Strategies and procedures to generate chimeric DNA polymerases for improved applications.

Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation. KEY POINTS: • Chimeric DNA polymerase is generated by rational design method. • Strategies include domain exchange and addition of proteins, domains, and motifs. • Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions.

Sialidase-Chimeric Bioengineered Bacteria for Tumor-Sialoglycan-Triggered Solid Tumor Therapy.

Adoptive cell therapies for solid tumors are usually limited by off-target antigens, incapable tissue infiltration, and cell function exhaustion. In contrast, bacterial cells possess the inherent competencies of preferential tumor targeting, deep tissue penetration, and high intratumoral bioactivity and represent promising alternatives to overcome these challenges. Here, a sialic-acid-responsive regulatory gene circuit is engineered into Escherichia coli MG1655 to express cytolysin of hemolysin E (HlyE). Furthermore, sialidases are bioorthogonally decorated onto the surface of azido-functionalized bioengineered bacteria for recognizing tumor sialoglycans and cleaving their sialosides into free sialic acids. As chemical inducers, sialic acids feedbackingly activate the bacterial gene circuit to produce HlyE and lyse tumor cells. This study mimics the tumor antigen-induced cytotoxin production and cell lysis that occurs in chimeric antigen receptor T (CAR-T) cells yet surmounts the intrinsic limitations of adoptive cell therapies. Moreover, sialidase-mediated tumor cell desialylation also reverses the immunosuppressive effect of glycoimmune checkpoints and further improves the therapeutic effect of solid tumors.

Protein engineering a PhotoRNR chimera based on a unifying evolutionary apparatus among the natural classes of ribonucleotide reductases.

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5'-di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C-terminal tail of the radical-generating ß subunit to render a chimeric RNR. Inspired by a recent cryo-EM structure, a [Re] photooxidant is located adjacent to Y356[ß], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y356• along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism-based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any ß metallocofactor highlights the adaptability of the 10-stranded αß barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs.

An enhanced activity and thermostability of chimeric Bst DNA polymerase for isothermal amplification applications.

Loop-mediated isothermal amplification (LAMP) is a widely used method for clinical diagnosis, customs quarantine, and disease prevention. However, the low catalytic activity of Bst DNA polymerase has made it challenging to develop rapid and reliable point-of-care testing. Herein, we developed a series of Bst DNA polymerase mutants with enhanced activity by predicting and analyzing the activity sites. Among these mutants, single mutants K431D and K431E showed a 1.93- and 2.03-fold increase in catalytic efficiency, respectively. We also created a chimeric protein by fusing the DNA-binding domain of DNA ligase from Pyrococcus abyssi (DBD), namely DBD-K431E, which enabled real-time LAMP at high temperatures up to 73 ℃ and remained active after heating at 70 ℃ for 8 h. The chimeric DBD-K431E remained active in the presence of 50 U/mL heparin, 10% ethanol, and up to 100 mM NaCl, and showed higher activity in 110 mM (NH4)2SO4, 110 mM KCl, and 12 mM MgSO4. Notably, it generated a fluorescence signal during the detection of Salmonella typhimurium at 2 × 102 ag/µL of genomic DNA and 1.24 CFU/mL of bacterial colony, outperforming the wild type and the commercial counterpart Bst 2.0. Our results suggest that the DBD-K431E variant could be a promising tool for general molecular biology research and clinical diagnostics. KEY POINTS: • Residue K431 is probably a key site of Bst DNA polymerase activity • The chimeric DBD-K431E is more inhibitor tolerant and thermostable than Bst-LF • The DBD-K431E variant can detect Salmonella typhimurium at 102 ag/µL or 100 CFU/mL.

Functional identification of two novel carbohydrate-binding modules of glucuronoxylanase CrXyl30 and their contribution to the lignocellulose saccharification.

