Parent Enzyme Research Articles

Investigating the antifungal potential of genetically modified hybrid chitinase enzymes derived from Bacillus subtilis and Serratia marcescens.

Chitinases are glycosyl hydrolase enzymes that break down chitin, an integral component of fungal cell walls. Bacteria such as Bacillus subtilis and Serratia marcescens produce chitinases with antifungal properties. In this study, we aimed to generate hybrid chitinase enzymes with enhanced antifungal activity by combining functional domains from native chitinases produced by B. subtilis and S. marcescens. Chitinase genes were cloned from both bacteria and fused together using overlap extension PCR. The hybrid constructs were expressed in E. coli and the recombinant enzymes purified. Gel electrophoresis and computational analysis confirmed the molecular weights and isoelectric points of the hybrid chitinases were intermediate between the parental enzymes. Antifungal assays demonstrated that the hybrid chitinases inhibited growth of the fungus Fusarium oxysporum significantly more than the native enzymes and also showed fungicidal activity against Candida albicans, Alternaria solani, and Rhizoctonia solani. The results indicate that hybrid bacterial chitinases are a promising approach to engineer novel antifungal proteins. This study provides insight into structure-function relationships of chitinases and strategies for generating biotherapeutics with enhanced bioactive properties. These hybrid chitinases result in a more potent and versatile antifungal agent.

Characterization of a λ-Carrageenase Mutant with the Generation of Long-Chain λ-Neocarrageenan Oligosaccharides

λ-carrageenan oligosaccharides can be widely applied in the food, pharmaceutical, medicine and cosmetic industries due to their abundant bioactivities, and they are important products for the high-value utilization of λ-carrageenan. However, oligosaccharides with different degrees of polymerization have different properties, and the final products of λ-carrageenase reported so far are mainly λ-neocarrabiose, λ-neocarratetraose and λ-neocarrahexaose without longer-chain oligosaccharides. Further research is consequently required. Herein, a mutant λ-carrageenase was constructed by deleting the pyrroloquinoline quinone-like domain of OUC-CglA derived from Maribacter vaceletii. Interestingly, it was discovered that the majority of final products of the mutant OUC-CglA-DPQQ were long-chain oligosaccharides with a polymerization degree of 10–20, which underwent significant changes compared to that of OUC-CglA. Additionally, without the pyrroloquinoline quinone-like domain, fewer inclusion bodies were produced throughout the expression process, and the yield of the λ-carrageenase increased about five-fold. However, compared to its parental enzyme, significant changes were made to its enzymatic properties. Its optimal temperature and pH were 15 °C and pH 7.0, and its specific activity was 51.59 U/mg. The stability of the enzyme decreased. Thus, it was found that the deleting domain was related to the formation of inclusion bodies, the stability of the enzyme, the activity of the enzyme and the composition of the products.

Evolving ω-amine transaminase AtATA guided by substrate-enzyme binding free energy for enhancing activity and stability against non-natural substrates.

In the field of chiral amine synthesis, ω-amine transaminase (ω-ATA) is one of the most established enzymes capable of asymmetric amination under optimal conditions. However, the applicability of ω-ATA toward more non-natural complex molecules remains limited due to its low transamination activity, thermostability, and narrow substrate scope. Here, by employing a combined approach of computational virtual screening strategy and combinatorial active-site saturation test/iterative saturation mutagenesis strategy, we have constructed the best variant M14C3-V5 (M14C3-V62A-V116S-E117I-L118I-V147F) with improved ω-ATA from Aspergillus terreus (AtATA) activity and thermostability toward non-natural substrate 1-acetylnaphthalene, which is the ketone precursor for producing the intermediate (R)-(+)-1-(1-naphthyl)ethylamine [(R)-NEA] of cinacalcet hydrochloride, showing activity enhancement of up to 3.4-fold compared to parent enzyme M14C3 (AtATA-F115L-M150C-H210N-M280C-V149A-L182F-L187F). The computational tools YASARA, Discovery Studio, Amber, and FoldX were applied for predicting mutation hotspots based on substrate-enzyme binding free energies and to show the possible mechanism with features related to AtATA structure, catalytic activity, and stability in silico analyses. M14C3-V5 achieved 71.8% conversion toward 50 mM 1-acetylnaphthalene in a 50 mL preparative-scale reaction for preparing (R)-NEA. Moreover, M14C3-V5 expanded the substrate scope toward aromatic ketone compounds. The generated virtual screening strategy based on the changes in binding free energies has successfully predicted the AtATA activity toward 1-acetylnaphthalene and related substrates. Together with experimental data, these approaches can serve as a gateway to explore desirable performances, expand enzyme-substrate scope, and accelerate biocatalysis.IMPORTANCEChiral amine is a crucial compound with many valuable applications. Their asymmetric synthesis employing ω-amine transaminases (ω-ATAs) is considered an attractive method. However, most ω-ATAs exhibit low activity and stability toward various non-natural substrates, which limits their industrial application. In this work, protein engineering strategy and computer-aided design are performed to evolve the activity and stability of ω-ATA from Aspergillus terreus toward non-natural substrates. After five rounds of mutations, the best variant, M14C3-V5, is obtained, showing better catalytic efficiency toward 1-acetylnaphthalene and higher thermostability than the original enzyme, M14C3. The robust combinational variant acquired displayed significant application value for pushing the asymmetric synthesis of aromatic chiral amines to a higher level.

