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1.
Expanding Substrate Promiscuity by Engineering a Novel Adenylating‐Methylating NRPS Bifunctional Enzyme 下载免费PDF全文
Dr. Sanjib K. Shrestha Dr. Sylvie Garneau‐Tsodikova 《Chembiochem : a European journal of chemical biology》2016,17(14):1328-1332
Nonribosomal peptides synthetases (NRPSs), which are multifunctional mega‐enzymes producing many biologically active metabolites, are ideal targets for enzyme engineering. NRPS adenylation domains play a critical role in selecting/activating the amino acids to be transferred to downstream NRPS domains in the biosynthesis of natural products. Both monofunctional and bifunctional A domains interrupted with an auxiliary domain are found in nature. Here, we show that a bifunctional interrupted A domain can be uninterrupted by deleting its methyltransferase auxiliary domain portion to make an active monofunctional enzyme. We also demonstrate that a portion of an auxiliary domain with almost no sequence identity to the original auxiliary domain can be insert into naturally interrupted A domain to develop a new active bifunctional A domain with increased substrate profile. This work shows promise for the creation of new interrupted A domains in engineered NRPS enzymes. 相似文献
2.
Howell EE 《Chembiochem : a European journal of chemical biology》2005,6(4):590-600
There are numerous examples of proteins that catalyze the same reaction while possessing different structures. This review focuses on two dihydrofolate reductases (DHFRs) that have disparate structures and discusses how the catalytic strategies of these two DHFRs are driven by their respective scaffolds. The two proteins are E. coli chromosomal DHFR (Ec DHFR) and a type II R-plasmid-encoded DHFR, typified by R67 DHFR. The former has been described as a very well evolved enzyme with an efficiency of 0.15, while the latter has been suggested to be a model for a "primitive" enzyme that has not yet been optimized by evolution. This comparison underlines what is important to catalysis in these two enzymes and concurrently highlights fundamental issues in enzyme catalysis. 相似文献
3.
Dale Glenn E.; Broger Clemens; Langen Hanno; Arcy Allan D'; Stber Dietrich 《Protein engineering, design & selection : PEDS》1994,7(7):933-939
In recent years resistance to the antibacterial agent trimethoprim(Tmp) has become more widespread and several Tmp-resistant (Tmprdihydrofolate reductases (DHFRs) have been described from Gram-negativebacteria. In staphylococci, however, only one Tmpr DHFR (typeS1 DHFR) has been found so far, and this is located on transposonTn4003. To help understand the mechanism of resistance, we areinterested in determining the 3-D structure of the recombinantenzyme produced in Escherichia coli. However, the productionlevel of the type S1 DHFR was very low and >95% of the totalrecombinant protein accumulated in inclusion bodies. Furthermore,as a result of an internal start of translation, a truncatedderivative of the enzyme that copurified with the full-lengthenzyme was produced. We were able to increase the expressionlevel 20-fold by changing 18 N-terminal codons and to eliminatethe internal start of translation. In addition, through molecularmodelling and subsequent site-directed mutagenesis to replacetwo amino acids, we constructed a biochemically similar butsoluble derivative of the type SI DHFR that, after productionin E.coli, resulted in a 264-fold increase in DHFR activity.The highly overproduced enzyme was purified to homogeneity,characterized biochemically and crystallized. 相似文献
4.
Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations 总被引:1,自引:0,他引:1
Schmitzer AR Lépine F Pelletier JN 《Protein engineering, design & selection : PEDS》2004,17(11):809-819
We have applied a global approach to enzyme active site exploration, where multiple mutations were introduced combinatorially at the active site of Type II R67 dihydrofolate reductase (R67 DHFR), creating numerous new active site environments within a constant framework. By this approach, we combinatorially modified all 16 principal amino acids that constitute the active site of this enzyme. This approach is fundamentally different from active site point mutation in that the native active site context is no longer accounted for. Among the 1536 combinatorially mutated active site variants of R67 DHFR we created, we selected and kinetically characterized three variants with highly altered active site compositions. We determined that they are of high fitness, as defined by a complex function consisting jointly of catalytic activity and resistance to trimethoprim. The k(cat) and K(M) values were similar to those for the native enzyme. The favourable Delta(DeltaG) values obtained (ranging from -0.72 to -1.08 kcal/mol) suggest that, despite their complex mutational pattern, no fundamental change in the catalytic mechanism has occurred. We illustrate that combinatorial active site mutagenesis can allow for the creation of compensatory mutations that could not be predicted and thus provides a route for more extensive exploration of functional sequence space than is allowed by point mutation. 相似文献
5.