BACKGROUND: Glycoside hydrolase (GH) family 30 xylanases are a distinct group of xylanases, most of which have a highly specific catalytic activity for glucuronoxylan. Since GH30 xylanases do not normally carry carbohydrate-binding modules (CBMs), our knowledge of the function of their CBMs is lacking. RESULTS: In this work, the CBM functions of CrXyl30 were investigated. CrXyl30 was a GH30 glucuronoxylanase containing tandem CBM13 (CrCBM13) and CBM2 (CrCBM2) at its C terminus, which was identified in a lignocellulolytic bacterial consortium previously. Both CBMs could bind insoluble and soluble xylan, with CrCBM13 having binding specificity for the xylan with L-arabinosyl substitutions, whereas CrCBM2 targeted L-arabinosyl side chains themselves. Such binding abilities of these two CBMs were completely different from other CBMs in their respective families. Phylogenetic analysis also suggested that both CrCBM13 and CrCBM2 belong to novel branches. Inspection of the simulated structure of CrCBM13 identified a pocket that just accommodates the side chain of 3(2)-alpha-L-arabinofuranosyl-xylotriose, which forms hydrogen bonds with three of the five amino acid residues involved in ligand interaction. The truncation of either CrCBM13 or CrCBM2 did not alter the substrate specificity and optimal reaction conditions of CrXyl30, whereas truncation of CrCBM2 decreased the kcat/Km value by 83% (± 0%). Moreover, the absence of CrCBM2 and CrCBM13 resulted in a 5% (± 1%) and a 7% (± 0%) decrease, respectively, in the amount of reducing sugar released by the synergistic hydrolysis of delignified corncob whose hemicellulose is arabinoglucuronoxylan, respectively. In addition, fusion of CrCBM2 with a GH10 xylanase enhanced its catalytic activity against the branched xylan and improved the synergistic hydrolysis efficiency by more than fivefold when delignified corncob was used as substrate. Such a strong stimulation of hydrolysis resulted from the enhancement of hemicellulose hydrolysis on the one hand, and the cellulose hydrolysis is also improved according to the lignocellulose conversion rate measured by HPLC. CONCLUSIONS: This study identifies the functions of two novel CBMs in CrXyl30 and shows the good potential of such CBMs specific for branched ligands in the development of efficient enzyme preparations.

Fluorometric biosensing of α-amylase using an artificial allosteric biosensor immobilized on nanostructured interface.

Protein biosensors hold a promise to transform the way we collect physiological data by enabling quantification of biomarkers outside of specialized laboratory environment. However, achieving high specificity and sensitivity in homogeneous assay format remains challenging. Here we report construction of fluorescent biosensor arrays based on artificial allosteric α-amylase-activated PQQ-dependent glucose dehydrogenase (Amy-GDH). Amy-GDH was covalently immobilized on silica nanoparticles that were then arrayed on fiberglass sheets. The activity of the biosensor was monitored using a smartphone camera via emergence of bright fluorescence (λex 365 nm) originating from reduced phenazine methosulfate upon glucose oxidation by Amy-GDH. We show that such biosensor arrays demonstrate an apparent Kd of 115 pM for α-amylase with a detection limit of 2 pM. Using the developed biosensor arrays, we were able to specifically and accurately quantify the concentration of α-amylase in biological fluids such as serum and saliva. We propose that the presented approach can enable construction of ultrasensitive point-of-care diagnostic arrays.

Improving Thermostability of Chimeric Enzymes Generated by Domain Shuffling Between Two Different Original Glucoamylases.

In order to improve enzymatic properties of glucoamylases, six recombinant genes GA1-GA6 were created by domain shuffling of glucoamylase genes GAA1 from Aspergillus niger Ld418AI and GATE from Talaromyces emersonii Ld418 TE using overlap extension PCR and were expressed in Saccharomyces cerevisiae W303-1B; only activities of GA1 and GA2 in the fermentation broth were higher than those of GAA1 but less than those of GATE. Further research results of GA1 and GA2 indicated that chimeric glucoamylases GA1 and GA2 revealed increased thermostability compared with GAA1 and GATE, although with a slight change in the activity and optimal temperature. However, GA1 had almost the same catalytic efficiency as GATE, whereas the catalytic efficiency of GA2 was slightly less than that of GATE, but still higher than that of GAA1. The structural analysis showed that the change of enzymatic properties could be caused by the increased and extended α-helix and ß-sheet, which change the secondary and tertiary structures of chimeric glucoamylases. These results demonstrated that domain shuffling was feasible to generate a chimeric enzyme with novel properties.

A cytochrome b-glucose dehydrogenase chimeric enzyme capable of direct electron transfer.

