Search

L-rhamnose isomerase (L-RhI) plays a key role in the microbial L-rhamnose metabolism by catalyzing the reversible isomerization of L-rhamnose to L-rhamnulose. Additionally, the enzyme exhibits activity on various other aldoses and ketoses, and its broad substrate specificity has attracted attention for its potential application in the production of rare sugars; however, improvement of the enzyme properties is desirable, such as thermal stability, enzymatic activity, and a pH optimum suitable for industrial usage. This review summarizes our current insights into L-RhIs with respect to their substrate recognition mechanism and their relationship with D-xylose isomerase (D-XI) based on structural and phylogenetic analyses. These two enzymes are inherently different, but recognize distinctly different substrates, and share common features that may be phylogenetically related. For example, they both have a flexible loop region that is involved in shaping active sites, and this region may also be responsible for various enzymatic properties of L-RhIs, such as substrate specificity and thermal stability. KEY POINTS: •L-RhIs share structural features with D-XI. •There are two types of L-RhIs: E. coli L-RhI-type and D-XI-type. •Flexible loop regions are involved in the specific enzyme properties., (© 2024. The Author(s).)

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-β-thia and β-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors., (© 2024 The Author(s).)

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum . Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar Pf DXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N -phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N -terminal domain. As shown for reverse carba and thia analogs, Pf DXR selectively binds the S -enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.

6-Deoxy-l-sorbose (6-DLS) is an imperative rare sugar employed in food, agriculture, pharmaceutical and cosmetic industeries. However, it is a synthetic and very expensive rare sugars, previously synthesized by chemo-enzymatic methods through a long chain of chemical processes. Recently, enzymatic synthesis of rare sugars has attracted a lot of attention due to its advantages over synthetic methods. In this work, a promising approach for the synthesis of 6-DLS from an inexpensive sugar l-fucose was identified. The genes for l-fucose isomerase from Paenibacillus rhizosphaerae (Pr-LFI) and genes for d-tagatose-3-epimerase from Caballeronia fortuita (Cf-DTE) have been used for cloning and co-expression in Escherichia coli, developed a recombinant plasmid harboring pANY1-Pr-LFI/Cf-DTE vector. The recombinant co-expression system exhibited an optimum activity at 50 °C of temperature and pH 6.5 in the presence of Co 2+ metal ion which inflated the catalytic activity by 6.8 folds as compared to control group with no metal ions. The recombinant co-expressed system was stable up to more than 50 % relative activity after 12 h and revealed a melting temperature (T m ) of 63.38 °C exhibiting half-life of 13.17 h at 50 °C. The co-expression system exhibited, 4.93, 11.41 and 16.21 g/L of 6-DLS production from initial l-fucose concentration of 30, 70 and 100 g/L, which equates to conversion yield of 16.44 %, 16.30 % and 16.21 % respectively. Generally, this study offers a promising strategy for the biological production of 6-DLS from an inexpensive substrate l-fucose in slightly acidic conditions with the aid of co-expression system harboring Pr-LFI and CF-DTE genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A novel l-arabinose isomerase (L-AI) from Arthrobacter psychrolactophilus (Ap L-AI) was successfully cloned and characterized. The enzyme catalyzes the isomerization of d-galactose into a rare sugar d-tagatose. The recombinant Ap L-AI had an approximate molecular weight of about 258 kDa, suggesting it was an aggregate of five 58 kDa monomers and became the first record as a homo-pentamer L-AI. The catalytic efficiency (k cat /Km) and Km for d-galactose were 0.32 mM -1  min -1 and 51.43 mM, respectively, while for l-arabinose, were 0.64 mM -1  min -1 and 23.41 mM, respectively. It had the highest activity at pH 7.0-7.5 and 60 °C in the presence of 0.250 mM Mn 2+ . Ap L-AI was discovered to be an outstanding thermostable enzyme that only lost its half-life value at 60 °C for >1000 min. These findings suggest that l-arabinose isomerase from Arthrobacter psychrolactophilus is a promising candidate for d-tagatose mass-production due to its industrially competitive temperature., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Glucose isomerase (GI) is extensively used in the food industry for production of high-fructose corn syrup and for the production of biofuels and other renewable chemicals. Structure-based studies on GI inhibitors are important for improving its efficiency in industrial applications. Here, we report the subatomic crystal structure of Streptomyces rubiginosus GI (SruGI) complexed with its inhibitor, xylitol, at 0.99 Å resolution. Electron density map and temperature factor analysis showed partial binding of xylitol to the M1 metal binding site of SruGI, providing two different conformations of the metal binding site and the substrate binding channel. The xylitol molecule induced a conformational change in the M2 metal ion-interacting Asp255 residue, which subsequently led to a conformational change in the side chain of Asp181 residue. This led to the positional shift of Pro25 by 1.71 Å and side chain rotation of Phe26 by 21°, where located on the neighboring protomer in tetrameric SruGI. The conformation change of these two residues affect the size of the substrate-binding channel of GI. Therefore, xylitol binding to M1 site of SruGI induces not only a conformational changes of the metal-binding site, but also conformational change of substrate-binding channel of the tetrameric SruGI. These results expand our knowledge about the mechanism underlying the inhibitory effect of xylitol on GI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

d-Allose is a low-calorie rare sugar with great application potential in the food and pharmaceutical industries. The production of d-allose has been accomplished using l-rhamnose isomerase (L-RI), but concomitantly increasing the enzyme's stability and activity remains challenging. Here, we rationally engineered an L-RI from Clostridium stercorarium to enhance its stability by comprehensive computation-aided redesign of its flexible regions, which were successively identified using molecular dynamics simulations. The resulting combinatorial mutant M2-4 exhibited a 5.7-fold increased half-life at 75 °C while also exhibiting improved catalytic efficiency. Especially, by combining structure modeling and multiple sequence alignment, we identified an α0 region that was universal in the L-RI family and likely acted as a "helix-breaker". Truncating this region is crucial for improving the thermostability of related enzymes. Our work provides a significantly stable biocatalyst with potential for the industrial production of d-allose.

