Chimeric Enzymes Research Articles

A predicted transmembrane region in plant diacylglycerol acyltransferase 2 regulates specificity toward very-long-chain acyl-CoAs

Triacylglycerols are the main constituent of seed oil. The specific fatty acid composition of this oil is strongly impacted by the substrate specificities of acyltransferases involved in lipid synthesis, such as the integral membrane enzyme diacylglycerol acyltransferase (DGAT). Two forms of DGAT, DGAT1 and DGAT2, are thought to contribute to the formation of seed oil, and previous characterizations of various DGAT2 enzymes indicate that these often are associated with the incorporation of unusual fatty acids. However, the basis of DGAT2's acyl-donor specificity is not known because of the inherent challenges of predicting structural features of integral membrane enzymes. The recent characterization of DGAT2 enzymes from Brassica napus reveals that DGAT2 enzymes with similar amino acid sequences exhibit starkly contrasting acyl-donor specificities. Here we have designed and biochemically tested a range of chimeric enzymes, substituting parts of these B. napus DGAT2 enzymes with each other, allowing us to pinpoint a region that dramatically affects the specificity toward 22:1-CoA. It may thus be possible to redesign the acyl-donor specificity of DGAT2 enzymes, potentially altering the fatty acid composition of seed oil. Further, the characterization of a DGAT2 chimera between Arabidopsis and B. napus demonstrates that the specificity regulated by this region is transferrable across species. The identified region contains two predicted transmembrane helices that appear to reoccur in a wide range of plant DGAT2 orthologues, suggesting that it is a general feature of plant DGAT2 enzymes.

The human mitochondrial enzyme BCO2 exhibits catalytic activity toward carotenoids and apocarotenoids

The enzyme β-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in Escherichia coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the 4-amino acid insertion into mouse Bco2 did not impede the chimeric enzyme's catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9',C10'. Thus, our study demonstrates that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues.

Group II Introns Generate Functional Chimeric Relaxase Enzymes with Modified Specificities through Exon Shuffling at Both the RNA and DNA Level.

Group II introns are large self-splicing RNA enzymes with a broad but somewhat irregular phylogenetic distribution. These ancient retromobile elements are the proposed ancestors of approximately half the human genome, including the abundant spliceosomal introns and non-long terminal repeat retrotransposons. In contrast to their eukaryotic derivatives, bacterial group II introns have largely been considered as harmful selfish mobile retroelements that parasitize the genome of their host. As a challenge to this view, we recently uncovered a new intergenic trans-splicing pathway that generates an assortment of mRNA chimeras. The ability of group II introns to combine disparate mRNA fragments was proposed to increase the genetic diversity of the bacterial host by shuffling coding sequences. Here, we show that the Ll.LtrB and Ef.PcfG group II introns from Lactococcus lactis and Enterococcus faecalis respectively can both use the intergenic trans-splicing pathway to catalyze the formation of chimeric relaxase mRNAs and functional proteins. We demonstrated that some of these compound relaxase enzymes yield gain-of-function phenotypes, being significantly more efficient than their precursor wild-type enzymes at supporting bacterial conjugation. We also found that relaxase enzymes with shuffled functional domains are produced in biologically relevant settings under natural expression levels. Finally, we uncovered examples of lactococcal chimeric relaxase genes with junctions exactly at the intron insertion site. Overall, our work demonstrates that the genetic diversity generated by group II introns, at the RNA level by intergenic trans-splicing and at the DNA level by recombination, can yield new functional enzymes with shuffled exons, which can lead to gain-of-function phenotypes.

Biochemical Characterization of a Bifunctional Enzyme Constructed by the Fusion of a Glucuronan Lyase and a Chitinase from Trichoderma sp.

