ABSTRACT
Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1-E2-Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.
Subject(s)
Bacterial Proteins , Bacteriophages , Escherichia , Ubiquitin-Activating Enzymes , Ubiquitin-Conjugating Enzymes , Ubiquitination , Ubiquitins , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacteriophages/chemistry , Bacteriophages/immunology , Bacteriophages/metabolism , Catalytic Domain , Crystallography, X-Ray , Cysteine/chemistry , Cysteine/metabolism , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/metabolism , Escherichia/chemistry , Escherichia/enzymology , Escherichia/immunology , Escherichia/virology , Evolution, Molecular , Lysine/chemistry , Lysine/metabolism , Models, Molecular , Operon/genetics , Protein Domains , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitins/metabolism , Ubiquitins/chemistry , Eukaryota/enzymology , Eukaryota/metabolismABSTRACT
Hyaluronidase, an enzyme that degrades hyaluronic acid (HA), is utilized in clinical settings to facilitate drug diffusion, manage extravasation, and address injection-related complications linked to HA-based fillers. In this study, a novel hyaluronate lyase EsHyl8 was cloned, expressed, and characterized from Escherichia sp. A99 of human intestinal origin. This lyase belongs to polysaccharide lyase (PL) family 8, and showed specific activity towards HA. EsHyl8 exhibited optimal degradation at 40 °C and pH 6.0. EsHyl8 exhibited a high activity of 376.32 U/mg among hyaluronidases of human gut microorganisms. EsHyl8 was stable at 37 °C and remained about 70 % of activity after incubation at 37 °C for 24 h, demonstrating excellent thermostability. The activity of EsHyl8 was inhibited by Zn2+, Cu2+, Fe3+, and SDS. EsHyl8 was an endo-type enzyme whose end-product was unsaturated disaccharide. This study enhances our understanding of hyaluronidases from human gut microorganisms.
Subject(s)
Cloning, Molecular , Polysaccharide-Lyases , Polysaccharide-Lyases/genetics , Polysaccharide-Lyases/chemistry , Polysaccharide-Lyases/isolation & purification , Polysaccharide-Lyases/metabolism , Humans , Recombinant Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/isolation & purification , Escherichia/genetics , Escherichia/enzymology , Hyaluronic Acid/chemistry , Hyaluronic Acid/metabolism , Enzyme Stability , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Hydrogen-Ion Concentration , Substrate SpecificityABSTRACT
BACKGROUND: L-Methioninase (EC 4.4.1.11; MGL) is a pyridoxal phosphate (PLP)-dependent enzyme that is produced by a variety of bacteria, fungi, and plants. L-methioninase, especially from Pseudomonas and Citrobacter sp., is considered as the efficient therapeutic enzyme, particularly in cancers such as glioblastomas, medulloblastoma, and neuroblastoma that are more sensitive to methionine starvation. OBJECTIVE: The low stability is one of the main drawbacks of the enzyme; in this regard, in the current study, different features of the enzyme, including phylogenetic, functional, and structural from Pseudomonas, Escherichia, Clostridium, and Citrobacter strains were evaluated to find the best bacterial L-Methioninase. METHODS: After the initial screening of L-Methioninase sequences from the above-mentioned bacterial strains, the three-dimensional structures of enzymes from Escherichia fergusonii, Pseudomonas fluorescens, and Clostridium homopropionicum were determined through homology modeling via GalaxyTBM server and refined by GalaxyRefine server. RESULTS AND CONCLUSION: Afterwards, PROCHECK, verify 3D, and ERRAT servers were used for verification of the obtained models. Moreover, antigenicity, allergenicity, and physico-chemical analysis of enzymes were also carried out. In order to get insight into the interaction of the enzyme with other proteins, the STRING server was used. The secondary structure of the enzyme is mainly composed of random coils and alpha-helices. However, these outcomes should further be validated by wet-lab investigations.