Fructose, which is produced by the isomerization of glucose isomerase, is a crucial precursor for the biosynthesis of rare sugars. In this study, thermophilic glucose isomerases (GI) from Caldicellulosiruptor acetigenus (CAGI), Thermoanaerobacter thermocopriae (TTGI), and Thermotoga petrophila (TPGI) were screened from GenBank database by a virtual probe and were successfully expressed in Escherichia coli BL21(DE3). The results of characterization demonstrated that the optimal pH for CAGI and TTGI were 8.0 and were maintained at 80% in a slightly acidic environment. The relative residual activities of CAGI and TTGI were found to be 40.6% and 52.6%, respectively, following an incubation period of 24 h at 90 ℃. Furthermore, CAGI and TTGI exhibited superior catalytic performance that their reaction equilibrium both reached only after an hour at 85 ℃ with 200 g/L glucose, and the highest conversion rates were 54.2% and 54.1%, respectively. This study identifies competitive enzyme candidates for fructose production in the industry with appreciable cost reduction., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli., (© 2023 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Xylose isomerase catalyzes the isomerization of D-xylose to D-xylulose with promiscuous activity for other saccharides including D-glucose, D-allose, and L-arabinose. The xylose isomerase from the fungus Piromyces sp. E2 (PirE2_XI) is used to engineer xylose usage by the fermenting yeast Saccharomyces cerevisiae, but its biochemical characterization is poorly understood with divergent catalytic parameters reported. We have measured the kinetic parameters of the PirE2_XI and analyzed its thermostability and pH-dependence towards different substrates. The PirE2_XI shows promiscuous activity towards D-xylose, D-glucose, D-ribose and L-arabinose with variable effects depending on different divalent ions and epimerizes D-xylose at C3 to produce D-ribulose in a substrate/product dependent ratio. The enzyme follows Michaelis-Menten kinetics for the substrates used and although K M values for D-xylose are comparable at 30 and 60 °C, the k cat /K M is three-fold greater at 60 °C. The purified PirE2_XI shows maximal activity at 65 °C in the pH range of 6.5-7.5 and is a thermostable enzyme, maintaining full activity over 48 h at 30 °C or 12 h at 60 °C. This is the first report demonstrating epimerase activity of the PirE2_XI and its ability to isomerize D-ribose and L-arabinose, and provides a comprehensive in vitro study of substrate specificity, effect of metal ions and temperature on enzyme activity and these findings advance the knowledge of the mechanism of action of this enzyme., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Methylthio-d-ribose-1-phosphate (MTR1P) isomerase (MtnA) catalyzes the reversible isomerization of the aldose MTR1P into the ketose methylthio-d-ribulose 1-phosphate. It serves as a member of the methionine salvage pathway that many organisms require for recycling methylthio-d-adenosine, a byproduct of S-adenosylmethionine metabolism, back to methionine. MtnA is of mechanistic interest because unlike most other aldose-ketose isomerases, its substrate exists as an anomeric phosphate ester and therefore cannot equilibrate with a ring-opened aldehyde that is otherwise required to promote isomerization. To investigate the mechanism of MtnA, it is necessary to establish reliable methods for determining the concentration of MTR1P and to measure enzyme activity in a continuous assay. This chapter describes several such protocols needed to perform steady-state kinetics measurements. It additionally outlines the preparation of [ 32 P]MTR1P, its use in radioactively labeling the enzyme, and the characterization of the resulting phosphoryl adduct., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Isomerases are enzymes that induce physical changes in a molecule without affecting the original molecular formula. Among this class of enzymes, xylose isomerases (XIs) are the most studied to date, partly due to their extensive application in industrial processes to produce high-fructose corn sirups. In recent years, the need for sustainable initiatives has triggered efforts to improve the biobased economy through the use of renewable raw materials. In this context, D-xylose usage is crucial as it is the second-most abundant sugar in nature. The application of XIs in biotransforming xylose, enabling downstream metabolism in several microorganisms, is a smart strategy for ensuring a low-carbon footprint and producing several value-added biochemicals with broad industrial applications such as in the food, cosmetics, pharmaceutical, and polymer industries. Considering recent advancements that have expanded the range of applications of XIs, this review provides a comprehensive and concise overview of XIs, from their primary sources to the biochemical and structural features that influence their mechanisms of action. This comprehensive review may help address the challenges involved in XI applications in different industries and facilitate the exploitation of xylose bioprocesses.

Protein crystallization is an astounding feat of nature. Even though proteins are large, anisotropic molecules with complex, heterogeneous surfaces, they can spontaneously group into two- and three-dimensional arrays with high precision. And yet, the biggest hurdle in this assembly process, the formation of a nucleus, is still poorly understood. In recent years, the two-step nucleation model has emerged as the consensus on the subject, but it still awaits extensive experimental verification. Here, we set out to reconstruct the nucleation pathway of the candidate protein glucose isomerase (GI), for which there have been indications that it may follow a two-step nucleation pathway under certain conditions. We find that the precursor phase present during the early stages of the reaction process is nanoscopic crystallites that have lattice symmetry equivalent to the mature crystals found at the end of a crystallization experiment. Our observations underscore the need for experimental data at a lattice-resolving resolution on other proteins so that a general picture of protein crystal nucleation can be formed., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

To improve the operational stability of glucose isomerase in E. coli TEGI-W139F/V186T, the immobilized cells were prepared with modified diatomite as a carrier and 74.1% activity of free cells was recovered after immobilization. Results showed that the immobilized cells still retained 86.2% of the initial transformational activity after intermittent reused 40 cycles and the yield of D-fructose reached above 42% yield at 60 °C. Moreover, the immobilized cells were employed in the continuous production of High Fructose Corn Syrup (HFCS) in a recirculating packed bed reactor for 603 h at a constant flow rate. It showed that the immobilized cells exhibited good operational stability and the yield of D-fructose retained above 42% within 603 h. The space-time yield of high fructose corn syrup reached 3.84 kg L -1  day -1 . The investigation provided an efficient immobilization method for recombinant cells expressing glucose isomerase with higher stability, and the immobilized cells are a promising biocatalyst for HFCS production.

Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles that become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not yet been observed for protein systems. Here we employ cryo-transmission electron microscopy (cryoEM) in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. As these nanocrystalline assemblies grow larger we witness imperfect docking events leading to oriented aggregation into mesocrystalline assemblies. These observations highlight the underappreciated role of the interaction between crystalline nuclei, and the impact of OA on the crystallization process of proteins.

Glutathione (GSH) is the most abundant non-protein thiol and its cellular concentration has been reported as 17 mM in Escherichia coli. This study introduces a label-free method to determine the binding affinity of GSH to proteins, utilizing the intrinsic fluorescence of proteins; the dissociation constants of GSH for d-arabinose 5-phosphate isomerase KdsD, fumarase C, malate dehydrogenase, and RNA polymerase subunit α have been determined as 96 ± 8, 246 ± 42, 292 ± 78, and 296 ± 97 μM, respectively. The dissociation constants, less than 2% of the cellular concentration of GSH, suggests that protein-GSH interactions are strong enough to make all of the GSH-binding sites occupied fully. The method described here may be applicable to other proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

It is important to reveal the exact cause of poor diffractivity in protein crystals in order to determine the accurate structure of protein molecules. It is shown that there is a large amount of local strain in subgrains of glucose isomerase crystals even though the overall crystal quality is rather high, as shown by clear equal-thickness fringes in X-ray topography. Thus, a large stress is exerted on the subgrains of protein crystals, which could significantly lower the resistance of the crystals to radiation damage. It is also demonstrated that this local strain can be reduced through the introduction of dislocations in the crystal. This suggests that the introduction of dislocations in protein crystals can be effective in enhancing the crystal quality of subgrains of protein crystals. By exploiting this effect, the radiation damage in subgrains could be decreased, leading to the collection of X-ray diffraction data sets with high diffractivity.

Glucose isomerase (GI) is an important enzyme that is widely used in industrial applications, such as in the production of high-fructose corn syrup or bioethanol. Studying inhibitor effects on GI is important to deciphering GI-specific molecular functions, as well as potential industrial applications. Analysis of the existing xylitol-bound GI structure revealed low metal occupancy at the M2 site; however, it remains unknown why this phenomenon occurs. This study reports the room-temperature structures of native and xylitol-bound GI from Streptomyces rubiginosus (SruGI) determined by serial millisecond crystallography. The M1 site of native SruGI exhibits distorted octahedral coordination; however, xylitol binding results in the M1 site exhibit geometrically stable octahedral coordination. This change results in the rearrangement of metal-binding residues for the M1 and M2 sites, the latter of which previously displayed distorted metal coordination, resulting in unstable coordination of Mg 2+ at the M2 site and possibly explaining the inducement of low metal-binding affinity. These results enhance the understanding of the configuration of the xylitol-bound state of SruGI and provide insights into its future industrial application.

Glucose/xylose isomerase catalyzes the reversible isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This enzyme is not only involved in sugar metabolism but also has industrial applications, such as in the production of high fructose corn syrup and bioethanol. Various crystal structures of glucose isomerase have shown the binding configuration of the substrate and its molecular mechanism; however, the metal binding mechanism required for the isomerization reaction has not been fully elucidated. To better understand the functional metal binding, the crystal structures of the metal-bound and metal-free states of Streptomyces rubiginosus glucose isomerase (SruGI) were determined at 1.4 Å and 1.5 Å resolution, respectively. In the meal-bound state of SruGI, Mg 2+ is bound at the M1 and M2 sites, while in the metal-free state, these sites are occupied by water molecules. Structural comparison between the metal binding sites of the metal-bound and metal-free states of SruGI revealed that residues Glu217 and Asp257 exhibit a rigid configuration at the bottom of the metal binding site, suggesting that they serve as a metal-binding platform that defined the location of the metal. In contrast, the side chains of Glu218, His220, Asp255, Asp257, and Asp287 showed configuration changes such as shifts and rotations. Notably, in the metal-free state, the side chains of these amino acids are shifted away from the metal binding site, indicating that the metal-binding residues exhibit a minimal open configuration, which allows metal binding without large conformational changes., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

M. tuberculosis GmhA enzyme catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-α-manno-heptose-7-phosphate in GDP-D-glycero-α-D-manno-heptose biosynthetic pathway. The D-glycero-α-D-manno-heptose is a major constituent of lipopolysaccharide and contributes to virulence and antibiotic resistance to mycobacteria. In current study, we have performed the structural and biochemical analysis of M. tuberculosis GmhA, the first enzyme involved in D-sedoheptulose 7-phosphate isomerization in GDP-D-α-D-heptose biosynthetic pathway. The MtbGmhA enzyme exits as tetramer and small angle X-ray scattering analysis also yielded tetrameric envelope in solution. The MtbGmhA enzyme binds to D-sedoheptulose 7-phosphate with K m  ~ 0.31 ± 0.06 mM -1 and coverts it to D-glycero-D-α-manno-heptose-7-phosphate with catalytic efficiency (k cat /K m ) ~ 1.45 mM -1  s -1 . The residues involved in D-sedoheptulose 7-phosphate and Zn 2+ binding were identified using modeled MtbGmhA + D-sedoheptulose 7-phosphate + Zn 2+ structure. To understand the role in catalysis, six site directed mutants of MtbGmhA were generated, which showed significant decrease in catalytic activity. The circular dichroism analysis showed ~ 46% α-helix, ~ 19% β-sheet and ~ 35% random coil structures of MtbGmhA enzyme and melting temperature ~ 53.5 °C. Small angle X-ray scattering analysis showed the tetrameric envelope, which fitted well with modeled MtbGmhA tetramer in closed conformation. The MtbGmhA dynamics involved in D-sedoheptulose 7-phosphate and Zn 2+ binding was identified using dynamics simulation and showed enhanced stability in presence of these ligands. Our biochemical data and structural knowledge have provided insight into mechanism of action of MtbGmhA enzyme, which can be targeted for novel antibiotics development against M. tuberculosis.