Engineering the Activity of a Newly Identified Arylalkylamine N-Acetyltransferase in the Acetylation of 5-Hydroxytryptamine.

Arylalkylamine N-acetyltransferase (AANAT) serves as a key enzyme in the biosynthesis of melatonin by transforming 5-hydroxytryptamine (5-HT) to N-acetyl-5-hydroxytryptamine (NAS), while its low activity may hinder melatonin yield. In this study, a novel AANAT derived from Sus scrofa (SsAANAT) was identified through data mining using 5-HT as a model substrate, and a rational design of SsAANAT was conducted in the quest to improving its activity. After four rounds of mutagenesis procedures, a triple combinatorial dominant mutant M3 was successfully obtained. Compared to the parent enzyme, the conversion of the whole-cell reaction bearing the best variant M3 exhibted an increase from 50 % to 99 % in the transformation of 5-HT into NAS. Additionally, its catalytic efficiency (kcat/Km) was enhanced by 2-fold while retaining the thermostability (Tm>45 °C). In the up-scaled reaction with a substrate loading of 50 mM, the whole-cell system incorporating variant M3 achieved a 99 % conversion of 5-HT in 30 h with an 80 % yield. Molecular dynamics simulations were ultilized to shed light on the origin of improved activity. This study broadens the repertoire of AANAT for the efficient biosynthesis of melatonin.

Identification of a CTX-M-255 β-lactamase containing a G239S substitution selectively conferring resistance to penicillin/β-lactamase inhibitor combinations.

An Escherichia coli isolate, WGS1363, showed resistance to piperacillin/tazobactam but susceptibility to cephalosporins and contained a previously unrecognized β-lactamase, CTX-M-255, as the only acquired β-lactamase. CTX-M-255 was identical to CTX-M-27 except for a G239S substitution. Here, we characterize the hydrolytic spectrum of CTX-M-255 and a previously reported β-lactamase, CTX-M-178, also containing a G239S substitution and compare it to their respective parental enzymes, CTX-M-27 and CTX-M-15. All β-lactamase genes were expressed in E. coli TOP10 and MICs to representative β-lactam-antibiotics were determined. Furthermore, blaCTX-M-15, blaCTX-M-27, blaCTX-M-178 and blaCTX-M-255 with C-terminal His-tag fusions were affinity purified for enzyme kinetic assays determining Michaelis-Menten kinetic parameters against representative β-lactam-antibiotics and IC50s of clavulanate, sulbactam, tazobactam and avibactam. TOP10-transformants expressing blaCTX-M-178 and blaCTX-M-255 showed resistance to penicillin/β-lactamase combinations and susceptibility to cephalothin and cefotaxime in contrast to transformants expressing blaCTX-M-15 and blaCTX-M-27. Determination of enzyme kinetic parameters showed that CTX-M-178 and CTX-M-255 both lacked hydrolytic activity against cephalosporins and showed impaired hydrolytic efficiency against penicillin antibiotics compared to their parental enzymes. Both enzymes appeared more active against piperacillin compared to benzylpenicillin and ampicillin. Compared to their parental enzymes, IC50s of β-lactamase-inhibitors were increased more than 1000-fold for CTX-M-178 and CTX-M-255. CTX-M-178 and CTX-M-255, both containing a G239S substitution, conferred resistance to piperacillin/tazobactam and may be characterized as inhibitor-resistant CTX-M β-lactamases. Inhibitor resistance was accompanied by loss of activity against cephalosporins and monobactams. These findings add to the necessary knowledge base for predicting antibiotic susceptibility from genotypic data.