The development of third generation biosensors depends on the availability of direct electron transfer (DET) capable enzymes. A successful strategy is to fuse a cytochrome domain to an enzyme to fulfil the function of a built-in redox mediator between the catalytic center and the electrode. In this study, we fused the cytochrome domain of Neurospora crassa CDH IIA (NcCYT) N-terminally to glucose dehydrogenase from Glomerella cingulata (GcGDH) to generate the chimeric enzyme NcCYT-GcGDH in a large amount for further studies. Heterologous expression in P. pastoris and chromatographic purification resulted in 1.8 g of homogeneous chimeric enzyme. Biochemical and electrochemical characterization confirmed that the chimeric enzyme is catalytically active, able to perform interdomain electron transfer (IET) and direct electron transfer (DET) via the fused cytochrome domain. The midpoint redox potential of the fused b-type cytochrome is 91 mV vs. SHE at pH 6.5 and the specific current obtained on a porous graphite electrode is 2.3 µA cm-2. The high current obtained on this simple, unmodified electrode at a rather low redox potential is a promising starting point for further optimization. The high yield of NcCYT-GcGDH and its high specific activity supports the application of the chimeric enzyme in bioelectrocatalytic applications.

Connecting Artificial Proteolytic and Electrochemical Signaling Systems with Caged Messenger Peptides.

Enzymatic polypeptide proteolysis is a widespread and powerful biological control mechanism. Over the last few years, substantial progress has been made in creating artificial proteolytic systems where an input of choice modulates the protease activity and thereby the activity of its substrates. However, all proteolytic systems developed so far have relied on the direct proteolytic cleavage of their effectors. Here, we propose a new concept where protease biosensors with a tunable input uncage a signaling peptide, which can then transmit a signal to an allosteric protein reporter. We demonstrate that both the cage and the regulatory domain of the reporter can be constructed from the same peptide-binding domain, such as calmodulin. To demonstrate this concept, we constructed a proteolytic rapamycin biosensor and demonstrated its quantitative actuation on fluorescent, luminescent, and electrochemical reporters. Using the latter, we constructed sensitive bioelectrodes that detect the messenger peptide release and quantitatively convert the recognition event into electric current. We discuss the application of such systems for the construction of in vitro sensory arrays and in vivo signaling circuits.

Construction and characterization of a functional chimeric laccase from metagenomes suitable as a biocatalyst.

Screening of gene-specific amplicons from metagenomes (S-GAM) is an efficient technique for the isolation of homologous genes from metagenomes. Using the S-GAM approach, we targeted multi-copper oxidase (MCO) genes including laccase and bilirubin oxidase (BOX) in soil and compost metagenomes, and successfully isolated novel MCO core regions. These core enzyme genes shared approximately 70% identity with that of the putative MCO from Micromonospora sp. MP36. According to the principle of S-GAM, the N- and C-terminal regions of the deduced products of the mature gene come from the known parent gene, which should be homologous and compatible with the target gene. We constructed two different MCO hybrid genes using Bacillus subtilis BOX and Micromonospora sp. MP36 MCO, to give Bs-mg-mco and Mic-mg-mco, respectively. The constructed chimeric MCO genes were fused with the maltose-binding protein (MBP) gene at the N-terminus for expression in Escherichia coli cells. We found that MBP-Mic-mg-MCO/Mic-mg-MCO possessed the characteristic properties of laccase, although MBP-Bs-mg-MCO had no activity. This novel laccase (Mic-mg-MCO) demonstrated unique substrate specificity, sufficient activity at neutral pH, and high thermal stability, which are suitable properties for its use as a laccase biocatalyst.

Biosynthesis of Random-Homo Block Copolymer Poly[Glycolate-ran-3-Hydroxybutyrate (3HB)]-b-Poly(3HB) Using Sequence-Regulating Chimeric Polyhydroxyalkanoate Synthase in Escherichia coli.