Subject(s)
Bacterial Proteins/genetics , Carbon-Sulfur Lyases/genetics , Bacterial Proteins/chemistry , Carbon-Sulfur Lyases/chemistry , Citrobacter/enzymology , Citrobacter/genetics , Clostridium/enzymology , Clostridium/genetics , Escherichia/enzymology , Escherichia/genetics , Patents as Topic , Phylogeny , Pseudomonas/enzymology , Pseudomonas/geneticsABSTRACT
BACKGROUND: Urinary tract infections caused by extended-spectrum beta-lactamase-producing bacteria are increasing worldwide. At our hospital, the number of pediatric patients hospitalized because of an upper urinary tract infection has dramatically increased since 2016. In total, 60.5% of urinary tract infections are caused by extended-spectrum beta-lactamase-producing Escherichia coli. Such a high prevalence of extended-spectrum beta-lactamase-producing E. coli has not been detected previously in Japan. Therefore, we evaluated the clinical and bacteriologic characteristics and efficacy of antibiotics against upper urinary tract infections caused by E. coli in children. METHODS: This retrospective study surveyed 152 patients who were hospitalized in the pediatric department of Shimane Prefectural Central Hospital because of upper urinary tract infections caused by E. coli. Medical records were reviewed to examine patient characteristics. O antigens, antibiotic susceptibility, gene typing, and pulse-field gel electrophoresis were studied at the Shimane Prefectural Institute of Public Health and Environmental Science. RESULTS: Urine sample analyses showed extended-spectrum beta-lactamase types such as CTX-M-9 and plural virulence genes. We changed the primary antibiotic treatment to flomoxef or cefmetazole to treat upper urinary tract infections caused by Gram-negative bacilli. After changing treatment, the time to fever alleviation was significantly shortened. CONCLUSION: Extended-spectrum beta-lactamase-producing E. coli should be suspected in community-acquired upper urinary tract infections. Therefore, when treating patients, it is necessary to focus on antibiotic susceptibility and the prevalence of extended-spectrum beta-lactamase-producing bacteria found in each area. Flomoxef and cefmetazole are useful primary treatments for upper urinary tract infections caused by extended-spectrum beta-lactamase-producing E. coli.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Cefmetazole/therapeutic use , Cephalosporins/therapeutic use , Community-Acquired Infections/drug therapy , Escherichia coli Infections/drug therapy , Escherichia/enzymology , Urinary Tract Infections/drug therapy , Adolescent , Child , Child, Preschool , Community-Acquired Infections/microbiology , Drug Resistance, Multiple, Bacterial/genetics , Escherichia/genetics , Escherichia coli Infections/microbiology , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Female , Genotype , Humans , Infant , Infant, Newborn , Male , O Antigens/metabolism , Retrospective Studies , Urinary Tract Infections/microbiology , Virulence/genetics , beta-Lactamases/biosynthesis , beta-Lactamases/geneticsABSTRACT
The important platform polysaccharide N-acetylglucosamine (GlcNAc) has great potential to be used in the fields of food, cosmetics, agricultural, pharmaceutical, medicine and biotechnology. This GlcNAc is being produced by traditional methods of environment-unfriendly chemical digestion with strong acids. Therefore, researchers have been paying more attention to enzymatic hydrolysis process for the production of GlcNAc. Hence, in this study, we isolated novel chitinase (Escherichia fergusonii) and chitosanase (Chryseobacterium indologenes, Comamonas koreensis) producing strains from Korean native calves feces, and developed the potential of an eco-friendly microbial progression for GlcNAc production from swollen chitin and chitosan by enzymatic degradation. Maximum chitinase (7.24±0.07U/ml) and chitosanase (8.42±0.09, 8.51±0.25U/ml) enzyme activity were reached in submerged fermentation at an optimal pH of 7.0 and 30°C. In this study, sucrose, yeast extract, (NH4)2SO4, and NaCl were found to be the potential enhancers of exo-chitinase activity and glucose, corn flour, yeast extract, soybean flour, (NH4)2SO4, NH4Cl and K2HPO4 were found to be the potential activator for exo-chitosanase activity. Optimum concentrations of the carbon sources for enhanced chitinase activity were 9.91, 3.21, 9.86, 1.66U/ml and chitosanase activity were 1.63, 1.13, 2.28, 3.71, 9.02, 4.93, and 2.14U/ml. These enzymes efficiently hydrolyzed swollen chitin and chitosan to N-acetylglucosamine were characterized by thin layer chromatography and were further confirmed by high-pressure liquid chromatography. From a commercial perspective, we isolated, optimized and characterized exochitinase from Escherichia fergusonii (HANDI 110) and chitosanase from Chryseobacterium indologenes (HANYOO), and Comamonas koreensis (HANWOO) for the large-scale production of GlcNAc facilitating its potential use in industrial applications.
