New insights into thermostable iron-containing/activated alcohol dehydrogenases from hyperthermophiles.

Alcohol dehydrogenase (ADH) is an important enzyme that catalyzes alcohol oxidation and/or aldehyde reduction. As one of NAD+-dependent ADH types, iron-containing/activated ADH (Fe-ADH) is ubiquitous in Bacteria, Archaea, and Eukaryotes, possessing a similar "tunnel-like" structure that is composed of a domain A in its N-terminus and a domain B in its C-terminus. A conserved "GGGS" sequence in the domain A of Fe-ADH associates with NAD+, and one conserved Asp residue and three conserved His residues in the domain B are its catalytic active sites by surrounding with Fe atom, suggesting that it might employ similar catalytic mechanism. Notably, all the biochemically characterized Fe-ADHs from hyperthermophiles that thrive in above 80 °C possess two unique characteristics that are absent in other Fe-ADHs: thermophilicity and thermostability, thereby demonstrating that they can oxidize alcohol and reduce aldehyde at high temperature. Considering these two unique characteristics, Fe-ADHs from hyperthermophiles are potentially industrial biocatalysts for alcohol and aldehyde biotransformation at high temperature. Herein, we reviewed structural and biochemical characteristics of Fe-ADHs from hyperthermophiles, focusing on similarity and difference between Fe-ADHs from hyperthermophiles and their homologs from non-hyperthermophiles, and between hyperthermophilic archaeal Fe-ADHs and bacterial homologs. Furthermore, we proposed future directions of Fe-ADHs from hyperthermophiles.

Archaeal DNA alkylation repair conducted by DNA glycosylase and methyltransferase.

Alkylated bases in DNA created in the presence of endogenous and exogenous alkylating agents are either cytotoxic or mutagenic, or both to a cell. Currently, cells have evolved several strategies for repairing alkylated base. One strategy is a base excision repair process triggered by a specific DNA glycosylase that is used for the repair of the cytotoxic 3-methyladenine. Additionally, the cytotoxic and mutagenic O6-methylguanine (O6-meG) is corrected by O6-methylguanine methyltransferase (MGMT) via directly transferring the methyl group in the lesion to a specific cysteine in this protein. Furthermore, oxidative DNA demethylation catalyzed by DNA dioxygenase is utilized for repairing the cytotoxic 3-methylcytosine (3-meC) and 1-methyladenine (1-meA) in a direct reversal manner. As the third domain of life, Archaea possess 3-methyladenine DNA glycosylase II (AlkA) and MGMT, but no DNA dioxygenase homologue responsible for oxidative demethylation. Herein, we summarize recent progress in structural and biochemical properties of archaeal AlkA and MGMT to gain a better understanding of archaeal DNA alkylation repair, focusing on similarities and differences between the proteins from different archaeal species and between these archaeal proteins and their bacterial and eukaryotic relatives. To our knowledge, it is the first review on archaeal DNA alkylation repair conducted by DNA glycosylase and methyltransferase. KEY POINTS: • Archaeal MGMT plays an essential role in the repair of O 6 -meG • Archaeal AlkA can repair 3-meC and 1-meA.

Biochemical and mutational studies of an endonuclease V from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A.

Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV's function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60 °C and at pH 7.0-9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg2+ is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8 kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding.

Biochemical and functional characterization of a thermostable RecJ exonuclease from Thermococcus gammatolerans.