Glycolate (GL)-containing polyhydroxyalkanoate (PHA) was synthesized in Escherichia coli expressing the engineered chimeric PHA synthase PhaC AR and coenzyme A transferase. The cells produced poly[GL-co-3-hydroxybutyrate (3HB)] with the supplementation of GL and 3HB, thus demonstrating that PhaC AR is the first known class I PHA synthase that is capable of incorporating GL units. The triad sequence analysis using 1H nuclear magnetic resonance indicated that the obtained polymer was composed of two distinct regions, a P(GL-ran-3HB) random segment and P(3HB) homopolymer segment. The random segment was estimated to contain a 71 mol% GL molar ratio, which was much greater than the value (15 mol%) previously achieved by using PhaC1 P s STQK. Differential scanning calorimetry analysis of the polymer films supported the presence of random copolymer and homopolymer phases. The solvent fractionation of the polymer indicated the presence of a covalent linkage between these segments. Therefore, it was concluded that PhaC AR synthesized a novel random-homo block copolymer, P(GL-ran-3HB)-b-P(3HB).

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.

High-level expression of Bacillus amyloliquefaciens laccase and construction of its chimeric variant with improved stability by domain substitution.

Large-scale application of bacterial laccases is usually limited by their low production, and their recombinant expression in Escherichia coli is prone to form inactive aggregates in the cytoplasm. In this work, we optimized the expression conditions of Bacillus amyloliquefaciens laccase (LacA) in E. coli, and obtained high yield for the extracellular production of LacA. The final activity reached 20,255 U/L for LacA, which is among one of the highest activities for recombinant bacterial laccases. Moreover, a chimeric enzyme (Lac3A/S) was designed based on LacA by domain substitution with a stable laccase from B. subtilis. The hybrid laccase could also be secreted into the culture medium with high expression level, and had higher thermal and alkaline stabilities than those of LacA. It was fully active after 10-day incubation at pH 9.0, and retained 47% of its initial activity after incubation at 70 °C for 5 h. Homology analysis of protein structure indicated Lac3A/S had a more packed structure in the copper-binding sites than LacA, which might lead to an enhancement in stability under harsh conditions.

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

Reactions catalyzed by artificial allosteric enzymes, chimeric proteins with fused biorecognition and catalytic units, were used to mimic multi-input Boolean logic systems. The catalytic parts of the systems were represented by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH). Two biorecognition units, calmodulin or artificial peptide-clamp, were integrated into PQQ-GDH and locked it in the OFF or ON state respectively. The ligand-peptide binding cooperatively with Ca2+ cations to a calmodulin bioreceptor resulted in the enzyme activation, while another ligand-peptide bound to a clamp-receptor inhibited the enzyme. The enzyme activation and inhibition originated from peptide-induced allosteric transitions in the receptor units that propagated to the catalytic domain. While most of enzymes used to mimic Boolean logic gates operate with two inputs (substrate and co-substrate), the used chimeric enzymes were controlled by four inputs (glucose - substrate, dichlorophenolindophenol - electron acceptor/co-substrate, Ca2+ cations and a peptide - activating/inhibiting signals). The biocatalytic reactions controlled by four input signals were considered as logic networks composed of several concatenated logic gates. The developed approach allows potentially programming complex logic networks operating with various biomolecular inputs representing potential utility for different biomedical applications.

Characterization of Thermostable and Chimeric Enzymes via Isopeptide Bond-Mediated Molecular Cyclization.

Mannooligosaccharides are released by mannan-degrading endo-ß-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability and resilience to aggregation and freeze-thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.

Evidence for the participation of an extra α-helix at ß-subunit surface in the thermal stability of Co-type nitrile hydratase.

Nitrile hydratase (NHase) has attracted considerable attention since it can efficiently catalyze the hydration of nitriles to valuable amides. However, the poor stability of NHase is one of the main drawbacks in the industrial application. In this study, we compared the structural difference between Fe-type and Co-type NHase and found that an extra α helix existed at the ß-subunit surface of Co-type NHase (defined as the ß-6th helix). Then, the effects of the ß-6th helix were investigated on the thermal stability and the catalytic kinetics of a Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229). When the ß-6th helix was deleted or disrupted, the thermal stability of NHase1229 was reduced to 17.6 and 12.9% of that of wild NHase1229, respectively. Thus, the ß-6th helix is important for the thermal stability of Co-type NHase. Based on the structural characteristics of Co-type NHase, the ß-6th helix may be interacted with another helix at the α-subunit (defined as the α-2nd helix) by hydrophobic network just as a "magnetic suction buckle" on the enzyme surface to stabilize the binding of α- and ß-subunits. The ß-6th helix is located at the mouth of the substrate and product tunnel, so it plays crucial roles in catalytic process. Furthermore, the ß-6th helix in NHase1229 was swapped with a thermophilic NHase fragment from Pseudonocardia thermophila JCM3095 (NHase1229-Swap). The thermal stability of NHase1229-Swap was significantly improved, and the half-life was approximately 2.4-fold at 40 °C than that of the wild NHase1229. The knowledge is useful for improving the stability of NHases by restriction fragment swapping.