Park H; Bradrick TD; Howell EE 《Protein engineering, design & selection : PEDS》1997,10(12):1415-1424
R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria
as a resistance mechanism to increasing clinical use of the antibacterial
drug trimethoprim. Type II DHFRs are not homologous in either sequence or
structure with chromosomal DHFRs. The crystal structure of R67 DHFR shows a
single active site pore that spans the length of the homotetramer. Related
sites (due to a 222 symmetry element at the center of the pore) are used to
bind ligands, i.e. each half of the pore can accommodate either the
substrate, dihydrofolate (DHF), or the cofactor, NADPH, although DHF and
NADPH are bound differently. To evaluate the role of glutamine 67 (and its
symmetry- related Q167, Q267 and Q367 residues which occur at the center of
the active site pore), a Q67H mutation was constructed. Binary binding of
dihydrofolate (DHF; monitored by isothermal titration calorimetry) displays
two identical sites with a Kd value of 0.04 microM, while binding of NADPH
shows two sites possessing negative cooperativity with Kd values of 0.027
and 0.62 microM. A comparison of ligand binding in Q67H versus wild-type
(wt) R67 DHFR indicates both ligands bind more tightly (80-6000-fold) and
DHF binding in Q67H R67 DHFR no longer displays positive cooperativity as
seen in wt R67 DHFR. Ternary complex binding in the Q67H mutant indicates a
total of two ligands can bind per pore. Substantial substrate and cofactor
inhibition are observed during catalysis, consistent with non-productive
binding of either two DHF or two NADPH molecules in Q67H R67 DHFR. Because
of the symmetry- related binding sites in the active site pore, the
accumulation of potentially positive mutations in R67 DHFR is limited by
the balance between tighter binding of ligands (and thus potentially
increased catalytic efficiency) and inhibition that arises upon tighter
binding of two identical ligands at symmetry-related sites.
相似文献
6.
Effects of insertions and deletions in a beta-bulge region of Escherichia coli dihydrofolate reductase 总被引:1,自引:0,他引:1
The role of a beta-bulge in Escherichia coli dihydrofolate reductase (DHFR)
has been explored by a series of insertion and deletion mutations.
Insertion of a seven amino acid sequence from a structurally equivalent
'beta-blowout' sequence from human DHFR destabilizes E. coli DHFR by 3.6
kcal/mol and decreases catalytic efficiency (kcat/K(m)) 34- fold. Deletion
of F137, delta 137, the looped out residue in the bulge, also destabilizes
E. coli DHFR by 2.8 kcal/mol but only decreases catalytic efficiency
threefold. Concurrent deletion of F137 and mutation of, V136 to proline to
try and maintain the strand twist associated with the beta-bulge decreases
protein stability by 3.4 kcal/mol and decreases catalytic efficiency
84-fold. These insertion/deletion mutations were also constructed in a D27S
DHFR background. The D27S mutation has been described previously and
proposed to remove the catalytic acid from the active site. The delta 137
mutation partially suppresses the effect of the D27S mutation as it
decreases the K(m) for substrate, dihydrofolate, twofold. Non-additive
effects are observed for the insertion/deletion mutations in wild-type
versus D27S DHFR backgrounds, consistent with structural changes.
相似文献
7.
Boll B Hennig S Xie C Sohng JK Heide L 《Chembiochem : a European journal of chemical biology》2011,12(7):1105-1114
The biosynthesis of aminocoumarin antibiotics requires two acyladenylate-forming enzymes: one for the activation of L-tyrosine as a precursor of the aminocoumarin moiety and another for the linkage of an acyl moiety to the aminocoumarin moiety. Unexpectedly, the biosynthetic gene cluster of the aminocoumarin antibiotic rubradirin was found to contain three genes for putative acyladenylate-forming enzymes of aminocoumarin biosynthesis and conjugation. We expressed, purified, and investigated these three proteins. Orf4 (55 kDa) was shown to be an active aminocoumarin acyl ligase. RubF6 (56 kDa) was inactive, but could be converted into an active enzyme by site-directed mutagenesis. RubC1 (138 kDa) was shown to be a unique bifunctional enzyme, comprising an aminocoumarin acyl ligase, and tyrosine-adenylation and peptidyl-carrier domains. This natural hybrid enzyme is unique among known proteins. A hypothesis is proposed as to how such an enzyme could offer a particularly effective machinery for aminocoumarin antibiotic biosynthesis. 相似文献
8.