Subject(s)
Acetylglucosamine/biosynthesis , Chitinases/biosynthesis , Chryseobacterium/enzymology , Comamonas/enzymology , Escherichia/enzymology , Glycoside Hydrolases/biosynthesis , Carbon/pharmacology , Chitin/metabolism , Chitosan/metabolism , Chromatography, Thin Layer , Hydrolysis , Nitrogen/pharmacology , Phylogeny , Salts/pharmacologyABSTRACT
The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to â¼66,000). Biotechnol. Bioeng. 2017;114: 2187-2195. © 2017 Wiley Periodicals, Inc.
Subject(s)
Acid Phosphatase/chemistry , Alcohols/chemistry , Escherichia/enzymology , Esters/chemical synthesis , Morganella morganii/enzymology , Phosphates/chemical synthesis , Acid Phosphatase/genetics , Enzyme Activation , Mutagenesis, Site-Directed , PhosphorylationABSTRACT
Antimicrobial resistance through extended-spectrum beta-lactamases (ESBLs) and transferable (plasmid-encoded) cephamycinases (pAmpCs) represents an increasing problem in human and veterinary medicine. The presence of ESBL-/pAmpC-producing commensal enterobacteria in farm animals, such as broiler chickens, is considered one possible source of food contamination and could therefore also be relevant for human colonization. Studies on transmission routes along the broiler production chain showed that 1-day-old hatchlings are already affected. In this study, ESBL-/pAmpC-positive broiler parent flocks and their corresponding eggs, as well as various environmental and air samples from the hatchery, were analyzed. The eggs were investigated concerning ESBL-/pAmpC-producing enterobacteria on the outer eggshell surface (before/after disinfection), the inner eggshell surface, and the egg content. Isolates were analyzed concerning their species, their phylogroup in the case of Escherichia coli strains, the respective resistance genes, and the phenotypical antibiotic resistance. Of the tested eggs, 0.9% (n = 560) were contaminated on their outer shell surface. Further analyses using pulsed-field gel electrophoresis showed a relationship of these strains to those isolated from the corresponding parent flocks, which demonstrates a pseudo-vertical transfer of ESBL-/pAmpC-producing enterobacteria into the hatchery. Resistant enterobacteria were also found in environmental samples from the hatchery, such as dust or surfaces which could pose as a possible contamination source for the hatchlings. All 1-day-old chicks tested negative directly after hatching. The results show a possible entry of ESBL-/pAmpC-producing enterobacteria from the parent flocks into the hatchery; however, the impact of the hatchery on colonization of the hatchlings seems to be low. IMPORTANCE: ESBL-/pAmpC-producing enterobacteria occur frequently in broiler-fattening farms. Recent studies investigated the prevalence and possible transmission route of these bacteria in the broiler production chain. It seemed very likely that the hatcheries play an important role in transmission and/or contamination events. There are only few data on transmission investigations from a grandparent or parent flock to their offspring. However, reliable data on direct or indirect vertical transmission events in the hatchery are not available. Therefore, we conducted our study and intensively investigated the broiler hatching eggs from ESBL-/pAmpC-positive broiler parent flocks as well as the hatchlings and the environment of the hatchery.
Subject(s)
Cephamycins/metabolism , Enterobacteriaceae Infections/veterinary , Escherichia coli/genetics , Escherichia/genetics , Infectious Disease Transmission, Vertical/veterinary , Poultry Diseases/transmission , beta-Lactamases/genetics , Animals , Animals, Domestic , Chickens/microbiology , Drug Resistance, Multiple, Bacterial/genetics , Eggs/microbiology , Enterobacteriaceae Infections/microbiology , Enterobacteriaceae Infections/transmission , Escherichia/drug effects , Escherichia/enzymology , Escherichia/isolation & purification , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Escherichia coli Infections/transmission , Escherichia coli Infections/veterinary , Farms , Humans , Plasmids , Poultry Diseases/microbiology , beta-Lactamases/biosynthesisABSTRACT
The development of synthetic biological devices has increased rapidly in recent years and the practical benefits of such biological devices are becoming increasingly clear. Here, we further improved the design of a previously reported high-throughput genetic enzyme screening system by investigating device-compatible biological components and phenol-mediated cell-cell communication, both of which increased the efficiency and practicality of the screening device without requiring the use of flow cytometry analysis. A sensor cell was designed to detect novel microbes with target enzyme activities on solid media by forming clear, circular colonies with fluorescence around the unknown microbes producing target enzymes. This mechanism of detection was enabled by the combination of pre-effector phenolic substrate treatment in the presence of target enzyme-producing microbes and control of the growth and fluorescence of remote sensor cells via phenol-mediated cell-cell communication. The sensor cells were applied to screen soil bacteria with phosphatase activity using phenyl phosphate as phenolic substrates. The sensor cells facilitated successful visualization of phosphatase activity in unknown microbes, which were identified by 16S rRNA analysis. Enzyme activity assays confirmed that the proposed screening technique was able to find 23 positive clones out of 33 selected colonies. Since many natural enzymatic reactions produce phenolic compounds from phenol-derived substrates, we anticipate that the proposed technique may have broad applications in the assessment and screening of novel microbes with target enzymes of interest. This method also can provide insights into the identification of novel enzymes for which screening assays are not yet available.