Bifunctional enzymes created by the fusion of a glucuronan lyase (TrGL) and a chitinase (ThCHIT42) from Trichoderma sp. have been constructed with the aim to validate a proof of concept regarding the potential of the chimera lyase/hydrolase by analyzing the functionality and the efficiency of the chimeric constructions compared to parental enzymes. All the chimeric enzymes, including or nor linker (GGGGS), were shown functional with activities equivalent or higher to native enzymes. The velocity of glucuronan lyase was considerably increased for chimeras, and may involved structural modifications at the active site. The fusion has induced a slightly decrease of the thermostability of glucuronan lyase, without modifying its catalytic activity regarding pH variations ranging from 5 to 8. The biochemical properties of chitinase seemed to be more disparate between the different fusion constructions suggesting an impact of the linkers or structural interactions with the linked glucuronan lyase. The chimeric enzymes displayed a decreased stability to temperature and pH variations, compared to parental one. Overall, TrGL-ThCHIT42 offered the better compromise in terms of biochemical stability and enhanced activity, and could be a promising candidate for further experiments in the field of fungi Cell Wall-Degrading Enzymes (CWDEs).

In vitro Validation of Chimeric β-Galactosylceramidase Enzymes With Improved Enzymatic Activity and Increased Secretion.

Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disease (LSD) caused by inherited defects of the β-galactosylceramidase (GALC) gene. The infantile forms display a rapid and aggressive central and peripheral nervous system (CNS and PNS) dysfunction. No treatments are available for GLD patients. Effective gene therapy (GT) strategies for GLD require a safe and widespread delivery of the functional GALC enzyme to all affected tissues/organs, and particularly to the CNS. The use of chimeric lysosomal enzymes with increased secretion and enhanced transport across the blood-brain barrier (BBB) that boost the efficacy of GT approaches in pre-clinical models of similar neurodegenerative LSDs may benefit GLD as well. Here, we tested the safety and biological efficacy of chimeric GALC enzymes engineered to express an alternative signal peptide (iduronate-2-sulfatase – IDSsp) and the low-density lipoprotein receptor (LDLr)-binding domain from the Apolipoprotein E II (ApoE II) in GLD murine neural and hematopoietic stem/progenitor cells and progeny, which are relevant cells types in the context of in vivo and ex vivo GT platforms. We show that the lentiviral vector-mediated expression of the chimeric GALC enzymes is safe and leads to supranormal enzymatic activity in both neural and hematopoietic cells. The IDSsp.GALC shows enhanced expression and secretion in comparison to the unmodified GALC. The chimeric GALC enzymes produced by LV-transduced cells reduce intracellular galactosylceramide (GalCer) storage and effectively cross-correct GLD murine neurons and glial cells, indicating that the transgenic enzymes are delivered to lysosomes, efficiently secreted, and functional. Of note, the expression of LDLr and LDLr-related proteins in GLD neurons and glial cells supports the exploitation of this system to enhance the GALC supply in affected CNS cells and tissues. These in vitro studies support the use of chimeric GALC enzymes to develop novel and more effective GT approaches for GLD.

Chimeric cytochrome P450 3A4 used for in vitro prediction of food-drug interactions.

Inhibition of cytochrome P450 (CYP)-mediated drug metabolism by dietary substances is the main cause of drug-food interactions in humans. The present study reports on the in vitro inhibition assays of human CYP3A4 genetically linked to the reductase domain of bacterial BM3 of Bacillus megaterium (BMR) resulting in the chimeric protein CYP3A4-BMR. The activity of this chimeric enzyme was initially measured colorimetrically with erythromycin as the substrate where KM values similar to published data were determined. Subsequently, the inhibition assays with three different dietary products, grapefruit juice, curcumin, and resveratrol, were carried out with the chimeric enzyme both in solution and immobilized on electrode surfaces. For the solution studies, nicotinamide adenine dinucleotide phosphate was added as the electron donor, whereas the need for this cofactor was obviated in the immobilized enzyme as it was supplied by the electrode. Inhibition of the N-demethylation of erythromycin by CYP3A4-BMR chimera was measured at increasing concentrations of the different dietary compounds with calculated IC50 values of 0.5%, 31μM, and 250μM for grapefruit juice, curcumin, and resveratrol measured in solution compared with 0.7%, 24μM, and 208μM measured electrochemically, respectively. These data demonstrate the feasibility of the use of both CYP3A4-BMR chimera as well as bioelectrochemistry for in vitro studies of not only drug-food interactions but also prediction of adverse drug reactions in this important P450 enzyme.