A single polypeptide chain containing two dihydrofolate reductase( D M ) sequences from Escherichia coli was constructed to determineif a repeat sequence fusion protein could be expressed in anactive form. The possibility that intersequence interactionscould play a significant role for this enzyme is suggested bythe results of Hall and Frieden (1989, Proc. NatlAcad. Sri.USA, 86, 3060-3064) who observed a substantial decrease in theyield of active enzyme when folded hi the presence of a largeC-terminal fragment. The fusion protein [DHFR(Cysl52Ghi)-De-DHFR(MetlGln)] was efficiently expressed in E.coli cells and hasan activity which is twice that of the wild-type enzyme in thestandard assay. The Michaelis constants of the fusion proteinfor the substrate, dihydrofolate and the cofactor, NADPH, areessentially unchanged from those of the wild-type protein. Theureainduced in vitro unfolding reaction of the fusion proteinat low concentrations was found to be fully reversible and followa three state model, suggesting that the two domains unfoldindependently. At higher protein concentrations the unfoldingtransition broadened and shifted to a higher urea concentration.Size-exclusion chromatography results are consistent with theformation of aggregates at the higher protein concentration,even in the absence of denaturant. 相似文献
9.
Effects of the length of a glycine linker connecting the N-and C- termini of a circularly permuted dihydrofolate reductase 总被引:1,自引:0,他引:1
An important consideration in the construction of active and stable
circularly permuted proteins is the connective sequence that links the
native N- and C-termini. For this reason, various lengths of polyglycine
linkers (two, three, four, five and six glycines) were employed to connect
the original N- and C-termini of a circularly permuted construct of
Escherichia coli dihydrofolate reductase (DHFR) in which the new N-terminus
was Met16. Examination of the circular dichroism (CD) spectra,
gel-filtration chromatography elution profiles, urea-induced unfolding
properties and enzyme kinetics revealed that, among the linkers tested, a
linker length of five glycines was the most favorable. The Vmax of the
circularly permuted variant with a five glycine linker (cpM16G5) was about
20% that of wild-type DHFR, although far UV CD spectra, gel filtration
elution time, conformational stability and Km for the substrate
dihydrofolate and Kd for the coenzyme NADPH were comparable in the two
proteins. Another circularly permuted DHFR with a five glycine linker in
which a new N-terminus was created at Leu24 (cpL24G5) was also constructed
and assayed. The Vmax of cpL24G5 was almost the same as the wild-type,
presumably due to the optimization of the glycine linker. The improved
activity of the Leu24 permutant is probably due to the disruption of a
catalytically important structure, the M20 loop, in the Met16 permutant.
相似文献
10.
Anke Matura Dorina Köpke Claudia Marschelke Julius Kramer Alla Synytska Marco Sallat 《化学,工程师,技术》2020,92(3):248-255
Mono- and bifunctional hybrid core-shell particles were used for enzyme immobilization. Ideal conditions for enzyme immobilization were evaluated based on monofunctional poly(2-dimethylamino)ethyl methacrylate-modified particles. Then, the obtained results were transferred to a bifunctional Janus particle system, which should simultaneously enable enzyme immobilization, switchable separation, and reuse of the enzyme. Finally, application of the system was exemplarily demonstrated by multiple discoloration of textile industry process water with laccase and a cost estimation is presented. 相似文献
11.
Julio Alfonso Felix Ortego Rosa Sanchez-Monge Gloria Garcia-Casado Merardo Pujol Pedro Castañera Gabriel Salcedo 《Journal of chemical ecology》1997,23(7):1729-1741
The α-amylase activity was determined throughout the larval development of Spodoptera frugiperda. Maximal activities with optimal pH in the range 8.5–9.5 were found in last instars. Protein preparations enriched in heterotetrameric inhibitors from wheat flour were active towards gut amylases from last instars, while those corresponding to homodimeric and monomeric inhibitors showed low inhibition levels. These results were further supported by testing purified members of each inhibitor type and by analyzing the effects of the inhibitors on the amylase isoenzyme pattern from native PAGE. High levels of trypsin-like activity were also found in gut extracts from last instars. Different genetic variants of the major barley trypsin inhibitor were active against this gut enzyme. None of the other larval digestive protease activities (chymotrypsin-like, elastase-like, leucine aminopeptidase-like, and carboxypeptidase A and B-like) were inhibited, indicating that the barley inhibitor is specific towards trypsin-like enzymes. 相似文献
12.