Subject(s)
Bacterial Proteins/genetics , Cell Communication , Genes, Bacterial , Trans-Activators/genetics , Aeromonas/enzymology , Aeromonas/genetics , Bacterial Proteins/metabolism , Chromatiaceae/enzymology , Chromatiaceae/genetics , DNA, Bacterial/isolation & purification , Escherichia/enzymology , Escherichia/genetics , Flow Cytometry , High-Throughput Screening Assays , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Pseudomonas/enzymology , Pseudomonas/genetics , RNA, Ribosomal, 16S/isolation & purification , Republic of Korea , Sequence Analysis, DNA , Serratia/enzymology , Serratia/genetics , Shigella flexneri/enzymology , Shigella flexneri/genetics , Soil Microbiology , Trans-Activators/metabolismABSTRACT
Escherichia coli L-asparaginases have great significance in the treatment of leukemia. Consequently, there is considerable interest in engineering this enzyme for improving its stability. In this work, the effect of surface charge on the stability of the enzyme l-asparaginase II was studied by site-directed mutagenesis of the cloned ansB gene from Escherichia sp. Replacement of two positively charged residues (K139 and K207) on the surface loops with neutral and reverse charges resulted in altered thermo stability in designed variants. Neutral charge substitutions (K139A and K207A) retained greater tolerance and stability followed by negative charge substitutions (K139D and K207D) compared to control mutant K139R and wild enzyme. From the results, it was concluded that the optimization of surface charge contributed much to the thermal properties of proteins without affecting the structure.
Subject(s)
Asparaginase/chemistry , Bacterial Proteins/chemistry , Escherichia/enzymology , Animals , Asparaginase/genetics , Bacterial Proteins/genetics , Base Sequence , Cattle , Cloning, Molecular , Escherichia coli , Feces/microbiology , Hot Temperature , Hydrogen-Ion Concentration , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Protein Denaturation , Protein Stability , Static Electricity , Surface PropertiesABSTRACT
In this work, we introduce an entirely automated enzyme assay based on capillary electrophoresis coupled to electrospray ionization mass spectrometry termed MINISEP-MS for multiple interfluent nanoinjections-incubation-separation-enzyme profiling using mass spectrometry. MINISEP-MS requires only nanoliters of reagent solutions and uses the separation capillary as a microreactor, allowing multiple substrates to be assayed simultaneously. The method can be used to rapidly profile the substrate specificity of any enzyme and to measure steady-state kinetics in an automated fashion. We used the MINISEP-MS assay to profile the substrate specificity of three aminotransferases (E. coli aspartate aminotransferase, E. coli branched-chain amino acid aminotransferase, and Bacillus sp. YM-1 D-amino acid aminotransferase) for 33 potential amino acid substrates and to measure steady-state kinetics. Using MINISEP-MS, we were able to recapitulate the known substrate specificities and to discover new amino acid substrates for these industrially relevant enzymes. Additionally, we were able to measure the apparent K(M) and k(cat) parameters for amino acid donor substrates of these aminotransferases. Because of its many advantages, the MINISEP-MS assay has the potential of becoming a useful tool for researchers aiming to identify or create novel enzymes for specific biocatalytic applications.