A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aim of this work was to develop a stable immobilized enzyme biocatalyst for the isomerization of d-galactose to d-tagatose at high temperature. l-Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was produced as a (His) 6 -tagged protein, immobilized on a copper-chelate epoxy support and subjected to several postimmobilization treatments aimed at increasing its operational and structural stability. Treatment with glutaraldehyde and ethylenediamine resulted in a more than twofold increase in the operational stability and in all enzyme subunits linked, directly or indirectly, to the support via covalent bonds. A postimmobilization treatment of the immobilized derivatives with mercaptoethanol for the removal of any remaining copper ions, determined a further increase of the operational biocatalytic activity. Immobilized derivatives subjected to both treatments were used for the bioconversion of 18 g/L d-galactose to d-tagatose at 80°C in a packed bed reactor in three repeated cycles and showed a better operational stability compared with the literature data. This study shows that a postimmobilization stabilization treatment with glutaraldehyde and ethylenediamine can stabilize the multi-subunit structure of an enzyme immobilized on a metal-chelate epoxy support with an increase of its operational stability, results that are not easily achievable with the sole immobilization on epoxy or metal chelate-epoxy supports in the case of complex multimeric enzymes with geometric incongruence with the support., (© 2020 American Institute of Chemical Engineers.)

The Calvin-Benson cycle is the key metabolic pathway of photosynthesis responsible for carbon fixation and relies on eleven conserved enzymes. Ribose-5-phosphate isomerase (RPI) isomerizes ribose-5-phosphate into ribulose-5-phosphate and contributes to the regeneration of the Rubisco substrate. Plant RPI is the target of diverse post-translational modifications including phosphorylation and thiol-based modifications to presumably adjust its activity to the photosynthetic electron flow. Here, we describe the first experimental structure of a photosynthetic RPI at 1.4 Å resolution. Our structure confirms the composition of the catalytic pocket of the enzyme. We describe the homo-dimeric state of the protein that we observed in the crystal and in solution. We also map the positions of previously reported post-translational modifications and propose mechanisms by which they may impact the catalytic parameters. The structural data will inform the biochemical modeling of photosynthesis.

Rare sugars have received increasing attention due to their important applications as sweeteners and building blocks. The substrate specificity and catalytic properties of ribose-5-phosphate isomerase A (RpiA) in isomerization of rare sugars have not been extensively explored. In this study, an RpiA from Ochrobactrum sp. CSL1 was cloned and expressed in Escherichia coli. The biochemical and reaction features were explored and its broad substrate specificity was identified. A higher reaction rate in isomerizing l-rhamnose to l-rhamnulose by OsRpiA, compared with OsRpiB found in the same strain indicated higher efficiency in preparing rare sugars, which was verified by kinetics study. The 2.8 Å resolution structure of OsRpiA was then solved and used in subsequent molecular dynamics experiments, providing a possible explanation for its distinct substrate specificity. The present study highlighted the unique role of microbial RpiA in preparing rare sugars, and its structural information provided a reliable reference for further reaction mechanism research and enzyme engineering work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ribose-5-phosphate isomerase (Rpi, EC 5.3.1.6) is widespread in microorganisms, animals, and plants. It has a pivotal role in the pentose phosphate pathway and responsible for catalyzing the isomerization between D-ribulose 5-phosphate and D-ribose 5-phosphate. In recent years, Rpi has received considerable attention as a multipurpose biocatalyst for production of rare sugars, including D-allose, L-rhamnulose, L-lyxose, and L-tagatose. Besides, it has been thought of as a potential drug target in the treatment of trypanosomatid-caused diseases such as Chagas' disease, leishmaniasis, and human African trypanosomiasis. Despite increased research activities, up to now, no systematic review of Rpi has been published. To fill this gap, this paper provides detailed information about the enzymatic properties of various Rpis. Furthermore, structural features, catalytic mechanism, and molecular modifications of Rpis are summarized based on extensive crystal structure research. Additionally, the applications of Rpi in rare sugar production and the role of Rpi in trypanocidal drug design are reviewed. Key points • Fundamental properties of various ribose-5-phosphate isomerases (Rpis). • Differences in crystal structure and catalytic mechanism between RpiA and RpiB. • Application of Rpi as a rare sugar producer and a potential drug target.

The insolubilization of a recombinant l-arabinose isomerase (l-AI) from Enterococcus faecium by cross-linked enzyme aggregates (CLEA) was investigated, aiming the biochemical production of d-tagatose from d-galactose. d-tagatose is a functional sweetener that has many health benefits, sweetening properties and lower calorific value. Different precipitants (ammonium sulfate, ethanol, acetone, polyethylene glycol 4000) were used in the first step of the protocol, in order to establish the precipitation conditions, and the best results of yield and activity were achieved with ammonium sulfate. In order to facilitate the recovery of the biocatalyst, a new strategy for immobilization of the multimeric enzyme l-arabinose isomerase was proposed. Magnetic cross-linked enzyme aggregates (m-CLEA) were obtained using ammonium sulfate as precipitant and magnetic nanoparticles (MNP) functionalized with APTES (3- Aminopropyltriethoxysilane). Another immobilization strategy was to immobilize the enzyme onto MNP-APTES, as a control. The best results were achieved when the m-CLEA was produced with 20 mg of MNP, 7.69 U. g -1 of enzymatic activity, 7.61 % of recovered activity, 99 % of yield of immobilization. On the other hand, the enzyme immobilized onto MNP-APTES, presented only 2.12 U. g -1 of enzymatic activity, 32.3 % of recovered activity, and 15 % of yield of immobilization., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recently, functional sugars, such as d-mannose, have attracted considerable attention due to their excellent physiological benefits for human health and wide applications in food and pharmaceutical industries. Therefore, d-mannose production using a sugar isomerase such as d-lyxose isomerase (d-LIase) has emerged as a research hotspot owing to its advantages over plant extraction and chemical synthesis methods. In this study, a putative d-LIase gene from Caldanaerobius polysaccharolyticus was cloned and expressed in Escherichia coli. Then, a biochemical characterization of the recombinant d-LIase was carried out and its potential use in d-mannose production also assessed. Results showed that d-LIase exhibited its maximum activity under these optimal conditions: temperature of 65 °C, a pH of 6.5, and the Mn 2+ metal ion. The d-LIase was active at pH 6.0-8.0; it was also quite thermostable up to 60 °C and approximately 85 % of its maximum activity was retained after incubating for 4 h. Further, our Nano-DSC analysis determined that its melting temperature (T m ) was 70.74 °C. Using 100, 300, and 500 g L -1 of d-fructose as substrate, 25.6, 74.4, and 115 g L -1 of d-mannose were produced respectively, corresponding to a conversion rate of 25.6 %, 24.8 %, and 23.0 % under optimal conditions. Taken together, our results provide evidence for a promising candidate d-LIase for producing d-mannose directly from d-fructose., Competing Interests: Declaration of Competing Interest The authors also declared no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Drug resistance is a major healthcare challenge, resulting in a continuous need to develop new inhibitors. The development of these inhibitors requires an understanding of the mechanisms of resistance for a critical mass of occurrences. Recent genome editing technologies based on high-throughput DNA synthesis and sequencing may help to predict mutations resulting in resistance by testing large mutagenesis libraries. Here we describe the rationale of this approach, with examples and relevance to drug development and resistance in malaria.