In an attempt to introduce a large peptide that is not normallytranslocated across membranes into the cytosol of eukaryoticcells, we created a new chimeric protein termed CEDH betweenPseudomonas aeruginosa exotoxin A (ETA) and a variant enzymeof Mus musculus dihydrofolate reductase (DHFR) with reducedaffinity for antifolates, ETA1413.DHFR1187.ETA609613.We have defined, genetically constructed and expressed the chimericprotein in Escherichia coli. We showed that the CEDH chimericprotein, purified to homogeneity on an immunoaffinity resin,confers a methotrexate-resistant phenotype to Chinese hamsterovary cells. Furthermore, the chimeric protein allowed the growthof dihydrofolate reductase-deficient Chinese hamster ovary cellsin the absence of hypoxanthine and thymidine. These resultsdemonstrated that the chimeric protein exhibited enzyme activityand possessed the tightly folded native structure, and thatthe DHFR protein can be selectively internalized and translocatedvia domains of exotoxin A. These data show that the ETA systemis an efficient system for the delivery of a variety of largepolypeptides into the cytosol without stress to the target cells,and extends the use of this delivery system to proteins thatare not normally translocated across membranes. 相似文献
13.
Victoria A. Roberts Pnina Dauber-Osguthorpe David J. Osguthorpe Eugene Levin Arnold T. Hagler 《Israel journal of chemistry》1986,27(2):198-210
In this paper, we report on studies of ligand binding to the enzyme dihydrofolate reductase (DHFR). Energy minimizations of four complexes of DHFR with the inhibitor trimethoprim, an antibiotic, and the cofactor NADPH have been carried out in order to investigate the energetics responsible for the 100,000-fold increase in binding affinity of trimethoprim to E. coli DHFR compared with chicken liver DHFR. Several factors suggested to be responsible for the enhanced binding in bacterial DHFR's were investigated in terms of intermolecular and intramolecular energetics. The strain energies of trimethoprim in the four complexes were calculated and found to be about 6 kcal mol−1 in all complexes of the two species. In the binary complex of chicken liver DHFR, where the largest variation was observed, 2 kcal mol−1 higher than in the other complexes, it was found that this increase was compensated for by the slightly more favorable intermolecular interaction of the trimethoxyphenyl moiety with the protein. Comparison of the minimized binary and ternary complexes of E. coli allowed us to investigate the cooperativity in the binding of trimethoprim and NADPH in the bacterial enzyme in terms of the underlying intermolecular forces. This cooperativity was found to be due to a direct trimethoprim - NADPH interaction in the E. coli enzyme rather than enhanced protein-inhibitor interactions induced upon binding of the cofactor. These interactions are not as favorable in the vertebrate enzyme, consistent with the significantly diminished cooperativity observed in this enzyme. 相似文献
14.
Sam Hay Dr. Rhiannon M. Evans Colin Levy Dr. E. Joel Loveridge Dr. Xi Wang Dr. David Leys Dr. Rudolf K. Allemann Prof. Dr. Nigel S. Scrutton Prof. 《Chembiochem : a European journal of chemical biology》2009,10(14):2348-2353
We report the crystal structure of dihydrofolate reductase (DHFR) from the psychropiezophilic bacterium Moritella profunda, which was isolated from the deep ocean at 2 °C and 280 bar. The structure is typical of a chromosomal DHFR and we were unable to identify any obvious structural features that would suggest pressure adaptation. In particular, the core regions of the enzyme are virtually identical to those of the DHFR from the mesophile Escherichia coli. The steady‐state rate at pH 9, which is limited by hydride transfer at atmospheric pressure, is roughly constant between 1 and 750 bar, falling at higher pressures. However, the value of KM increases with increasing pressure, and as a result kcat/KM decreases over the entire pressure range studied. Isotope effect studies showed that increasing the pressure causes a change in the rate‐limiting step of the reaction. We therefore see no evidence of pressure adaptation in either the structure or the activity of this enzyme. 相似文献
15.