“Mix and match” auto-assembly of glycosyltransferase domains delivers biocatalysts with improved substrate promiscuity

Glycosyltransferases (GT) catalyse the glycosylation of bioactive natural products, including peptides and proteins, flavonoids, sterols, and have been extensively used as biocatalysts to generate glycosides. However, the often narrow substrate specificity of wild-type GTs requires engineering strategies to expand it. The GT-B structural family is constituted by GTs that share a highly conserved tertiary structure, in which the sugar donor and acceptor substrates bind in dedicated domains. Here, we have used this selective binding feature to design an engineering process to generate chimeric glycosyltransferases that combine auto-assembled domains from two different GT-B enzymes. Our approach enabled the generation of a stable dimer with broader substrate promiscuity than the parent enzymes that was related to relaxed interactions between domains in the dimeric GT-B. Our findings provide a basis for the development of a novel class of heterodimeric GTs with improved substrate promiscuity for applications in biotechnology and natural product synthesis.

Laboratory Evolution of Metalloid Reductase Substrate Recognition and Nanoparticle Product Size.

Glutathione reductase-like metalloid reductase (GRLMR) is an enzyme that reduces selenodiglutathione (GS-Se-SG), forming zerovalent Se nanoparticles (SeNPs). Error-prone polymerase chain reaction was used to create a library of ∼10,000 GRLMR variants. The library was expressed in BL21Escherichia coli in liquid culture with 50 mM of SeO32- present, under the hypothesis that the enzyme variants with improved GS-Se-SG reduction kinetics would emerge. The selection resulted in a GRLMR variant with two mutations. One of the mutations (D-E) lacks an obvious functional role, whereas the other mutation is L-H within 5 Å of the enzyme active site. This mutation places a second H residue within 5 Å of an active site dicysteine. This GRLMR variant was characterized for NADPH-dependent reduction of GS-Se-SG, GSSG, SeO32-, SeO42-, GS-Te-SG, and TeO32-. The evolved enzyme demonstrated enhanced reduction of SeO32- and gained the ability to reduce SeO42-. This variant is named selenium reductase (SeR) because of its emergent broad activity for a wide variety of Se substrates, whereas the parent enzyme was specific for GS-Se-SG. This study overall suggests that new biosynthetic routes are possible for inorganic nanomaterials using laboratory-directed evolution methods.

Asymmetric α-benzylation of cyclic ketones enabled by concurrent chemical aldol condensation and biocatalytic reduction

Chemoenzymatic cascade catalysis has emerged as a revolutionary tool for streamlining traditional retrosynthetic disconnections, creating new possibilities for the asymmetric synthesis of valuable chiral compounds. Here we construct a one-pot concurrent chemoenzymatic cascade by integrating organobismuth-catalyzed aldol condensation with ene-reductase (ER)-catalyzed enantioselective reduction, enabling the formal asymmetric α-benzylation of cyclic ketones. To achieve this, we develop a pair of enantiocomplementary ERs capable of reducing α-arylidene cyclic ketones, lactams, and lactones. Our engineered mutants exhibit significantly higher activity, up to 37-fold, and broader substrate specificity compared to the parent enzyme. The key to success is due to the well-tuned hydride attack distance/angle and, more importantly, to the synergistic proton-delivery triade of Tyr28-Tyr69-Tyr169. Molecular docking and density functional theory (DFT) studies provide important insights into the bioreduction mechanisms. Furthermore, we demonstrate the synthetic utility of the best mutants in the asymmetric synthesis of several key chiral synthons.