RecJ is ubiquitous in bacteria and Archaea, and play an important role in DNA replication and repair. Currently, our understanding on biochemical function of archaeal RecJ is incomplete due to the limited reports. The genome of the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes one putative RecJ protein (Tga-RecJ). Herein, we report biochemical characteristics and catalytic mechanism of Tga-RecJ. Tga-RecJ can degrade ssDNA in the 5'-3' direction at high temperature as observed in Thermococcus kodakarensis RecJ and Pyrococcus furiosus RecJ, the two closest homologs of the enzyme. In contrasted to P. furiosus RecJ, Tga-RecJ lacks 3'-5' ssRNA exonuclease activity. Furthermore, maximum activity of Tga-RecJ is observed at 50 °C ~ 70 °C and pH 7.0-9.0 with Mn2+, and the enzyme is the most thermostable among the reported RecJ proteins. Additionally, the rates for hydrolyzing ssDNA by Tga-RecJ were estimated by kinetic analyses at 50 °C ~ 70 °C, thus revealing its activation energy (10.5 ± 0.6 kcal/mol), which is the first report on energy barrier for ssDNA degradation by RecJ. Mutational studies showed that the mutations of residues D36, D83, D105, H106, H107 and D166 in Tga-RecJ to alanine almost completely abolish its activity, thereby suggesting that these residues are essential for catalysis.

Efficiently Enantioselective Hydrogenation Photosynthesis of (R)-1-[3,5-Bis(trifluoromethyl)phenyl] ethanol over a CLEs-TiO2 Bioinorganic Hybrid Materials.

Engineering of biological pathways with man-made materials provides inspiring blueprints for sustainable drug production. (R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethanol [(R)-3,5-BTPE], as an important artificial chiral intermediate for complicated pharmaceutical drugs and biologically active molecules, is often synthesized through a hydrogenation reaction of 3,5-bis(trifluoromethyl)acetophenone (3,5-BTAP), in which enantioselectivity and sufficient active hydrogen are the key to restricting the reaction. In this work, a biohybrid photocatalytic hydrogenation system based on an artificial cross-linked enzymes (CLEs)-TiO2-Cp*Rh(bpy) photoenzyme is developed through a bottom-up engineering strategy. Here, TiO2 nanotubes in the presence of Cp*Rh(bpy) are used to transform NADP+ to NADPH during the formation of chiral alcohol intermediates from the catalytic reduction of a ketone substrate by alcohol dehydrogenase CLEs. Hydrogen and electrons, provided by water and photocatalytic systems, respectively, are transferred to reduce NADP+ to NADPH via [Cp*Rh(bpy)(H2O)]2+. With the resulting NADPH, [(R)-3,5-BTPE] is synthesized using our efficient CLEs obtained from the cell lysate by nonstandard amino acid modification. Through this biohybrid photocatalytic system, the photoenzyme-catalyzed combined reductive synthesis of [(R)-3,5-BTPE] has a yield of 41.2% after reaction for 24 h and a very high enantiomeric excess value (>99.99%). In the case of reuse, this biohybrid system retained nearly 95% of its initial catalytic activity for synthesizing the above chiral alcohol. The excellent reusability of the CLEs and TiO2 nanotubes hybrid catalytic materials highlights the environmental friendliness of (R)-3,5-BTPE production.

Succinic anhydride-based chemical modification making laccase@Cu3(PO4)2 hybrid nanoflowers robust in removing bisphenol A in wastewater.

To prepare a robust biocatalyst and enhance the removal of bisphenol A in wastewater, succinic anhydride was reacted with laccase to obtain succinic anhydride-modified laccase (SA-laccase) and then co-crystallized with Cu3(PO4)2 to form SA-laccase@Cu3(PO4)2 hybrid nanoflowers (hNFs). The activity of SA-laccase@Cu3(PO4)2 reached 5.27 U/mg, 1.86-, 2.88- and 2.15-fold those of bare laccase@Cu3(PO4)2, laccase@Ca3(PO4)2 and laccase@epoxy resin, respectively. Compared with free laccase, the obtained hNFs present enhanced activity and tolerance to pH and high temperature in the removal of BPA. Under the optimum conditions of pH 6.0 and 35 °C, BPA removal reached 93.2% using SA-laccase@Cu3(PO4)2 hNFs, which was 1.21-fold of that using free laccase. In addition, the obtained SA-laccase@Cu3(PO4)2 hNFs retained nearly 90% of their initial catalytic activity for BPA removal after 8 consecutive batch cycles. This efficient method for preparing immobilized laccase can also be further developed and improved to acquire green biocatalysts for removing persistent organic pollutants in wastewater.