A new bifunctional reactive disperse dye containing a temporarily anionic sulphatoethylsulphone and a nonionic disulphide bis(ethylsulphone) groups was synthesised and applied to nylon 6 fabric by the exhaust dyeing at a variety of pH and temperature conditions. A monofunctional reactive disperse dye containing only nonionic disulphide bis(ethylsulphone) group was also synthesised and its dyeing behaviour was compared with the bifunctional dye. The bifunctional reactive disperse dye exhibited high exhaustion and fixation values at pH 6 and 120 °C. The results also indicate that the combination of temporarily anionic and nonionic reactive groups of the bifunctional dye provided great enhancement in dyeing performance compared to that of the monofunctional dye. The dyes also showed very good levelling and fastness properties on nylon 6 fabric. 相似文献
16.
Japrung D Chusacultanachai S Yuvaniyama J Wilairat P Yuthavong Y 《Protein engineering, design & selection : PEDS》2005,18(10):457-464
Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications. 相似文献
17.
Yuko Hamabe Ryoko Matsuura Akinori Jyo Masao Tamada Akio Katakai 《Reactive and Functional Polymers》2009,69(1):1-8
Bifunctional fibers containing phosphonate and sulfonate were derived from chloromethylstyrene grafted polyolefin fibers (PPPE-g-CMS) by phosphorylation and subsequent sulfonation reactions. It was clarified that phosphorylation of PPPE-g-CMS by Arbusov reaction is more suitable than one by the reaction with PCl3 in the presence of AlCl3, because the latter damaged fibers and gave phosphinate groups in addition to phosphonate ones. Then, bifunctional fibers containing phosphonate and sulfonate groups were prepared by sulfonation of monofunctional phosphonate fibers obtained via Arbusov reaction with chlorosulfonic acid. The metal ion selectivity of the bifunctional fibers was governed by both phosphonate and sulfonate groups. In addition, bifunctional fibers gave much more excellent kinetic performances in column-mode uptake of Cu(II) than the monofunctional phosphonate fibers and resin. 相似文献
18.
Dale Glenn E.; Schnfeld Hans-Joachim; Langen Hanno; Stieger Martin 《Protein engineering, design & selection : PEDS》1994,7(7):925-931
The production of the trimethoprim-resistant type SI dihydrofolatereductase (DHFR) from Staphylococcus aureus in Escherichia colicells overproducing the chaperonins GroEL and GroES is described.The simultaneous overproduction of the chaperonins with DHFRresults in an increased solubility of the enzyme. We comparethe time course of production of active type SI DHFR by measuringenzyme activity in cells overproducing or not overproducingthe chaperonins. Although co-overproduction of the chaperoninsreduces the total production level of type S1 DHFR, the amountof soluble and active DHFR is increased several-fold in comparisonwith cells producing only DHFR. Thus, the higher concentrationsof GroES and GroEL in cells overproducing the chaperonins partiallyprotect DHFR from aggregation, resulting in higher concentrationsof soluble and active DHFR in the cell. Furthermore, we alsodemonstrate that the chaperonins can improve in vitro refoldingyields of type S1 DHFR. These results suggest that it is possibleto purify suitable amounts of trimethoprim-res stant type SIDHFR for X-ray crystallographic studies. 相似文献
19.
Dr. Thembaninkosi G. Gaule Dr. Mark A. Smith Prof. Arwen R. Pearson Prof. Peter F. Knowles Prof. Michael J. McPherson 《Chembiochem : a European journal of chemical biology》2015,16(4):559-564
For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild‐type monomer and an inactive monomer in which an active‐site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme. 相似文献
20.
A bifunctional reactive disperse dye containing two temporarily anionic sulphatoethylsulphone groups was synthesised and applied to nylon 6 fabric by exhaust dyeing at a variety of pH levels and temperatures. A monofunctional reactive disperse dye containing one temporarily anionic sulphatoethylsulphone group was also synthesised, and its dyeing behaviour was compared with the bifunctional dye. The bifunctional reactive disperse dye exhibited high exhaustion and total fixation yield under alkaline conditions. The results also indicate that the introduction of two temporarily anionic sulphatoethylsulphone groups of the bifunctional dye gave an enhancement in dyeing performance compared with that of the monofunctional dye. The dyes also showed very good levelling and fastness properties on nylon 6 fabric. 相似文献