Sequence - dynamics - function relationships in protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) are crucial regulators of cellular signaling. Their activity is regulated by the motion of a conserved loop, the WPD-loop, from a catalytically inactive open to a catalytically active closed conformation. WPD-loop motion optimally positions a catalytically critical residue into the active site, and is directly linked to the turnover number of these enzymes. Crystal structures of chimeric PTPs constructed by grafting parts of the WPD-loop sequence of PTP1B onto the scaffold of YopH showed WPD-loops in a wide-open conformation never previously observed in either parent enzyme. This wide-open conformation has, however, been observed upon binding of small molecule inhibitors to other PTPs, suggesting the potential of targeting it for drug discovery efforts. Here, we have performed simulations of both enzymes and show that there are negligible energetic differences in the chemical step of catalysis, but significant differences in the dynamical properties of the WPD-loop. Detailed interaction network analysis provides insight into the molecular basis for this population shift to a wide-open conformation. Taken together, our study provides insight into the links between loop dynamics and chemistry in these YopH variants specifically, and how WPD-loop dynamic can be engineered through modification of the internal protein interaction network.

Tuning the peroxidase activity of artificial P450 peroxygenase by engineering redox-sensitive residues.

Cytochrome P450 monooxygenases (P450s) are well recognized as versatile bio-oxidation catalysts. However, the catalytic functions of P450s are highly dependent on NAD(P)H and redox partner proteins. Our group has recently reported the use of a dual-functional small molecule (DFSM) for generating peroxygenase activity of P450BM3, a long-chain fatty acid hydroxylase from Bacillus megaterium. The DFSM-facilitated P450BM3 peroxygenase system exhibited excellent peroxygenation activity and regio-/enantioselectivity for various organic substrates, such as styrenes, thioanisole, small alkanes, and alkylbenzenes. Very recently, we demonstrated that the DFSM-facilitated P450BM3 peroxygenase could be switched to a peroxidase by engineering the redox-sensitive tyrosine residues in P450BM3. Given the great potential of P450 peroxidase for C-H oxyfunctionalization, we herein report scrutiny of the effect of mutating redox-sensitive residues on peroxidase activity by deeply screening all redox-sensitive residues of P450BM3, namely methionines, tryptophans, cysteines, and phenylalanines. As a result, six beneficial mutations at positions M212, F81, M112, F173, M177, and F77 were screened out from 78 constructed mutants, and significantly enhanced the peroxidase activity of P450BM3 in the presence of Im-C6-Phe, a typical DFSM molecule. Further combination of the beneficial mutations resulted in a more than 100-fold improvement in peroxidase activity compared with that of the combined parent enzyme and DFSM, comparable to or better than most natural peroxidases. In addition, mutations of redox-sensitive residues even dramatically increased, by more than 300-fold, the peroxidase activity of the starting F87A enzyme in the absence of the DFSM, despite the far lower apparent catalytic turnover number compared with the DFSM-P450 system. This study provides new insights and a potential strategy for regulating the catalytic promiscuity of P450 enzymes for multiple functional oxidations.

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation

AAA+ proteases degrade intracellular proteins in a highly specific manner. E. coli ClpXP, for example, relies on a C-terminal ssrA tag or other terminal degron sequences to recognize proteins, which are then unfolded by ClpX and subsequently translocated through its axial channel and into the degradation chamber of ClpP for proteolysis. Prior cryo-EM structures reveal that the ssrA tag initially binds to a ClpX conformation in which the axial channel is closed by a pore-2 loop. Here, we show that substrate-free ClpXP has a nearly identical closed-channel conformation. We destabilize this closed-channel conformation by deleting residues from the ClpX pore-2 loop. Strikingly, open-channel ClpXP variants degrade non-native proteins lacking degrons faster than the parental enzymes in vitro but degraded GFP-ssrA more slowly. When expressed in E. coli, these open channel variants behave similarly to the wild-type enzyme in assays of filamentation and phage-Mu plating but resulted in reduced growth phenotypes at elevated temperatures or when cells were exposed to sub-lethal antibiotic concentrations. Thus, channel closure is an important determinant of ClpXP degradation specificity.

Production of highly water-soluble genistein α-diglucoside using an engineered O-α-glycoligase with enhanced transglycosylation activity and altered substrate specificity

Genistein is one of isoflavones, showing various biological functions for human health. MalA-D416A, termed O-α-glycoligase, is an acid/base catalytic residue-deficient mutant of a α-glucosidase from Sulfolobus solfataricus, synthesizing genistein 7-O-α-glucoside using α-glucosyl fluoride as the donor substrate. Through mutagenesis toward MalA-D416A, an O-α-glycoligase variant with two mutations (D416R and Q450S) was identified as a biocatalyst with a 58.8-fold enhanced catalytic efficiency for genistein compared to the parent enzyme. The use of a 2:1 ratio of α-glucosyl fluoride and genistein at pH 9 facilitated the synthesis of genistein 7,4′-O-α-diglucoside by MalA-D416R/Q450S. The α-diglucoside exhibited 2,459-fold improved water solubility compared to genistein itself as well as facile deglycosylation by the intestinal α-glucosidase from rat, suggesting the potential of the α-diglucoside for improved bioavailability in human intestine. Through molecular docking analyses the modulation of the active site conformation by these mutations was expected for proper binding of both genistein and the monoglucoside.

Characterization and designing of an SAM riboswitch to establish a high-throughput screening platform for SAM overproduction in Saccharomyces cerevisiae.

S-adenosyl- l-methionine (SAM)is a high-value compound widely used in the treatment of various diseases. SAM can be produced through fermentation, but further enhancing the microbial production of SAM requires novel high-throughput screening methods for rapid detection and screening of mutant libraries. In this work, anSAM-OFF riboswitch capable of responding to the SAM concentration was obtained and a high-throughput platform for screening SAM overproducers was established. SAM synthase was engineered by semirational design and directed evolution, which resulted in the SAM2S203F,W164R,T251S,Y285F,S365R mutant with almost twice higher catalytic activity than the parental enzyme. The best mutant was then introduced into Saccharomyces cerevisiae BY4741, and the resulting strain BSM8 produced a sevenfold higher SAM titer in shake-flask fermentation, reaching 1.25 g L-1 . This work provides a reference for designing biosensors to dynamically detect metabolite concentrations for high-throughput screening and the construction of effective microbial cell factories.

Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fusca

The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) was prepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostable protein (e.g., FDH).

Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches.

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.

Improved thermostability of type I pullulanase from Bacillus thermoliquefaciens by error-prone PCR

Pullulanase (PulB) is a starch-debranching enzyme. In order to improve its catalytic performance, random mutagenesis was performed on the pullulanase gene derived from Bacillus thermoliquefaciens. Two rounds of error-prone PCR were carried out. Mutant T252S was screened in the first round of error-prone library, which had the highest catalytic activity. During the second round of mutations, mutant enzyme G250P/T252S/G253T/N255K was screened, which had further improved catalytic activity and the best thermostability. Compared with the parent enzyme, the specific activity of mutant enzyme G250P/T252S/G253T/N255K increased by 1.9 times, Km decreased by 22.7 %, kcat increased by 28.7 %, and kcat/Km increased by 68.4 %. The thermostability of the mutant enzyme improved significantly, showing that the half-life at 60 °C was extended to 7.5 h, which was 87.5 % higher than that of the parent enzyme. The mutation sites in these two rounds were concentrated in the 250–255 regions, indicating that this region was an important region affecting the catalytic activity and Thermostability. The reasons for the change of enzymtic properties was also preliminarily analyzed through three-dimensional simulation.

Pocket Modification of ω-Amine Transaminase AtATA for Overcoming the Trade-Off Between Activity and Stability Toward 1-Acetonaphthone

Amine transaminases (ATAs) catalyze the asymmetric amination of prochiral ketones or aldehydes to their corresponding chiral amines. However, the trade-off between activity and stability in enzyme engineering represents a major obstacle to the practical application of ATAs. Overcoming this trade-off is important for developing robustly engineered enzymes and a universal approach for ATAs. Herein, we modified the binding pocket of ω-ATA from Aspergillus terreus (AtATA) to identify the key amino acid residues controlling the activity and stability of AtATA toward 1-acetonaphthone. We discovered a structural switch comprising four key amino acid sites (R128, V149, L182, and L187), as well as the “best” mutant (AtATA_D224K/V149A/L182F/L187F; termed M4). Compared to the parent enzyme AtATA_D224K (AtATA-Pa), M4 increased the catalytic efficiency (kcat/Km1-acetonaphthone, where kcat is the constant of catalytic activities and is 10.1 min−1, Km1-acetonaphthone is Michaelis-Menten constant and is 1.7 mmol·L–1) and half-life (t1/2) by 59-fold to 5.9 L·min−1·mmol−1 and by 1.6-fold to 46.9 min, respectively. Moreover, using M4 as the biocatalyst, we converted a 20 mmol·L–1 aliquot of 1-acetonaphthone in a 50 mL scaled-up system to the desired product, (R)-(+)-1(1-naphthyl)ethylamine ((R)-NEA), with 78% yield and high enantiomeric purity (R > 99.5%) within 10 h. M4 also displayed significantly enhanced activity toward various 1-acetonaphthone analogs. The related structural properties derived by analyzing structure and sequence information of robust ATAs illustrated their enhanced activity and thermostability. Strengthening of intramolecular interactions and expansion of the angle between the substrate-binding pocket and the pyridoxal 5′-phosphate (PLP)-binding pocket contributed to synchronous enhancement of ATA thermostability and activity. Moreover, this pocket engineering strategy successfully transferred enhanced activity and thermostability to three other ATAs, which exhibited 8%–22% sequence similarity with AtATA. This research has important implications for overcoming the trade-off between ATA activity and thermostability.

Improvement in salt-tolerance of Aspergillus oryzae γ-glutamyl transpeptidase via protein chimerization with Aspergillus sydowii homolog

γ-Glutamyl transpeptidase is one of the key enzymes involved in glutamate production during high-salt fermentation of soy sauce and miso by koji mold, Aspergillus oryzae. However, the activity of γ-glutamyl transpeptidase from A. oryzae (AOggtA) is markedly reduced in the presence of NaCl, thus classifying it as a non-salt-tolerant enzyme. In contrast, the homologous protein from the xerophilic mold, A. sydowii (ASggtA) maintains its activity under high-salt conditions. Therefore, in this study, a chimeric enzyme, ASAOggtA, was designed and engineered to improve salt-tolerance in AOggtA by swapping the N-terminal region, based on sequence and structure comparisons between salt-tolerant ASggtA and non-salt-tolerant AOggtA. The parental AOggtA and ASggtA and their chimera, ASAOggtA, were heterologously expressed in A. oryzae and purified. The chimeric enzyme inherited the superior activity and stability from each of the two parent enzymes. ASAOggtA showed > 2-fold greater tolerance than AOggtA in the presence of 18% NaCl. In addition, the chimera showed a broader range of pH stability and greater thermostability than ASggtA. AOggtA and ASAOggtA were sy over the range pH 3.0 to pH 10.5. Thermal stability was found to be in the order AOggtA (57.5 °C, t1/2 = 32.5 min) > ASAOggtA (55 °C, t1/2 = 20.5 min) > ASggtA (50 °C, t1/2 = 12.5 min). The catalytic and structural characteristics indicated that non-salt-tolerant AOggtA would not undergo irreversible structural changes in the presence of NaCl, but rather a temporary conformational change, which might result in reducing the substrate binding and catalytic activity, on the basis of kinetic properties. In addition, the chimeric enzyme showed hydrolytic activity toward L-glutamine that was as high as that of AOggtA. The newly-designed chimeric ASAOggtA might have potential applications in high-salt fermentation, such as miso and shoyu, to increase the content of the umami-flavor amino acid, L-glutamate.

The Crystal Structure of Engineered Nitroreductase NTR 2.0 and Impact of F70A and F108Y Substitutions on Substrate Specificity.

Bacterial nitroreductase enzymes that convert prodrugs to cytotoxins are valuable tools for creating transgenic targeted ablation models to study cellular function and cell-specific regeneration paradigms. We recently engineered a nitroreductase ("NTR 2.0") for substantially enhanced reduction of the prodrug metronidazole, which permits faster cell ablation kinetics, cleaner interrogations of cell function, ablation of previously recalcitrant cell types, and extended ablation paradigms useful for modelling chronic diseases. To provide insight into the enhanced enzymatic mechanism of NTR 2.0, we have solved the X-ray crystal structure at 1.85 Angstroms resolution and compared it to the parental enzyme, NfsB from Vibrio vulnificus. We additionally present a survey of reductive activity with eight alternative nitroaromatic substrates, to provide access to alternative ablation prodrugs, and explore applications such as remediation of dinitrotoluene pollutants. The predicted binding modes of four key substrates were investigated using molecular modelling.

A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol.

R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation-reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from -17.0kJ/mol to -29.1kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation-reduction cascade at larger scales.