From Graphite to Laccase Biofunctionalized Few-Layer Graphene: A "One Pot" Approach Using a Chimeric Enzyme.

A chimeric enzyme based on the genetic fusion of a laccase with a hydrophobin domain was employed to functionalize few-layer graphene, previously exfoliated from graphite in the presence of the hydrophobin. The as-produced, biofunctionalized few-layer graphene was characterized by electrochemistry and Raman spectroscopy, and finally employed in the biosensing of phenols such as catechol and dopamine. This strategy paves the way for the functionalization of nanomaterials by hydrophobin domains of chimeric enzymes and their use in a variety of electrochemical applications.

Assessment of combination of pretreatment of Sorghum durra stalk and production of chimeric enzyme (β-glucosidase and endo β-1,4 glucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) for saccharification to produce bioethanol

Optimization of pretreatment and saccharification of Sorghum durra stalk (Sds) was carried out. The chimeric enzyme (CtGH1-L1-CtGH5-F194A) having β-glucosidase (CtGH1) and endo β-1,4 glucanase activity (CtGH5-F194A) and cellobiohydrolase (CtCBH5A) from Clostridium thermocellum were used for saccharification. Chimeric enzyme will save production cost of two enzymes, individually. Stage 2 pretreatment by 1% (w/v) NaOH assisted autoclaving + 1.5% (v/v) dilute H2SO4 assisted oven heating gave lower total sugar yield (366.6 mg/g of pretreated Sds) and total glucose yield (195 mg/g of pretreated Sds) in pretreated hydrolysate with highest crystallinity index 55.6% than the other stage 2 pretreatments. Optimized parameters for saccharification of above stage 2 pretreated biomass were 3% (w/v) biomass concentration, enzyme (chimera: cellobiohydrolase) ratio, 2:3 (U/g) of biomass, total enzyme loading (350 U/g of pretreated biomass), 24 h and 30 °C. Best stage 2 pretreated Sds under optimized enzyme saccharification conditions gave maximum total reducing sugar yield 417 mg/g and glucose yield 285 mg/g pretreated biomass in hydrolysate. Best stage 2 pretreated Sds showed significantly higher cellulose, 71.3% and lower lignin, 2.0% and hemicellulose, 12.2% (w/w) content suggesting the effectiveness of method. This hydrolysate upon SHF using Saccharomyces cerevisiae under unoptimized conditions produced ethanol yield, 0.12 g/g of glucose. Abbreviation: Ct-Clostridium thermocellum, Sds-Sorghum durra stalk, TRS-Total reducing sugar, HPLC-High performance liquid chromatography, RI-Refractive index, ADL-acid insoluble lignin, GYE-Glucose yeast extract, MGYP-Malt glucose yeast extract peptone, SHF-separate hydrolysis and fermentation, OD-Optical density, PVDF-Poly vinylidene fluoride, TS-total sugar, FESEM-Field emission scanning electron microscopy, XRD-X-ray diffraction, FTIR-Fourier transform infra-red spectroscopy and CrI-Crystallinity index.

PQQ-Glucose Dehydrogenase-Calmodulin Chimera Enzyme: Different Triggered Activation for Multipurpose Biosensors