Subject(s)
Biological Assay , Transaminases/metabolism , Biocatalysis , Electrophoresis, Capillary , Escherichia/classification , Escherichia/enzymology , Mass Spectrometry , Substrate Specificity , Transaminases/chemistryABSTRACT
5'-Nucleotides including 5'-inosinic acid have characteristic taste and important application in various foods as flavour potentiators. The selective nucleoside acid phosphatase/phosphotransferase (AP/PTase) can catalyse the synthesis of 5'-nucleotides by transfer of phosphate groups. In this study, a 747-bp gene encoding AP/PTase from Escherichia blattae was synthesised. After expression, the recombinant AP/PTase was purified using nickel-NTA. The optimal temperature and pH of this enzyme were 30°C and 5.0, respectively. The activity was partially inhibited by metal ions such as Hg(2+), Ag(+) and Cu(2+), but not by chelating reagents such as EDTA. The values of K(m) and V(max) for inosine were 40 mM and 3.5 U/mg, respectively. Using this purified enzyme, 16.83 mM of 5'-IMP was synthesised from 37 mM of inosine and the molar yield reached 45.5%. Homology modelling and docking simulation were discussed.
Subject(s)
Acid Phosphatase/metabolism , Bacterial Proteins/metabolism , Escherichia/enzymology , Inosine Monophosphate/metabolism , Phosphotransferases/metabolism , Acid Phosphatase/chemistry , Acid Phosphatase/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Enzyme Stability , Escherichia/genetics , Escherichia/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Kinetics , Phosphotransferases/chemistry , Phosphotransferases/geneticsABSTRACT
In the design of inhibitors of phosphosugar metabolizing enzymes and receptors with therapeutic interest, malonate has been reported in a number of cases as a good and hydrolytically-stable surrogate of the phosphate group, since both functions are dianionic at physiological pH and of comparable size. We have investigated a series of malonate-based mimics of the best known phosphate inhibitors of class II (zinc) fructose-1,6-bis-phosphate aldolases (FBAs) (e.g., from Mycobacterium tuberculosis), type I (zinc) phosphomannose isomerase (PMI) from Escherichia coli, and phosphoglucose isomerase (PGI) from yeast. In the case of FBAs, replacement of one phosphate by one malonate on a bis-phosphorylated inhibitor (1) led to a new compound (4) still showing a strong inhibition (K(i) in the nM range) and class II versus class I selectivity (up to 8×10(4)). Replacement of the other phosphate however strongly affected binding efficiency and selectivity. In the case of PGI and PMI, 5-deoxy-5-malonate-D-arabinonohydroxamic acid (8) yielded a strong decrease in binding affinities when compared to its phosphorylated parent compound 5-phospho-D-arabinonohydroxamic acid (2). Analysis of the deposited 3D structures of the kinetically evaluated enzymes complexed to the phosphate-based inhibitors indicate that malonate could be a good phosphate surrogate only if phosphate is not tightly bound at the enzyme active site, such as in position 7 of compound 1 for FBAs. These observations are of importance for further design of inhibitors of phosphorylated-compounds metabolizing enzymes with therapeutic interest.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Fructose-Bisphosphate Aldolase/antagonists & inhibitors , Glucose-6-Phosphate Isomerase/antagonists & inhibitors , Malonates/chemical synthesis , Mannose-6-Phosphate Isomerase/antagonists & inhibitors , Animals , Catalytic Domain , Enzyme Activation/drug effects , Escherichia/enzymology , Humans , Inhibitory Concentration 50 , Malonates/chemistry , Malonates/pharmacology , Models, Biological , Molecular Structure , Yeasts/enzymologyABSTRACT
Extended-spectrum-beta-lactamase (ESBL)-producing organisms have captured the attention of clinicians and laboratorians and are agents of nosocomial and community onset infections (J. D. Pitout and K. B. Laupland, Lancet Infect. Dis. 8:159-166, 2008). ESBLs in many enterobacteriaceae and in nonfermenting Gram-negative organisms have been described (K. Bush and G. A. Jacoby, Antimicrob. Agents Chemother. 54:969-976, 2010). We present the first case of a clinical isolate of multidrug-resistant Escherichia fergusonii expressing an extended-spectrum-beta-lactamase (ESBL).
Subject(s)
Cystitis/microbiology , Drug Resistance, Multiple, Bacterial , Enterobacteriaceae Infections/diagnosis , Escherichia/classification , Escherichia/enzymology , beta-Lactamases/biosynthesis , Aged , Anti-Bacterial Agents/pharmacology , Enterobacteriaceae Infections/microbiology , Escherichia/drug effects , Escherichia/isolation & purification , Female , Humans , Microbial Sensitivity TestsABSTRACT
Chiral aryl vicinal diols were obtained in high ee and yield by asymmetric dihydroxylation of aryl olefins with tandem biocatalysts: one contains an enantioselective styrene monooxygenase, and the other contains a regioselective epoxide hydrolase.