Background: Enzymatic isomerization is a promising strategy to solve the problem of xylose fermentation and, consequently, to leverage the production of advanced biofuels and biochemicals. In a previous work, our research group discovered a new strain of Streptomyces with great biotechnological potential due to its ability to produce a broad arsenal of enzymes related to lignocellulose degradation., Methods: We applied a multidisciplinary approach involving enzyme kinetics, biophysical methods, small angle X-ray scattering and X-ray crystallography to investigate two novel xylose isomerases, XylA1F1 and XylA2F1, from this strain., Results: We showed that while XylA1F1 prefers to act at lower temperatures and relatively lower pH, XylA2F1 is extremely stable at higher temperatures and presents a higher turnover number. Structural analysis revealed that XylA1F1 exhibits unique properties in the active site not observed in classical XylAs from classes I and II nor in its ortholog XylA2F1. It encompasses the natural substitutions, M86A and T93K, that create an extra room for substrate accommodation and narrow the active-site entrance, respectively. Such modifications may contribute to the functional differentiation of these enzymes., Conclusions: We have characterized two novel xylose isomerases that display distinct functional behavior and harbor unprecedented amino-acid substitutions in the catalytic interface., General Significance: Our findings contribute to a better understanding of the functional and structural aspects of xylose isomerases, which might be instrumental for the valorization of the hemicellulosic fraction of vegetal biomass., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Metal-organic frameworks (MOFs) are a class of porous materials with versatile properties. In this study, ZIF-8 was employed to establish a two-enzyme system by encapsulating permeabilized Bacillus subtilis cells coated with glucose isomerase. B. subtilis was constructed by introducing the shuttle plasmid PMA5 associated with the overexpression of trehalose synthase. Using this two-enzyme system, trehalose was produced by trehalose synthase and the byproduct glucose was converted to fructose with the help of glucose isomerase. The decrease in glucose production not only relieved the inhibition of the entire reaction chain but also increased the final yield of trehalose. The highest trehalose production rate reached 67.7% and remained above 50% after 20 batches. In addition, the toxicity of the ZIF-8 coating for B. subtilis was investigated by fluorescence microscopy and was found to be negligible. By simulating an extreme environment, the ZIF-8 coating was demonstrated to have a protective effect on the cells and enzymes. This study provides a theoretical basis for the application of MOFs in the immobilization of microorganisms and enzymes.

Glucose isomerase (GI) that catalyzes the conversion of D-glucose to D-fructose is one of the most important industrial enzymes for the production of high-fructose corn syrup (HFCS). In this study, a novel GI (CbGI) was cloned from Caldicellulosiruptor bescii and expressed in Escherichia coli . The purified recombinant CbGI (rCbGI) showed neutral and thermophilic properties. It had optimal activities at pH 7.0 and 80°C and retained stability at 85°C. In comparison with other reported GIs, rCbGI exhibited higher substrate affinity ( K m = 42.61 mM) and greater conversion efficiency (up to 57.3% with 3M D-glucose as the substrate). The high catalytic efficiency and affinity of this CbGI is much valuable for the cost-effective production of HFCS., Competing Interests: The authors declare no conflict of interest regarding the publication of this paper., (Copyright © 2020 Chenxia Dai et al.)