In the last decade, the rise of synthetic biology has driven the efforts to construct artificial allosteric protein switches in order to detected and quantify natural and artificial chemistries in vitro and in vivo. Typically, this involves construction of chimeric enzymes via insertion of a regulatory receptor domain into the biocatalytic reporter domain. Construction of such chimeric enzymes utilizes the recombinant DNA technology that is a core technology of protein engineering [1].Herein, we report on the bioelectrocatalytic properties of pyrroloquinoline quinone-dependent glucose dehydrogenase fusion with calmodulin (PQQ-GDH-CaM). This protein is catalytically inactive in its ground form but can be activated by the addition of calmodulin binding peptide that induces its conformational transition and activation. The PQQ-GDH-CaM was immobilized onto highly porous gold (hPG) produced electrochemically [2] by using a bifunctional linker, namely 4-mercaptobenzoic acid further activated through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (EDC/NHS) coupling to covalent bind the PQQ-GDH-CaM chimeric enzyme [3].At first, we characterized the switchable features of PQQ-GDH-CaM by simply exposing it to the activating peptide. Subsequently we devised an approach to use proteolytic activation of a caged-peptide to generate activating peptide for PQQ-GDH-CaM. Furthermore, the system was utilized to study different pathways for PQQ-GDH-CaM triggered activation according to different logic gates in order to realize multipurpose biosensors (e.g., glucose detection, peptide detection etc.) [4]. References Gamella, M., Guo, Z., Alexandrov, K., Katz, E. (2019). ChemElectroChem, 6(3), 638-645.Bollella, P., Hibino, Y., Kano, K., Gorton, L., Antiochia, R. (2018). Analytical chemistry, 90(20), 12131-12136.Koushanpour, A., Gamella, M., Guo, Z., Honarvarfard, E., Poghossian, A., Schöning, M. J., Alexandrov, K., Katz, E. (2017). The Journal of Physical Chemistry B, 121(51), 11465-11471.Bollella, P., Katz, E. (2019). International Journal of Unconventional Computing, 14(3-4), 181-198.

(Invited) Chimeric and Single-Site Mutant Enzymes for the Design of Nanostructured Bioelectrodes in Enzymatic Fuel Cells and Sensors

Control over redox enzyme immobilization and electrical interfacing at electrode is a major challenge when building an electrode for enzymatic sensor or fuel cell. An adequate environment has to be created in order to maximize the number of active enzymes and minimize electron transfer distances between enzyme active sites and conductive surfaces or redox partners. In this respect, the production of specific mutants is a powerful way to introduce a level of control over orientation and organization of redox enzymes at the electrode. In addition, the combination of these enzymes with nanomaterials is aimed at maximizing the number of wired enzymes per surface unit while also providing rapid electron transfer pathways and enhancing electrocatalytic current densities.1 A chimeric protein can be designed by combining a prion domain and a small iron-sulfur redox protein such as rubredoxin.2 Thanks to the ability of the prion domain to self-assemble in amyloid fibers, protein-only redox nanowires can be produced. These protein nanowires are able to entrap redox enzymes such as multicopper enzymes or [NiFe] hydrogenases, while ensuring interprotein electron transport between enzyme active sites and electrode surface.2,3 Chimeric laccases was also designed by combining this multicopper enzyme with a hydrophobin domain. In this case, hydrophobin strongly interacts with the pi-extended network of chemically-exfoliated graphene or carbon nanotubes. These biofunctionalized nanomaterials were used in electrochemical sensors for polyaromatic and phenolic compounds.4 Single-site mutation at the surface of enzymes is also a powerful means to control the immobilization and orientation of enzymes at the surface of nanostructured electrodes. We have especially investigated the covalent and non-covalent functionalization of single-site mutant laccases for the rational immobilization and direct wiring of the enzymes at the surface of carbon nanotubes and gold nanoparticles. 5 These nanomaterials are able to provide novel self-assembled nanostructured redox bioassembly or favor heterogeneous electron transfer to the enzyme by promoting specific orientations.1 A. Le Goff and M. Holzinger, Sustainable Energy Fuels, 2018, 2, 2555–2566.2 L. Altamura, C. Horvath, S. Rengaraj, A. Rongier, K. Elouarzaki, C. Gondran, A. L. B. Maçon, C. Vendrely, V. Bouchiat, M. Fontecave, D. Mariolle, P. Rannou, A. Le Goff, N. Duraffourg, M. Holzinger and V. Forge, Nat Chem, 2017, 9, 157–163.3 S. Rengaraj, R. Haddad, E. Lojou, N. Duraffourg, M. Holzinger, A. Le Goff and V. Forge, Angew. Chem. Int. Ed., 2017, 56, 7774–7778.4 I. Sorrentino, I. Stanzione, Y. Nedellec, A. Piscitelli, P. Giardina and A. Le Goff, submitted.5 N. Lalaoui, P. Rousselot-Pailley, V. Robert, Y. Mekmouche, R. Villalonga, M. Holzinger, S. Cosnier, T. Tron and A. Le Goff, ACS Catal., 2016, 6, 1894–1900.