Subject(s)
Alkenes/chemistry , Epoxide Hydrolases/chemistry , Oxygenases/chemistry , Biocatalysis , Escherichia/enzymology , Hydroxylation , Molecular Structure , Sphingomonas/enzymology , StereoisomerismABSTRACT
Chemo-enzymatic methods for covalently crosslinking carrier proteins with partner enzymes within modular synthases hold promise for elucidating and engineering metabolic pathways. Our efforts to crystallize the ACP-KS complexes of fatty acid synthases have been complicated by difficulties in the purification of the crosslinked complex from the other proteins in the reaction. Here we present a solution that employs an orthogonal purification strategy to achieve the quantity and level of purity necessary for further studies of this complex.
Subject(s)
Acrylates/chemistry , Acyl Carrier Protein/chemistry , Fatty Acid Synthases/chemistry , Fatty Acid Synthases/isolation & purification , Pantetheine/analogs & derivatives , Pantetheine/chemistry , Polyketide Synthases/chemistry , Polyketide Synthases/isolation & purification , Acyl Carrier Protein/isolation & purification , Coenzyme A/chemistry , Coenzyme A/genetics , Coenzyme A/isolation & purification , Cross-Linking Reagents/chemistry , Escherichia/enzymology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Fatty Acid Synthases/genetics , Models, Molecular , Polyketide Synthases/genetics , Polymerase Chain Reaction , Protein Conformation , Protein Structure, TertiaryABSTRACT
N(6)-Methyladenosine 1618 of Escherichia coli 23 S rRNA is located in a cluster of modified nucleotides 12 A away from the nascent peptide tunnel of the ribosome. Here, we describe the identification of gene ybiN encoding an enzyme responsible for methylation of A1618. Knockout of the ybiN gene leads to loss of modification at A1618. The modification is restored if ybiN knock-out strain has been co-transformed with a plasmid expressing the ybiN gene. On the basis of these results we suggest that ybiN gene should be renamed to rlmF in accordance with the accepted nomenclature for rRNA methyltransferases. Recombinant YbiN protein is able to methylate partially deproteinized 50 S ribosomal subunit, so-called 3.5 M LiCl core particle in vitro, but neither the completely assembled 50 S subunits nor completely deproteinized 23 S rRNA. Both lack of the ybiN gene and it's over-expression leads to growth retardation and loss of cell fitness comparative to the parental strain. It might be suggested that A1618 modification could be necessary for the exit tunnel interaction with some unknown regulatory peptides.
Subject(s)
Adenine/metabolism , Escherichia/genetics , Genes, Bacterial , Methyltransferases/metabolism , RNA, Bacterial/chemistry , RNA, Ribosomal, 23S/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia/enzymology , Escherichia/growth & development , Methylation , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Mutation , Plasmids , Protein Structure, Secondary , RNA, Bacterial/metabolism , RNA, Ribosomal, 23S/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Combining tunable transcription with an enzyme-degradation tag affords an effective means to reduce intracellular enzyme concentrations from high to very low levels. Such fine-tuned control allows selection pressure to be systematically increased in directed-evolution experiments. This facilitates identification of mutants with wild-type activity, as shown here for an engineered chorismate mutase. Numerous selection formats and cell-based screening methodologies may benefit from the large dynamic range afforded by this easily implemented strategy.
Subject(s)
Directed Molecular Evolution/methods , Selection, Genetic , Chorismate Mutase/genetics , Chorismate Mutase/metabolism , Escherichia/enzymology , Genetic Complementation Test , Genetic Engineering , Mutagenesis , Tetracycline , Transcription, GeneticABSTRACT
A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminoglycoside phosphotransferase gene (aph) from Tn903, a lacZ'' gene for alpha-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DH5alpha and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.
Subject(s)
Escherichia/genetics , Genetic Vectors/genetics , Kanamycin Kinase/genetics , Lac Operon , Pseudomonas/genetics , Anti-Bacterial Agents/pharmacology , Cloning, Molecular , Drug Resistance, Bacterial , Escherichia/enzymology , Genetic Complementation Test , Kanamycin/pharmacology , Plasmids , Pseudomonas/enzymologySubject(s)
Glucokinase/chemistry , Glucokinase/metabolism , Glucose/metabolism , Carbohydrate Sequence , Escherichia/enzymology , Glucokinase/genetics , Glucose/analogs & derivatives , Glucose/chemical synthesis , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5'-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5'-inosinic acid (5'-IMP) productivity and lower 5'-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5'-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.