An enzyme, l-ribose isomerase (l-RI), mostly catalyzes the isomerization of l-ribose and l-ribulose. These so-called rare sugars are essential for the treatment of cancer and other viral diseases. In the present study, l-ribose isomerase produced from a bacterium, Mycetocola miduiensis (Mm-LRIse), by using l-ribose as a carbon source. The recombinant l-ribose isomerase gene was cloned and overexpressed from M. miduiensis and purified with an exclusive band of 32 kDa by nickel-affinity chromatography. This gene possessed 267 amino acids protein having an estimated molecular weight of 29,568.17 Da. The native molecular weight of Mm-LRIse estimated by HPLC was 134.84 kDa. The recombinant l-ribose isomerase was highly active in sodium phosphate (50 mM) buffer at 40 °C and pH 7.5, showing the specific activity up to 47.40 U mg -1 . Mm-LRIse showed no significant enhancement in activity with metallic ions except Mn 2+ and Co 2+ . The values of Km, K ca t , K cat /Km and V max of Mm-LRIse against l-ribose substrate were 42.48 mM, 9259.26 min -1 , 217.43 min -1  mM -1 , and 277.78 U mg -1 respectively. At equilibrium, the l-ribulose transformation rate was nearly 32 % (6.34 g L -1 ) using 20 g L -1 of l-ribose. The results revealed that the Mm-LRIse enzyme has a potential for L-ribulose production from l-ribose., Competing Interests: Declaration of Competing Interest The authors declare that they do not have any conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glucose isomerase (GIase), an efficient enzyme in the isomerization of d-glucose to d-fructose, has been widely used in food processing. In this study, an efficient expression system for a Thermobifida fusca GIase (GIase Tfus ) in Escherichia coli was firstly designed via a two-stage feeding strategy for improving expression level. The cultivation strategy was performed at an exponential feeding rate during the pre-induction phase, followed by a gradient-decreasing feeding rate at the induction phase in a 3-L fermenter. During this process, the effect of induction conditions and the complex nitrogen supplementation in feeding solutions on GIase Tfus production were investigated and optimized. Under the optimal conditions, the yield of GIase Tfus reached 124.1 U/mL, which is the highest expression level of GIase by recombinant E. coli reported to date. Additionally, the obtained GIase Tfus was performed to produce high fructose corn syrup (HFCS) with conversion approacing 55 % from glucose (45 %, w/v) to fructose. According to the molecular dynamic simulation, a number of hydrogen bonds existed in the enzyme-substrate complex could stablilize the transient states, and a appreciate reaction distance of M1 catalytic site and oxygen atom of glucose make the reaction proceed easily, thus resulting in the efficient biosynthesis of HFCS. The function of GIase Tfus renders it a valuable catalyst for HFCS-55 (containing 55 % d-fructose) manufacturing, the most favorable industrial product of HFCS. The efficient expression of GIase Tfus and its efficient HFCS production lays the foundation for its proming industrial application., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Trypanosoma cruzi ribose-5-phosphate isomerase B (TcRpiB) is a crucial piece in the pentose phosphate pathway and thus is a potential drug target for treatment of Chagas' disease. TcRpiB residues, such as Cys69, Asp45, Glu149 and Pro47, have confirmed their roles in substrate recognition, catalytic reaction and binding site conformation. However, the joint performance of His11 and His102, in the D-ribose-5-phosphate (R5P) in the catalysis is not well understood. In this work, we probed the influence of different protonation states of His11 and His102 on the behavior of the ligand R5P using molecular dynamics simulations, network analysis and thermodynamic integration. Simulations revealed that a protonated His11 combined with a neutral His102 (His11 + ‒His102) was able to stabilize the ligand R5P in the binding site. Moreover, calculated relative free energy differences showed that when protonated His11 was coupled to a neutral His102 an exergonic process takes place. On the other hand, neutral His11 combined with a protonated His102 (His11‒His102 + ), sampled conformations that resembled the catalyzed product D-ribulose-5-phosphate (Ru5P). Network analysis also demonstrated some peculiarities for these systems with some negatively correlated nodes in the binding site for His11‒His102 + , and exclusive suboptimal paths for His11 + ‒His102. Therefore, the combined approach presented in this paper proposes two suitable protonation states for the TcRpiB catalytic mechanism, where an extra proton in either histidines might favor R5P binding or influence isomerization reaction to Ru5P. Our results may guide further in silico drug discovery studies. Communicated by Ramaswamy H. Sarma.

Tuberculosis is one of the deadliest diseases worldwide affecting approximately 10 million people in 2018. This classifies tuberculosis as epidemic in several countries and leads to an increasing number of multidrug-resistant strains. Thus, the development of new drugs is essential to effective treatments. A potential drug target is the ribose-5-phosphate isomerase, a ubiquitous enzyme important to nucleotide and cofactor biosynthesis. Here, we report the backbone assignment of ribose-5-phosphate isomerase of Mycobacterium tuberculosis (MtRpiB) that has been performed by triple resonance sequential approach using a [ 13 C, 15 N, 2 H]-labeled protein. This is the first ribose-5-phosphate isomerase, an enzyme previously classified as highly druggable, to be assigned. These data will be important to further screening studies to find inhibitors and determine their interaction with MtRpiB.

D-Allose is a rare sugar, can be used as an ingredient in a range of foods and dietary supplements, has alimentary activities, especially excellent anti-cancer effects and used in assisting cancer chemotherapy and radiotherapy, etc. To develop a simple and low-cost process for D-allose production, a one-pot enzymatic process using the substrate of D-fructose, and the recombinant enzymes of D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) was developed. These enzymes were cloned from Ruminococcus sp. and B. subtilis, respectively, successfully expressed in E. coli, extracted and immobilized using anion exchange resin and amino resin, respectively. The mass ratio of D-fructose, D-psicose and D-allose was 6.6:2.4:1.0 when the reaction reached equilibrium after 5 h of reaction. Using the low-cost substrate of D-fructose, the reusable immobilized enzymes and the one-pot reaction, the production process is simplified and the production cost is decreased. In addition, to simplify the enzyme extraction and immobilization processes, new methods for enzyme capture and immobilization were developed especially for DPE immobilization. This is the first report for one-pot D-allose production using immobilized L-RhI and DPE.

The glucose isomerase GICA from Caldicoprobacter algeriensis was immobilized by ionic adsorption on polymethacrylate carriers (Sepabeads EC-EA and EC-HA) or covalent attachment to glyoxal agarose. The Sepabeads EC-HA yielded the highest recovery of activity (89%). The optimum temperature and pH of immobilized GICA were 90 °C and 7.0, respectively, similar to the corresponding values of free enzyme. Nevertheless, the adsorbed enzyme displayed higher relative activity at acidic pH, greater thermostability, and better storage stability, compared to the free form. Moreover, the immobilized enzyme showed an excellent operational stability, in 15 successive 3 h reaction cycles at 85 °C under a batch reactor, preserving 83% of its initial activity. Interestingly, a continuous process for High Fructose Syrup (HFS) production was established with the adsorbed GICA using a packed bed reactor during eleven days at 70 °C. HPAEC-PAD analysis showed a maximum bioconversion rate of 49% after 48 h of operation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

d-Tagatose is a rare monosaccharide that is used in products in the food industry as a low-calorie sweetener. To facilitate biological conversion of d-tagatose, the agarolytic enzyme complexes based on the principle of the cellulosome structure were constructed through dockerin-cohesin interaction with the scaffoldin. The construction of agarolytic complexes composed of l-arabinose isomerase caused efficient isomerization activity on the agar-derived sugars. In a trienzymatic complex, the chimeric β-agarase (cAgaB) and anhydro-galactosidase (cAhgA) from Zobellia galactanivorans could synergistically hydrolyze natural agar substrates and l-arabinose isomerase (LsAraA Doc) from Lactobacillus sakei 23K could convert d-galactose into d-tagatose. The trienzymatic complex increased the concentration of d-tagatose from the agar substrate to 4.2 g/L. Compared with the monomeric enzyme, the multimeric enzyme showed a 1.4-fold increase in tagatose production, good thermostability, and reusability. A residual activity of 75% remained, and 52% of conversion was noted after five recycles. These results indicated that the dockerin-fused chimeric enzymes on the scaffoldin successfully isomerized d-galactose into d-tagatose with synergistic activity. Thus, the results demonstrated the possibility of advancing efficient strategies for utilizing red algae as a biomass source to produce d-tagatose in the industrial food field that uses marine biomass as the feedstock.