Construction and characterization of a chimeric enzyme of swollenin and xylanase to improve soybean straw hydrolysis

A study was carried out to produce a fusion protein (Swo-Xyn) using the Trichoderma reesei swollenin and Lentinula edodes xylanase and to investigate its characteristics and application in degrading soybean straw. In parallel, L. edodes xylanase (Xyn) alone was used as a control protein in application tests. The Swo-Xyn was recombined, expressed and produced by Pichia pastoris and had the maximum activity at 40 °C and pH 3.0 using xylan as substrate. The Swo-Xyn exhibited preferential hydrolysis of Xylan. The Swo-Xyn had slight low Km value (23.90 vs. 25.36 mg/ml) but significantly low Vmax value (162.4 vs. 227.2 μmol/mg·min) and specific activity (18.82 vs. 38.97 U/mg) relative to the Xyn. The Swo-Xyn activity was enhanced by Zn2+ in dose dependent manners with the peak activity at 30 mM of Zn2+. The Swo-Xyn could tolerate 15% of methanol, ethanol, aceton, and DMSO with >60% residual activity. The Swo-Xyn had the greater tolerance to SDS, EDTA, 2-ME than the Xyn and could be activated by DTT, Triton X-100, and Tween 20. Compared with the Xyn, the hydrolysis and sequent cellulose enzymolysis of soybean straw could be better improved by the Swo-Xyn. The Swo-Xyn should be more useful for improving the utilization of soybean straw.

Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals.

The front cover artwork is provided by groups of Prof. Evgeny Katz and Prof. Artem Melman (Clarkson University, NY, USA) as well as Prof. Kirill Alexandrov (Queensland University of Technology, Brisbane, Australia). The image shows activation/inhibition of a chimeric enzyme with biomolecular signals and a corresponding logic network - the artistic vision. Read the full text of the Communication at 10.1002/cphc.201901050.

Front Cover: Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals (ChemPhysChem 7/2020)

Front Cover: Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals (ChemPhysChem 7/2020)

Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10-13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

Molecular insights into the mechanism of substrate recognition of Streptomyces transglutaminases.

The microbial TGase from Streptomyces mobaraensis has used in various food industries. However, the detailed substrate specificities of TGases from the Streptomyces species toward the natural peptides remains to be unclear. In this study, we conducted the comparison of two different TGases from Streptomyces mobaranensis (SMTG) and Streptomyces cinnamoneus (SCTG). To clarify the region associated with the characteristics of enzymes, we constructed a chimeric enzyme of CM, of which is consisted of N-terminal half of SCTG and C-terminal half of SMTG. To reveal the differences in the substrate specificity between SCTG and SMTG toward natural peptides, we investigated the time dependence of TGase activity on the productivity of cross-linking peptide with tryptic casein and lysine by using LC-MS. We identified two peptides of "VLPVPQK" and "AVPYPQR" as substrates for both of the TGases.

The Protective Effect of a Chimeric PSH Antioxidant Enzyme in Renal Ischemia–Reperfusion Injury

Abstract—A rapid increase in the concentration of free radicals and reactive oxygen species at the reperfusion stage is the most dangerous stage of ischemia–reperfusion injury. An avalanche-like increase in the level of reactive oxygen species and secondary products of free radical oxidation of biological macromolecules leads to the development of oxidative stress. The use of exogenous antioxidants can reduce the concentration of reactive oxygen species in the affected tissues and suppress or correct the course of oxidative stress; thus, it significantly reduces the severity of ischemia–reperfusion injury. Acute ischemic renal failure is one of the most important social problems in a comprehensive list of pathologies associated with ischemia–reperfusion. The nephroprotective effect of a chimeric PSH antioxidant enzyme that includes human peroxiredoxin 6 and Mn-containing superoxide dismutase of Escherichia coli has been shown on an animal model of bilateral ischemia–reperfusion renal injury. The recombinant chimeric PSH protein was able to neutralize a maximally possible wide range of reactive oxygen species due to the superoxide dismutase and peroxidase activities. It has been shown with histological, biochemical, and molecular biological methods that the preliminary administration of the chimeric PSH protein before ischemia–reperfusion significantly reduced the degree of renal tissue injury and led to a quick normalization of their structural and functional state. In addition, the administration of the PSH enzyme increased the survival of experimental animals by a factor of more than 1.5. The use of the recombinant chimeric PSH enzyme can be an effective approach in the prevention and treatment of renal ischemia–reperfusion injury, as well as for maintaining an isolated kidney during transplantation.