Stable immobilization of aldehyde ketone reductase mutants containing nonstandard amino acids on an epoxy resin via strain-promoted alkyne-azide cycloaddition.

To avoid random chemical linkage and achieve precisely directed immobilization, mutant enzymes were obtained and immobilized using an incorporated reactive nonstandard amino acid (NSAA). For this purpose, aldehyde ketone reductase (AKR) was used as a model enzyme, and 110Y, 114Y, 143Y, 162Q and 189Q were each replaced with p-azido-l-phenylalanine (pAzF). Then, the mutant AKR was coupled to the functionalized support by strain-promoted alkyne-azide cycloaddition (SPAAC). The effects of the incorporation number and site of NSAAs on the loading and thermal stability of the immobilized AKR were examined. The results show that the mutant enzymes presented better specific activity than the wild type, except for AKR-110Y, and AKR-114Y showed 1.16-fold higher activity than the wild type. Moreover, the half-life (t 1/2) of the five-point immobilized AKR reached 106 h and 45 h, 13 and 7 times higher than that of the free enzyme at 30 °C and 60 °C, respectively. Comparison of these three types of enzymes shows that multi-point immobilization provides improved loading and thermal stability and facilitates one-step purification. We expect this platform to facilitate a fundamental understanding of precisely oriented and controllable covalent immobilization and enable bio-manufacturing paradigms for fine chemicals and pharmaceuticals.

Enhanced reductive removal of ciprofloxacin in pharmaceutical wastewater using biogenic palladium nanoparticles by bubbling H2.

To treat waste with waste and efficiently remove the organic pollutant, waste palladiums(ii) were adsorbed and reduced on microorganism surface to catalyze the reductive removal of ciprofloxacin in pharmaceutical wastewater. By optimizing conditions such as pH and temperature, the amount of biogenic palladium adsorbed and reduced on E. coli reached 139.48 mg g-1 (Pd/microorganisms). Moreover, most of the Pd(ii) was reduced to nanometer-sized Pd(0) as characterized by TEM and SEM with EDXA. Using the obtained biogenic palladium, the reductive removal of ciprofloxacin is up to 87.70% at 25 °C, 3.03 folds of that achieved in the absence of H2. The results show that waste E. coli microorganisms can efficiently adsorb and remove waste Pd(ii) and produce Bio-Pd nanoparticle catalysts in the presence of H2. This biogenic palladium presents high catalytic activity and great advantages in the reductive degradation of ciprofloxacin. Our method can also be applied to other waste metal ions to prepare the biogenic metals, facilitate their recovery and reuse in degrading organic pollutants in wastewater to achieve "treating waste using waste".

Biochemical characterization and mutational studies of a thermostable uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Uracil DNA glycosylases (UDGs) play an important role in removing uracil from DNA to initiate DNA base excision repair. Here, we characterized biochemically a thermostable UDG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba UDG), and probed its mechanism by mutational analysis. The recombinant Tba UDG cleaves exclusively uracil-containing ssDNA and dsDNA at 65°C. The enzyme displays an optimal cleavage activity at 70-75°C. Tba UDG cleaves DNA over a wide pH spectrum ranging from 4.0 to 11.0 with an optimal pH of 7.0-9.0. In addition, Tba UDG activity is independent on a divalent metal ion; however, both Zn2+ and Cu2+ completely inhibit the enzyme activity. Tba UDG activity is also inhibited by high NaCl concentration. Tba UDG removes uracil from DNA with the following preference: U≈U/G>U/T≈U/C>U/A. Kinetic results showed that Tba UDG cleaves uracil-containing ssDNA and dsDNA at a similar rate. The mutational studies showed that the E118A, N159A and H216A mutants completely abolish cleavage activity and retain compromised binding activity while the Y127A mutant displays similar cleavage and binding activities with the wild-type protein, suggesting that residues E118, N159 and H216 are essential for uracil removal and necessary for uracil recognition.