L-fuculose is a rare sugar that is useful for the agriculture and medicine industries. L-fucose isomerase (E.C.5.3.1.25), which is an aldose-ketose isomerase, plays a significant role in producing rare sugars. A recommended L-fucose isomerase gene was cloned from Caldanaerobius polysaccharolyticus and purified with a single band of 65 kDa using nickel-affinity chromatography, with a specific activity of 108.23 U mg -1 . The native molecular mass existed with 214 kDa was a trimer. The purified enzyme showed a maximum activity in 1 mM Mn 2+ at 55 °C and pH 6.5 with a melting temperature (T m ) of 80.3 °C in the presence of one molecule per monomer. L-fucose isomerase from C. polysaccharolyticus (Capo-LfIase) exhibited the highest activity of L-fucose with K m , k cat and K cat /k m values of 94.2 mM, 23854 min -1 and 253.3 min -1 mM -1 , respectively. Capo-LfIase showed more than 50% thermostability after 20 h of incubation at 45, 55, 65, 75 and 85 °C. The 9 putative active site residues of the L-fucose substrate were described using a homology model, and the results showed that Tyr440, Met185, Trp499 and Asn527 are the candidates of metal-binding residues, while Ser393, Glu337, Glu302, His528 and Asp361 would be involved in substrate binding. The conversion rate of L-fuculose from L-fucose was almost 28.2%, with 80 g L -1 L-fucose, and no byproduct was found. To the best of our knowledge, Capo-LfIase produces high yield of L-fuculose from L-fucose by enzymatic methods., Competing Interests: Declaration of Competing Interest The authors declare that they do not have any conflict of interest regarding the publication of this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

d-Ribulose and l-fuculose are potentially valuable rare sugars useful for anticancer and antiviral drugs in the agriculture and medicine industries. These rare sugars are usually produced by chemical methods, which are generally expensive, complicated and do not meet the increasing demands. Furthermore, the isomerization of d-arabinose and l-fucose byDd-arabinose and l-fucose by d-arabinose isomerase from bacterial sources for the production of d-ribulose and l-fuculose have not yet become industrial due to the shortage of biocatalysts, resulting in poor yield and high cost of production. In this study, a thermostable d-ribulose- and l-fuculose producing d-arabinose isomerase from the bacterium Thermanaeromonas toyohensis was characterized. The recombinant d-arabinose isomerase from T. toyohensis (Thto-DaIase) was purified with a single band at 66 kDa using His-trap affinity chromatography. The native enzyme existed as a homotetramer with a molecular weight of 310 kDa, and the specific activities for both d-arabinose and l-fucose were observed to be 98.08 and 85.52 U mg -1 , respectively. The thermostable recombinant Thto-DaIase was activated when 1 mM Mn 2+ was added to the reactions at an optimum pH of 9.0 at 75 °C and showed approximately 50% activity for both d-arabinose and l-fucose at 75 °C after 10 h. The Michaelis-Menten constant (K m ), the turnover number (k cat ) and catalytic efficiency (k cat /K m ) for d-arabinose/l-fucose were 111/81.24 mM, 18,466/10,688 min -1 , and 166/132 mM -1  min -1 , respectively. When the reaction reached to equilibrium, the conversion rates of d-ribulose from d-arabinose and l-fuculose from l-fucose were almost 27% (21 g L -1 ) and 24.88% (19.92 g L -1 ) from 80 g L -1 of d-arabinose and l-fucose, respectively., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In contrast to twinning by merohedry, the reciprocal lattices of the different domains of non-merohedral twins do not overlap exactly. This leads to three kinds of reflections: reflections with no overlap, reflections with an exact overlap and reflections with a partial overlap of a reflection from a second domain. This complicates the unit-cell determination, indexing, data integration and scaling of X-ray diffraction data. However, with hindsight it is possible to detwin the data because there are reflections that are not affected by the twinning. In this article, the successful solution and refinement of one mineral, one organometallic and two protein non-merohedral twins using a common strategy are described. The unit-cell constants and the orientation matrices were determined by the program CELL_NOW. The data were then integrated with SAINT. TWINABS was used for scaling, empirical absorption corrections and the generation of two different data files, one with detwinned data for structure solution and refinement and a second one for (usually more accurate) structure refinement against total integrated intensities. The structures were solved by experimental phasing using SHELXT for the first two structures and SHELXC/D/E for the two protein structures; all models were refined with SHELXL., (open access.)

In the present contribution, multicomplex-based pharmacophore studies were carried out on the structural proteome of Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase. Among the constructed models, a representative model with complementary features, accountable for the inhibition was used as a primary filter for the screening of database molecules. Auxiliary evaluations of the screened molecules were performed via drug-likeness and molecular docking studies. Subsequently, the stability of the docked inhibitors was envisioned by molecular dynamics simulations, principle component analysis, and molecular mechanics-Poisson-Boltzmann surface area-based free binding energy calculations. The stability assessment of the hits was done by comparing with the reference (beta-substituted fosmidomycin analog, LC5) to prioritize more potent candidates. All the complexes showed stable dynamic behavior while three of them displayed higher binding free energy compared with the reference. The work resulted in the identification of the compounds with diverse scaffolds, which could be used as initial leads for the design of novel PfDXR inhibitors., (© 2019 Wiley Periodicals, Inc.)