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity.

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNAAsp and tRNAAsn generating a correctly charged Asp-tRNAAsp and an erroneous Asp-tRNAAsn . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNAAsp over tRNAAsn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNAAsp and was unable to aspartylate tRNAAsn . The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.

Trienzymatic Complex System for Isomerization of Agar-Derived d-Galactose into d-Tagatose as a Low-Calorie Sweetener.

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.

Designing chimeric enzymes inspired by fungal cellulosomes

Cellulosomes are synthesized by anaerobic bacteria and fungi to degrade lignocellulose via synergistic action of multiple enzymes fused to a protein scaffold. Through templating key protein domains (cohesin and dockerin), designer cellulosomes have been engineered from bacterial motifs to alter the activity, stability, and degradation efficiency of enzyme complexes. Recently a parts list for fungal cellulosomes from the anaerobic fungi (Neocallimastigomycota) was determined, which revealed sequence divergent fungal cohesin, dockerin, and scaffoldin domains that could be used to expand the available toolbox to synthesize designer cellulosomes. In this work, multi-domain carbohydrate active enzymes (CAZymes) from 3 cellulosome-producing fungi were analyzed to inform the design of chimeric proteins for synthetic cellulosomes inspired by anaerobic fungi. In particular, Piromyces finnis was used as a structural template for chimeric carbohydrate active enzymes. Recombinant enzymes with retained properties were engineered by combining thermophilic glycosyl hydrolase domains from Thermotoga maritima with dockerin domains from Piromyces finnis. By preserving the protein domain order from P. finnis, chimeric enzymes retained catalytic activity at temperatures over 80 °C and were able to associate with cellulosomes purified from anaerobic fungi. Fungal cellulosomes harbor a wide diversity of glycoside hydrolases, each representing templates for the design of chimeric enzymes. By conserving dockerin domain position within the primary structure of each protein, the activity of both the catalytic domain and dockerin domain was retained in enzyme chimeras. Taken further, the domain positioning inferred from native fungal cellulosome proteins can be used to engineer multi-domain proteins with non-native favorable properties, such as thermostability.

Evolution-guided engineering of non-heme iron enzymes involved in nogalamycin biosynthesis.

Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. The key to the formation of this chemical diversity has been the rapid evolution of secondary metabolism. Many enzymes residing on these metabolic pathways have acquired atypical catalytic properties in comparison with their counterparts found in primary metabolism. The biosynthetic pathway of the anthracycline nogalamycin contains two such proteins, SnoK and SnoN, belonging to nonheme iron and 2-oxoglutarate-dependent mono-oxygenases. In spite of structural similarity, the two proteins catalyze distinct chemical reactions; SnoK is a C2-C5″ carbocyclase, whereas SnoN catalyzes stereoinversion at the adjacent C4″ position. Here, we have identified four structural regions involved in the functional differentiation and generated 30 chimeric enzymes to probe catalysis. Our analyses indicate that the carbocyclase SnoK is the ancestral form of the enzyme from which SnoN has evolved to catalyze stereoinversion at the neighboring carbon. The critical step in the appearance of epimerization activity has likely been the insertion of three residues near the C-terminus, which allow repositioning of the substrate in front of the iron center. The loss of the original carbocyclization activity has then occurred with changes in four amino acids near the iron center that prohibit alignment of the substrate for the formation of the C2-C5″ bond. Our study provides detailed insights into the evolutionary processes that have enabled Streptomyces soil bacteria to become the major source of antibiotics and antiproliferative agents. ENZYMES: EC number 1.14.11.