A single amino acid residue regulates the substrate affinity and specificity of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme that catalyses the oxidative cleavage of L-Trp. The ciliate Blepharisma stoltei has four IDO genes (IDO-I, -II, -III and -IV), which seem to have evolved via two sequential gene duplication events. Each IDO enzyme has a distinct enzymatic property, where IDO-III has a high affinity for L-Trp, whereas the affinity of the other three isoforms for L-Trp is low. IDO-I also exhibits a significant catalytic activity with another indole compound: 5-hydroxy-l-tryptophan (5-HTP). IDO-I is considered to be an enzyme that is involved in the biosynthesis of the 5-HTP-derived mating pheromone, gamone 2. By analysing a series of chimeric enzymes based on extant and predicted ancestral enzymes, we identified Asn131 in IDO-I and Glu132 in IDO-III as the key residues responsible for their high affinity for each specific substrate. These two residues were aligned in an identical position as the substrate-determining residue (SDR). Thus, the substrate affinity and specificity are regulated mostly by a single amino acid residue in the Blepharisma IDO-I and IDO-III enzymes.

Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes.

In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V max and catalytic efficiency (k cat/K M) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.

High l-Trp affinity of indoleamine 2,3-dioxygenase 1 is attributed to two residues located in the distal heme pocket.

Indoleamine 2, 3-dioxygenase (IDO) catalyzes the oxidative cleavage of the pyrrole ring of l-Trp to generate N-formyl-kynurenine. Two IDO genes, IDO1 and IDO2, are found in vertebrates. Mammalian IDO1s are high-affinity, l-Trp-degrading enzymes, whereas IDO2s generally have a relatively low affinity. It has been suggested that the distal-Ser (corresponding to Ser167 of human IDO1) was crucial for improvement in the affinity for l-Trp but this idea was insufficient to explain the high affinity shown by mammalian IDO1. In this study, the amino acid sequences of vertebrate ancestral IDO1 and ancestral IDO2 were inferred, and bacterially expressed ancestral IDOs were characterized. Although the amino acid sequences of the enzymes shared high identity (86%) with each other, they showed distinct enzymatic properties. In analyses of a series of ancestral IDO1/IDO2 chimeric enzymes and their variants, the distal-Tyr (corresponding to Tyr126 of human IDO1) was detected as another and was probably the most crucial residue for high l-Trp affinity. The two amino acid substitutions (distal-Ser to Thr and distal-Tyr to His) drastically decreased the l-Trp affinity and catalytic efficiency of IDO1s. Conversely, two substitutions (distal-Thr to Ser and distal-His to Tyr) were sufficient to bestow IDO1-like high affinity on ancestral and chicken IDO2.

An unusual chimeric amylosucrase generated by domain-swapping mutagenesis.

Amylosucrase (ASase; EC 2.4.1.4) synthesizes α-1,4-glucans using sucrose as a sole substrate. The aim of this study was to compare the enzymatic properties of four recombinant ASase genes to determine the underlying mechanisms thereof. Following cloning and expression in Escherichia coli, we determined that the ASase enzyme from Deinococcus geothermalis (DGAS) had the highest thermostability whereas ASase from Neisseria polysaccharea (NPAS) showed the greatest polymerization activity. Chimeric ASases were constructed using dgas and npas genes by overlap extension polymerase chain reaction. Two of the six chimeric ASases generated, NPAS-B' and DGAS-B, showed ASase activity using sucrose as the sole substrate. However, DGAS-B was not able to produce longer α-1,4-glucans; the highest degree of polymerization was <12. In the kinetic study, not only the substrate binding affinity but also the production rate of DGAS-B was greater than those of DGAS. Molecular dynamic computational simulation suggested that DGAS-B could not synthesize longer glucan chains because of the change in flexibilities of loops 4, 7, and 8as compared to those of DGAS.