Xylose isomerase (XI) is an industrially important metalloprotein studied for decades. Its reaction mechanism has been postulated to involve movement of the catalytic metal cofactor to several different conformations. Here, a dose-dependent approach was used to investigate the radiation damage effects on XI and their potential influence on the reaction mechanism interpreted from the X-ray derived structures. Radiation damage is still one of the major challenges for X-ray diffraction experiments and causes both global and site-specific damage. In this study, consecutive high-resolution data sets from a single XI crystal from the same wedge were collected at 100 K and the progression of radiation damage was tracked over increasing dose (0.13-3.88 MGy). The catalytic metal and its surrounding amino acid environment experience a build-up of free radicals, and the results show radiation-damage-induced structural perturbations ranging from an absolute metal positional shift to specific residue motions in the active site. The apparent metal movement is an artefact of global damage and the resulting unit-cell expansion, but residue motion appears to be driven by the dose. Understanding and identifying radiation-induced damage is an important factor in accurately interpreting the biological conclusions being drawn.

A novel glucose isomerase gene from the thermophilic Caldicoprobacter algeriensis, encoding a polypeptide of 438 residues, was identified, cloned and successfully expressed in E. coli. The purified enzyme (GICA) was a homotetramer of about 200 kDa displaying the highest activity at pH 7.0 and 90 °C and retaining 97% of its maximum activity at pH 6.5. The enzyme showed an excellent thermostability with a half-life of 6 min at 100 °C. Interestingly, GICA had a very high affinity of 40 mM and catalytic efficiency of 194 min -1  mM -1 toward d-glucose at 90 °C. A maximum of 54.7% d-glucose to d-fructose conversion was achieved by GICA at 85 °C making it an attractive candidate for HFCS-55 production. The primary sequence inspection and molecular modeling studies revealed that the thermal stability of GICA could be attributed to the presence of extra charged residues at the surface like E108 and Q408 increasing surface charge interactions. Moreover, a serine at position 56 near to P58 could establish hydrogen bond strengthening the dimer attachment. The high catalytic efficiency and affinity of GICA could be ascribed to the presence of amino acid like E108 and K62 that created more charges around the catalytic site entry., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We have developed a handling-free mounting method for X-ray crystallography of protein crystals at room temperatures-the glass capillary method. In this method, crystals were nucleated and grown on the capillary walls, and then, growth solutions were gently removed. The procedures for collecting data on the crystals were conducted by simply setting the capillary on the goniometer of a synchrotron beamline without touching the crystals. Crystal quality was characterized using mosaicity, resolution at I/σ(I) = 2, I/σ(I) at resolution = 2.0 Å, R merge , and completeness. Wilson plots were also used to characterize the quality of crystals. In particular, all samples showed very low mosaicity; the handling-free method successfully retained their low mosaicity and effectively maintained the crystal quality.

The rare sugar l-ribulose is produced from the abundant sugar l-arabinose by enzymatic conversion. An l-arabinose isomerase (AI) from Geobacillus thermodenitrificans was efficiently expressed and encapsulated in Saccharomyces cerevisiae spores. Deletion of the yeast OSW2 gene, which causes a mild defect in the integrity of the spore wall, substantially improved the activity of encapsulated AI, without damaging its superior enzymatic properties of thermostability, pH tolerance,and resistance toward SDS and proteinase treatments. In a 10 mL reaction, 100 mg of dry AI encapsulated in spores produced 250 mg of l-ribulose from 1 g of l-arabinose, indicating a 25% conversion rate. Notably, the product of l-ribulose was directly purified from the reaction solution with an approximately 91% recovery using a Ca 2+ ion exchange column. Our results describe not only a facile approach for the production of l-ribulose but also a useful strategy for the enzymatic conversion of rare sugars in "Izumoring".

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI ( Pb XI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that Pb XI is not a cold-active enzyme, but displays optimal activity at 60°C. We solved the crystal structure of Pb XI at 1.94-Å resolution to investigate the origin of its thermostability. The Pb XI structure shows a (β/α) 8 -barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the β4-α6 loop region, was newly identified and is thought to be important for the stability of Pb XI. Compared with previously determined thermostable and mesophilic XI structures, the β1-α2 loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible β1-α2 loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate lowtemperature purpose-specific XI enzymes.

N-Acetylglucosamine-6-phosphate deacetylase (NagA) and glucosamine-6-phosphate deaminase (NagB) are branch point enzymes that direct amino sugars into different pathways. For Staphylococcus aureus NagA, analytical ultracentrifugation and small-angle X-ray scattering data demonstrate that it is an asymmetric dimer in solution. Initial rate experiments show hysteresis, which may be related to pathway regulation, and kinetic parameters similar to other bacterial isozymes. The enzyme binds two Zn 2+ ions and is not substrate inhibited, unlike the Escherichia coli isozyme. S. aureus NagB adopts a novel dimeric structure in solution and shows kinetic parameters comparable to other Gram-positive isozymes. In summary, these functional data and solution structures are of use for understanding amino sugar metabolism in S. aureus, and will inform the design of inhibitory molecules., (© 2018 Federation of European Biochemical Societies.)

L-arabinose isomerase (L-AI) (EC 5. 3. 1. 4. L-AI) that mediates the isomerization of D-galactose to D-tagatose was isolated from Lactobacillus brevis (MF 465792), and was further purified and characterized. Pure enzyme with molecular weight of 60.1 kDa was successfully obtained after the purification using Native-PAGE gel extraction method, which was a monomer in solution. The L-AI was found to be stable at 45-75 °C, and at pH 7.0-9.0. Its optimum temperature and pH was determined as 65 °C and 7.0, respectively. Besides, we found that Ca 2+ , Cu 2+ , and Ba 2+ ions inhibited the enzyme activity, whereas the enzyme activity was significantly enhanced in the presence of Mg 2+ , Mn 2+ , or Co 2+ ions. The optimum concentration of Mn 2+ and Co 2+ was determined to be 1 mM. Furthermore, we characterized the kinetic parameters for L-AI and determined the K m (129 mM) and the V max (0.045 mM min - 1 ) values. Notably, L. brevisL-AI exhibited a high bioconversion yield of 43% from D-galactose to D-tagatose under the optimal condition, and appeared to be a more efficient catalyst compared with other L-AIs from various organisms.