in the Perspective by A. R. Alcántara et al.

EDITORIAL article Front. Bioeng. Biotechnol., 09 November 2022Sec. Bioprocess Engineering Volume 10 - 2022 | https://doi.org/10.3389/fbioe.2022.1010174

Lipolytic enzymes are essential biocatalysts in food processing as well as pharmaceutical and pesticide industries, catalyzing the cleavage of ester bonds in a variety of acyl chain substrates. Here, we report the crystal structure of an esterase from the deep-sea hydrothermal vent of the East Pacific Rise (EprEst). The X-ray structure of EprEst in complex with the ligand, acetate, has been determined at 2.03 Å resolution. The structure reveals a unique spatial arrangement and orientation of the helix cap domain and α/β hydrolase domain, which form a substrate pocket with preference for short-chain acyl groups. Molecular docking analysis further demonstrated that the active site pocket could accommodate p-nitrophenyl (pNP) carboxyl ligands of varying lengths (≤6 C atoms), with pNP-butyrate ester predicted to have the highest binding affinity. Additionally, the semirational design was conducted to improve the thermostability of EprEst by enzyme engineering based on the established structure and multiple sequence alignment. A mutation, K114P, introduced in the hinge region of the esterase, which displayed increased thermostability and enzyme activity. Collectively, the structural and functional data obtained herein could be used as basis for further protein engineering to ultimately expand the scope of industrial applications of marine-derived lipolytic enzymes.

Osmolytes protect microbial cells against temperature, osmolarity and other stresses. The osmolyte cyclic 2,3-diphosphoglycerate, originally isolated from the thermophilic archaeon Methanothermus fervidus, naturally protects cellular proteins under extreme conditions. The biosynthetic pathway for cyclic 2,3-diphosphoglycerate has been introduced into the thermophilic bacterium Thermus thermophilus. The two enzymes in this synthetic pathway, 2-phosphoglycerate kinase and cyclic diphosphoglycerate synthetase, were incorporated into a newly designed modular BioBricks vector. The expression of this two-enzyme cascade resulted in the whole cell production of cyclic 2,3 diphosphoglycerate. In vivo production of cyclic 2,3-diphosphoglycerate was confirmed by mass spectrometry to a concentration up to 650 µM. This study demonstrates the feasibility of using this well studied thermophilic bacterium as a host in a whole-cell factory approach to produce cyclic 2,3 diphosphoglycerate. This raises the potential for commercialisation of cDPG for cosmetic and healthcare applications. Our work demonstrates the application of Thermus thermophilus as an alternative host for other high value small organic molecules of industrial interest.

A novel D-lyxose isomerase has been identified within the genome of a hyperthermophilic archaeon belonging to the Thermofilum species. The enzyme has been cloned and over-expressed in Escherichia coli and biochemically characterised. This enzyme differs from other enzymes of this class in that it is highly specific for the substrate D-lyxose, showing less than 2% activity towards mannose and other substrates reported for lyxose isomerases. This is the most thermoactive and thermostable lyxose isomerase reported to date, showing activity above 95°C and retaining 60% of its activity after 60 min incubation at 80°C. This lyxose isomerase is stable in the presence of 50% (v/v) of solvents ethanol, methanol, acetonitrile and DMSO. The crystal structure of the enzyme has been resolved to 1.4–1.7 A. resolution in the ligand-free form and in complexes with both of the slowly reacting sugar substrates mannose and fructose. This thermophilic lyxose isomerase is stabilised by a disulfide bond between the two monomers of the dimeric enzyme and increased hydrophobicity at the dimer interface. These overall properties of high substrate specificity, thermostability and solvent tolerance make this lyxose isomerase enzyme a good candidate for potential industrial applications.

SUMMARY The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium‐dependent membrane‐binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5‐Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca 2+ ‐binding region in C2A domain is longer than classical C2 domains and a novel Ca 2+ binding site in the vWA domain. The structure of BON1 bound to Mn 2+ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid‐binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca 2+ . These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo .