ABSTRACT
Background: The emergence and spread of multidrug-resistant Staphylococcus aureus strains demonstrates the urgent need for new antimicrobials. Xanthorrhizol, a plant-derived sesquiterpenoid compound, has a rapid killing effect on methicillin-susceptible strains and methicillin-resistant strains of S. aureus achieving the complete killing of staphylococcal cells within 2 min using 64 µg/mL xanthorrhizol. However, the mechanism of its action is not yet fully understood. Methods: The S. aureus cells treated with xanthorrhizol were studied using optical diffraction tomography. Activity of xanthorrhizol against the wild-type and mscL null mutant of S. aureus ATCC 29213 strain was evaluated in the time-kill assay. Molecular docking was conducted to predict the binding of xanthorrhizol to the SaMscL protein. Results: Xanthorrhizol treatment of S. aureus cells revealed a decrease in cell volume, dry weight, and refractive index (RI), indicating efflux of the cell cytoplasm, which is consistent with the spontaneous activation of the mechanosensitive MscL channel. S. aureus ATCC 29213ΔmscL was significantly more resistant to xanthorrhizol than was the wild-type strain. Xanthorrhizol had an enhanced inhibitory effect on the growth and viability of exponentially growing S. aureus ATCC 29213ΔmscL cells overexpressing the SaMscL protein and led to a noticeable decrease in their viability in the stationary growth phase. The amino acid residues F5, V14, M23, A79, and V84 were predicted to be the residues of the binding pocket for xanthorrhizol. We also showed that xanthorrhizol increased the efflux of solutes such as K+ and glutamate from S. aureus ATCC 29213ΔmscL cells overexpressing SaMscL. Xanthorrhizol enhanced the antibacterial activity of the antibiotic dihydrostreptomycin, which targets the MscL protein. Conclusion: Our findings indicate that xanthorrhizol targets the SaMscL protein in S. aureus cells and may have important implications for the development of a safe antimicrobial agent.
ABSTRACT
The importance of protein engineering in the research and development of biopharmaceuticals and biomaterials has increased. Machine learning in computer-aided protein engineering can markedly reduce the experimental effort in identifying optimal sequences that satisfy the desired properties from a large number of possible protein sequences. To develop general protein descriptors for computer-aided protein engineering tasks, we devised new protein descriptors, one sequence-based descriptor (PCgrades), and three structure-based descriptors (PCspairs, 3D-SPIEs_5.4 Å, and 3D-SPIEs_8Å). While the PCgrades and PCspairs include general and statistical information in physicochemical properties in single and pairwise amino acids respectively, the 3D-SPIEs include specific and quantum-mechanical information with parameterized quantum mechanical calculations (FMO2-DFTB3/D/PCM). To evaluate the protein descriptors, we made prediction models with the new descriptors and previously developed descriptors for diverse protein datasets including protein expression and binding affinity change in SARS-CoV-2 spike glycoprotein. As a result, the newly devised descriptors showed a good performance in diverse datasets, in which the PCspairs showed the best performance ( R 2 = 0.783 for protein expression and R 2 = 0.711 for binding affinity). As a result, the newly devised descriptors showed a good performance in diverse datasets, in which the PCspairs showed the best performance. Similar approaches with those descriptors would be promising and useful if the prediction models are trained with sufficient quantitative experimental data from high-throughput assays for industrial enzymes or protein drugs.
ABSTRACT
The transgalactosylase activity of ß-galactosidase produces galacto-oligosaccharides (GOSs) with prebiotic effects similar to those of major oligosaccharides in human milk. ß-Galactosidases from Bacillus circulans ATCC 31382 are important enzymes in industrial-scale GOS production. Here, we show the high GOS yield of ß-galactosidase II from B. circulans (ß-Gal-II, Lactazyme-B), compared to other commercial enzymes. We also determine the crystal structure of the five conserved domains of ß-Gal-II in an apo-form and complexed with galactose and an acceptor sugar, showing the heterogeneous mode of transgalactosylation by the enzyme. Truncation studies of the five conserved domains reveal that all five domains are essential for enzyme catalysis, while some truncated constructs were still expressed as soluble proteins. Structural comparison of ß-Gal-II with other ß-galactosidase homologues suggests that the GOS linkage preference of the enzyme might be quite different from other enzymes. The structural information on ß-Gal-II might provide molecular insights into the transgalactosylation process of the ß-galactosidases in GOS production.
Subject(s)
Lactose , Oligosaccharides , Bacillus/chemistry , Bacillus/enzymology , Galactose , Models, Structural , beta-Galactosidase/geneticsABSTRACT
Xanthorrhizol, isolated from the Indonesian Java turmeric Curcuma xanthorrhiza, displays broad-spectrum antibacterial activity. We report herein the evidence that mechanism of action of xanthorrhizol may involve FabI, an enoyl-(ACP) reductase, inhibition. The predicted Y156V substitution in the FabI enzyme promoted xanthorrhizol resistance, while the G93V mutation originally known for triclosan resistance was not effective against xanthorrhizol. Two other mutations, F203L and F203V, conferred FabI enzyme resistance to both xanthorrhizol and triclosan. These results showed that xanthorrhizol is a food-grade antimicrobial compound targeting FabI but with a different mode of binding from triclosan.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli/enzymology , Food Additives/pharmacology , Phenols/pharmacology , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/metabolism , Escherichia coli/drug effects , Escherichia coli Infections/drug therapy , Escherichia coli Infections/microbiology , Escherichia coli Proteins/metabolism , Fatty Acid Synthase, Type II/antagonists & inhibitors , Fatty Acid Synthase, Type II/metabolism , Humans , Molecular Docking SimulationABSTRACT
Superoxide dismutase 1 (SOD1) binds copper and zinc ions and is one of three superoxide dismutases responsible for destroying free superoxide radicals in the body. Reactive oxygen species (ROS), including free superoxide radicals, play important roles in colitis. However, the role of SOD1 in oxidative stress under colitis remains unclear. Here, we examined the role of SOD1 in the DSS-induced mouse model of colitis. SOD1 deficiency resulted in severe oxidative stress with body weight loss, epithelial barrier disruption and decreased antioxidant enzyme activities. The levels of neutrophils, monocytes, pro-inflammatory CD11c+ macrophages and CD11b+CD103- dendritic cells (DCs) were increased, while anti-inflammatory CD206+ macrophages and CD11b-CD103+ DCs were decreased, in DSS-treated SOD1-knockout (KO) mice compared to DSS-treated wild-type mice. Furthermore, rescue of SOD activity in SOD1-KO mice by oral gavage of B. amyloliquefaciens SOD (BA SOD) significantly ameliorated enhanced DSS-induced colitis in these mice by suppressing p38-MAPK/NF-κB signaling, which can induce inflammation and apoptosis. Taken together, our results suggest that SOD1-mediated inhibitory responses play a crucial role in limiting the development of DSS-induced colitis, and that BA SOD is a promising candidate for treating colitis.
Subject(s)
Colitis , Oxidative Stress , Superoxide Dismutase-1 , Animals , Colitis/chemically induced , Colitis/genetics , Dextran Sulfate , Immunity , Mice , Mice, Inbred C57BL , Superoxide Dismutase-1/geneticsABSTRACT
Phloretin, the major polyphenol compound in apples and apple products, is interesting because it shows beneficial effects on human health. It is mainly found as a form of glucoside, phlorizin. However, the metabolic pathway of phloretin in humans has not been reported. Therefore, identifying phloretin metabolites made in human liver microsomes and the human cytochrome P450 (P450) enzymes to make them is interesting. In this study, the roles of human liver P450s for phloretin oxidation were examined using human liver microsomes and recombinant human liver P450s. One major metabolite of phloretin in human liver microsomes was 3-OH phloretin, which is the same product of a bacterial CYP102A1-catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed kcat values of 3.1 and 5.8 min-1, respectively. However, CYP3A4 has a 3.3-fold lower Km value than CYP2C19. The catalytic efficiency of a CYP3A4-catalyzed reaction is 1.8-fold higher than a reaction catalyzed by CYP2C19. Whole-cell biotransformation with CYP3A4 was achieved 0.16 mM h-1 productivity for 3-OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent inhibitor of CYP3A4-catalyzed testosterone 6ß-hydroxylation activity. Antibodies against CYP3A4 inhibited up to 90% of the microsomal activity of phloretin 3-hydroxylation. The immunoinhibition effect of anti-2C19 is much lower than that of anti-CYP3A4. Thus, CYP3A4 majorly contributes to the human liver microsomal phloretin 3-hydroxylation, and CYP2C19 has a minor role.
ABSTRACT
BACKGROUND: Bacillus anthracis is the causative agent of anthrax, a disease of both humans and various animal species, and can be used as a bioterror agent. Effective vaccines are available, but those could benefit from improvements, including increasing the immunity duration, reducing the shot frequency and adverse reactions. In addition, more sophisticated antigen delivery and potentiation systems are urgently required. The protective antigen (PA), one of three major virulence factors associated with anthrax was displayed on the surface of Bacillus subtilis spores, which is a vaccine production host and delivery vector with several advantages such as a low production cost, straightforward administration as it is safe for human consumption and the particulate adjuvanticity. Mice were immunized orally (PO), intranasally (IN), sublingually (SL) or intraperitoneally (IP) with the PA displaying probiotic spore vaccine. Clinical observation, serological analysis and challenge experiment were conducted to investigate the safety and efficacy of the vaccine. RESULTS: A/J mice immunized with the PA spore vaccine via PO, IN, SL, and IP were observed to have increased levels of active antibody titer, isotype profiles and toxin neutralizing antibody in sera, and IgA in saliva. The immunized mice were demonstrated to raise protective immunity against the challenge with lethal B. anthracis spores. CONCLUSIONS: In this study, we developed a B. subtilis spore vaccine that displays the PA on its surface and showed that the PA-displaying spore vaccine was able to confer active immunity to a murine model based on the results of antibody isotype titration, mucosal antibody identification, and a lethal challenge experiment.
Subject(s)
Anthrax Vaccines/pharmacology , Antigens, Bacterial/immunology , Bacillus subtilis/immunology , Bacterial Toxins/immunology , Animals , Anthrax/prevention & control , Anthrax Vaccines/administration & dosage , Antibodies, Neutralizing/blood , Bacillus anthracis , Immunization , Immunoglobulin A , Male , Mice , Saliva/immunology , Spores, Bacterial/immunology , Vaccines, SyntheticABSTRACT
A new plasmid containing a mutated fabL gene from Bacillus subtilis as a triclosan selection marker was developed as a useful B. subtilis/E. coli shuttle vector. The pHT-FabL40 plasmid is stable in both gram-positive and gram-negative hosts with increased plasmid DNA yield in E. coli.
ABSTRACT
A region suffering from an attack of a nerve agent requires not only a highly sorptive material but also a fast-acting catalyst to decontaminate the lethal chemical present. The product should be capable of high sorptive capacity, selectivity and quick response time to neutralize the long lasting harmful effects of nerve agents. Herein, we have utilized organophosphorus hydrolase (OPH) as a non-toxic bio-catalytic material held in with the supporting matrix of poly-ß-cyclodextrin (PCD) as a novel sorptive reinforced self-decontaminating material against organophosphate intoxication. OPH coated PCD (OPH-PCD) will not only be providing support for holding enzyme but also would be adsorbing methyl paraoxon (MPO) used as a simulant, in a host-guest inclusion complex formation. Sorption trend for PCD revealed preference towards the more hydrophobic MPO against para-nitrophenol (pNP). The results show sorption capacity of 1.26 mg/g of 100 µM MPO with PCD which was 1.7 times higher compared to pNP. The reaction rate with immobilized OPH-PCD was found to be 23% less compared to free enzyme. With the help of OPH-PCD, continuous hydrolysis (100%) of MPO into pNP was observed for a period of 24 h through packed bed reactor with good reproducibility and stability of enzyme. The long-term stability also confirmed its stable nature for the investigation period of 4 days where it maintained activity. Combined with its fast and reactive nature, the resulting self-decontaminating regenerating material provides a promising strategy for the neutralization of nerve agents and preserving the environment.
Subject(s)
Aryldialkylphosphatase/chemistry , Chemical Warfare Agents/chemistry , Cholinesterase Inhibitors/chemistry , Decontamination/methods , Enzymes, Immobilized/chemistry , Insecticides/chemistry , Paraoxon/analogs & derivatives , beta-Cyclodextrins/chemistry , Adsorption , Biocatalysis , Hydrogen-Ion Concentration , Paraoxon/chemistryABSTRACT
The genome sequence of the commercial probiotic strain "Bacillus polyfermenticus" GF423 was determined. Comparison of the 4.1-Mb genome sequence revealed Bacillus velezensis FZB42 as its closest relative. Based on the genome sequence, we propose that this probiotic strain be renamed Bacillus velezensis variant polyfermenticus.
ABSTRACT
BACKGROUND AND AIMS: Commercial superoxide dismutase [SOD] is derived from melon extract and has a potential as a dietary supplement due to its beneficial antioxidative effects. We aimed to improve the productivity of SOD compared with plant SOD by using a generally regarded as safe [GRAS] microorganism, Bacillus amyloliquefaciens, and assess its antioxidative effect using γ-radiation- and dextransulphate sodium [DSS]-induced oxidative models in mice. METHODS: We identified the sodA gene encoding manganese-containing SODs [Mn-SOD] in B. amyloliquefaciens, constructed a Mn-SOD deficient mutant, and screened a high-SOD-producing strain. We compared the antioxidative effect of orally administered enteric-coated SOD protein partially purified from B. amyloliquefaciens with wild-type and high-SOD-producing strain spores. The effect of SOD on DSS-induced colitis was also investigated. Colonic inflammation was assessed using disease activity index, macroscopic and histological damage scores, antioxidant enzyme activities, and inflammatory cytokines. RESULTS: The SOD activity of B. amyloliquefaciens is derived from secreted Mn-SOD encoded by the sodA gene, as shown by comparing sodA knock-out mutant spores with wild-type and high-SOD-producing spores. Enteric-coated SOD of B. amyloliquefaciens appears to be effective in reducing oxidative stress in γ-radiation- and DSS-induced mouse models. Co-administration of SOD with wild-type B. amyloliquefaciens or high-SOD-producer strain spores showed a synergistic effect. SOD enzyme and B. amyloliquefaciens spores contribute to the reduction of oxidative stress and inflammatory response in DSS-induced colitis. CONCLUSIONS: Mn-SOD of B. amyloliquefaciens could be another source of SOD supplement and may be useful to prevent and treat ulcerative colitis.
Subject(s)
Bacillus amyloliquefaciens/metabolism , Colitis, Ulcerative/drug therapy , Free Radical Scavengers/pharmacology , Oxidative Stress/drug effects , Superoxide Dismutase/pharmacology , Adaptor Proteins, Signal Transducing , Animals , Bacillus amyloliquefaciens/genetics , Bacterial Proteins/genetics , Catalase/blood , Colitis, Ulcerative/blood , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/pathology , Cucurbitaceae/metabolism , Dextran Sulfate , Dietary Supplements , Female , Free Radical Scavengers/therapeutic use , Gamma Rays/adverse effects , Glutathione Peroxidase/blood , Interleukin-1beta/blood , Interleukin-6/blood , Intracellular Signaling Peptides and Proteins , Mice , Oxidative Stress/radiation effects , Proteins/metabolism , Superoxide Dismutase/biosynthesis , Superoxide Dismutase/genetics , Superoxide Dismutase/therapeutic useABSTRACT
In Bacillus subtilis, large genomic deletions have been carried out for genome reduction, antibiotic overproduction, and heterologous protein overexpression. In view of the eco-friendliness of B. subtilis, it is critical that engineering preserves its food-grade status and avoids leaving foreign DNA in the genome. Existing methods of generating large genomic deletions leave antibiotic resistance markers or display low mutation efficiency. In this study, we introduced a clustered regularly interspaced short palindromic repeat-derived genome engineering technique to develop a highly efficient method of generating large genomic deletions in B. subtilis without any trace of foreign DNA. Using our system, we produced 38 kb plipastatin-synthesizing pps operon deletion with 80% efficiency. The significant increase in mutation efficiency was due to plasmids-delivered Streptococcus pyogenes-originated SpCas9, target-specific sgRNA and a donor DNA template, which produces SpCas9/sgRNA endonuclease complex continuously for attacking target chromosome until the mutagenic repair occurs. Our system produced single-gene deletion in spo0A (â¼100%), point mutation (â¼68%) and GFP gene insertion (â¼97%) in sigE and demonstrated its broad applicability for various types of site-directed mutagenesis in B. subtilis.
ABSTRACT
Xanthorrhizol is a potent antimicrobial compound isolated from the rhizome of Curcuma xanthorrhiza. However, the mechanism of xanthorrhizol action is unknown. To screen for probable target(s), we introduced the ASKA pooled-plasmid library into Escherichia coli W3110 imp4213 and enriched the library for resistant clones with increasing concentrations of xanthorrhizol. After three rounds of enrichment, we found nine genes that increased xanthorrhizol resistance. The resistant clones were able to grow in LB medium containing 256 µg/mL xanthorrhizol, representing a 16-fold increase in the minimum inhibitory concentration. Subsequent DNA sequence analysis revealed that overexpression of tadA, galU, fucU, ydeA, ydaC, soxS, nrdH, yiiD, and mltF genes conferred increased resistance towards xanthorrhizol. Among these nine genes, tadA is the only essential gene. tadA encodes a tRNA-specific adenosine deaminase. Overexpression of E. coli W3110 imp4213 (pCA24N-tadA) conferred resistance to xanthorrhizol up to 128 µg/mL. Moreover, overexpression of two tadA mutant enzymes (A143V and F149G) led to a twofold increase in the MIC. These results suggest that the targets of xanthorrhizol may include tadA, which has never before been explored as an antibiotic target.
Subject(s)
Adenosine Deaminase/metabolism , Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Escherichia coli/genetics , Phenols/pharmacology , RNA, Transfer/genetics , Adenosine Deaminase/genetics , Microbial Sensitivity TestsSubject(s)
Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Escherichia coli/drug effects , Polymyxin B/agonists , Pseudomonas aeruginosa/drug effects , Pyridones/pharmacology , Acinetobacter baumannii/growth & development , Anti-Bacterial Agents/chemistry , Ciclopirox , Drug Resistance, Multiple, Bacterial/drug effects , Drug Synergism , Escherichia coli/growth & development , Microbial Sensitivity Tests , Polymyxin B/pharmacology , Pseudomonas aeruginosa/growth & developmentABSTRACT
Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications.
Subject(s)
Bacillus subtilis/genetics , Gene Regulatory Networks , Genes, Synthetic , Genetic Engineering/methods , Bacillus subtilis/drug effects , Base Sequence , Chloramphenicol Resistance/genetics , DNA, Bacterial/genetics , Genetic Markers , Genome, Bacterial , Molecular Sequence Data , Mutagenesis , Operon , Plasmids/geneticsABSTRACT
Bacterial persisters are a small subpopulation of cells that exhibit multi-drug tolerance without genetic changes. Generally, persistence is associated with a dormant state in which the microbial cells are metabolically inactive. The bacterial response to unfavorable environmental conditions (heat, oxidative, acidic stress) induces the accumulation of aggregated proteins and enhances formation of persister cells in Escherichia coli cultures. We have found that methionine supplementation reduced the frequency of persisters at mild (37°C) and elevated (42°C) temperatures, as well as in the presence of acetate. Homoserine-o-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway, is prone to aggregation under many stress conditions, resulting in a methionine limitation in E. coli growth. Overexpression of MetA induced the greatest number of persisters at 42°C, which is correlated to an increased level of aggregated MetA. Substitution of the native metA gene on the E. coli K-12 WE chromosome by a mutant gene encoding the stabilized MetA led to reduction in persisters at the elevated temperature and in the presence of acetate, as well as lower aggregation of the mutated MetA. Decreased persister formation at 42°C was confirmed also in E. coli K-12 W3110 and a fast-growing WErph+ mutant harboring the stabilized MetA. Thus, this is the first study to demonstrate manipulation of persister frequency under stressful conditions by stabilization of a single aggregation-prone protein, MetA.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Homoserine O-Succinyltransferase/metabolism , Stress, Physiological/genetics , Drug Resistance, Multiple/genetics , Escherichia coli/physiology , Escherichia coli Proteins/genetics , Homoserine/genetics , Homoserine/metabolism , Homoserine O-Succinyltransferase/genetics , Methionine/genetics , Methionine/metabolismABSTRACT
In principle, protein display is enabled by fusing target proteins to naturally secreted, surface-anchored protein motifs. In this work, we developed a method of native protein display on the Bacillus spore surface that obviates the need to construct fusion proteins to display a motif. Spore coat proteins are expressed in the mother cell compartment and are subsequently assembled and deposited on the surface of spores. Therefore, target proteins overexpressed in the mother cell compartment during the late sporulation phase were expected to be targeted and displayed on the spore surface. As a proof of principle, we demonstrated the display of carboxymethylcellulase (CMCase) in its native form on the spore surface. The target protein, CMCase, was expressed under the control of the cry1Aa promoter, which is controlled by σ(E) and σ(K) and is expressed in the mother cell compartment. The correct display was confirmed using enzyme activity assays, flow cytometry, and immunogold electron microscopy. In addition, we demonstrated the display of a ß-galactosidase tetramer and confirmed its correct display using enzyme activity assays and protein characterization. This native protein display system, combined with the robust nature of Bacillus spores, will broaden the range of displayable target proteins. Consequently, the applications of display technology will be expanded, including high-throughput screening, vaccines, biosensors, biocatalysis, bioremediation, and other innovative bioprocesses.
Subject(s)
Bacillus subtilis/metabolism , Cell Surface Display Techniques , Cellulase/metabolism , Membrane Proteins/metabolism , Spores/metabolism , beta-Galactosidase/metabolism , Bacillus subtilis/genetics , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Cellulase/genetics , Endotoxins/genetics , Flow Cytometry , Gene Expression , Hemolysin Proteins/genetics , Membrane Proteins/genetics , Microscopy, Immunoelectron , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spores/genetics , beta-Galactosidase/geneticsABSTRACT
Human drug metabolites produced by cytochrome P450 enzymes are critical for safety testing and may themselves act as drugs or leads in the drug discovery and development process. Here, highly active chimeric fusion proteins (chimeras) were obtained by reductase domain swapping of mutants at key catalytic residues of the heme domain with that of a natural variant (CYP102A1.2) of P450 BM3 (CYP102A1.1) from Bacillus megaterium. Random mutagenesis at the heme domain of the chimera was also used to generate chimeric mutants that were more active and diverse than the chimeras themselves. To determine whether the chimeras and several mutants of the highly active chimera displayed enhanced catalytic activity and, more importantly, whether they acquired activities of biotechnological importance, we measured the oxidation activities of the chimeras and chimeric mutants toward human P450 substrates, mainly drugs. Some of the chimeric mutants showed high activity toward typical human P450 substrates including drugs. Statin leads, especially chiral products, with inhibitory effects toward HMG-CoA reductase could be obtained from metabolites of statin drugs generated using these chimeric mutants. This study reveals the critical role of the reductase domain for the activity of P450 BM3 and shows that chimeras generated by domain swapping can be used to develop industrial enzymes for the synthesis of human metabolites from drugs and drug leads.
Subject(s)
Bacillus megaterium/enzymology , Cytochrome P-450 Enzyme System/metabolism , Pharmaceutical Preparations/metabolism , Biotransformation , Cytochrome P-450 Enzyme System/genetics , Kinetics , Mutagenesis , Mutant Proteins/genetics , Mutant Proteins/metabolism , Oxidation-Reduction , Protein Engineering , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Acetate-mediated growth inhibition of Escherichia coli has been found to be a consequence of the accumulation of homocysteine, the substrate of the cobalamin-independent methionine synthase (MetE) that catalyzes the final step of methionine biosynthesis. To improve the acetate resistance of E. coli, we randomly mutated the MetE enzyme and isolated a mutant enzyme, designated MetE-214 (V39A, R46C, T106I, and K713E), that conferred accelerated growth in the E. coli K-12 WE strain in the presence of acetate. Additionally, replacement of cysteine 645, which is a unique site of oxidation in the MetE protein, with alanine improved acetate tolerance, and introduction of the C645A mutation into the MetE-214 mutant enzyme resulted in the highest growth rate in acetate-treated E. coli cells among three mutant MetE proteins. E. coli WE strains harboring acetate-tolerant MetE mutants were less inhibited by homocysteine in l-isoleucine-enriched medium. Furthermore, the acetate-tolerant MetE mutants stimulated the growth of the host strain at elevated temperatures (44 and 45°C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (Tm) but an enhanced in vivo stability. Thus, we demonstrate improved E. coli growth in the presence of acetate or at elevated temperatures solely due to mutations in the MetE enzyme. Furthermore, when an E. coli WE strain carrying the MetE mutant was combined with a previously found MetA (homoserine o-succinyltransferase) mutant enzyme, the MetA/MetE strain was found to grow at 45°C, a nonpermissive growth temperature for E. coli in defined medium, with a similar growth rate as if it were supplemented by l-methionine.
Subject(s)
Acetates/metabolism , Escherichia coli K12/drug effects , Escherichia coli K12/radiation effects , Growth Inhibitors/metabolism , Methyltransferases/metabolism , Microbial Viability/drug effects , Microbial Viability/radiation effects , Escherichia coli K12/genetics , Escherichia coli K12/growth & development , Homocysteine/metabolism , Methyltransferases/genetics , Mutagenesis , TemperatureABSTRACT
BACKGROUND: The growth of Escherichia coli at elevated temperatures is limited due to the inherent instability of homoserine o-succinyltransferase, MetA, which is the first enzyme in the methionine biosynthesis pathway. MetA is also unstable under other stressful conditions, such as weak organic acids and oxidative stress. The MetA protein unfolds, even at 25°C, forms considerable aggregates at 37°C and completely aggregates at 44°C. RESULTS: We extended the MetA mutation studies using a consensus concept based on statistics and sequence database analysis to predict the point mutations resulting in increased MetA stability. In this study, four single amino acid substitutions (Q96K, I124L, I229Y and F247Y) in MetA designed according to the consensus concept and using the I-mutant2.0 modeling tool conferred accelerated growth on the E. coli strain WE at 44°C. MetA mutants that enabled E. coli growth at higher temperatures did not display increased melting temperatures (Tm) or enhanced catalytic activity but did show improved in vivo stability at mild (37°C) and elevated (44°C) temperatures. Notably, we observed that the stabilized MetA mutants partially recovered the growth defects of E. coli mutants in which ATP-dependent proteases or the DnaK chaperone was deleted. These results suggest that the impaired growth of these E. coli mutants primarily reflect the inherent instability of MetA and, thus, the methionine supply. As further evidence, the addition of methionine recovered most of the growth defects in mutants lacking either ATP-dependent proteases or the DnaK chaperone. CONCLUSIONS: A collection of stable single-residue mutated MetA enzymes were constructed and investigated as background for engineering the stabilized mutants. In summary, the mutations in a single gene, metA, reframe the window of growth temperature in both normal and mutant E. coli strains.