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Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.
Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient.
Various DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis.
The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.
The protein cross-reactive material 197 (CRM197) is known to catalyze the hydrolytic cleavage of DNA (DNase activity). A suspected metal-binding site (S109, T111, and E112) and suspected DNA-binding motif (T89, K90, and V91) were predicted within the CRM197 protein X-ray crystal structure (4AE0) using METSITE and DNABindProt, respectively. Between these two predicted sites is a groove (K103, E116, T120, E122, F123, and R126) that may assist in DNase activity. Alanine scanning was performed at these sites to determine which amino acids might be important for DNase activity. These mutations individually or in combination either maintained or increased the overall DNase activity compared to the unmodified CRM197. Mutation at the suspected metal-binding site showed similar fluctuations to the overall DNase activity whether the DNase assays were run with Mg2+ and Ca2+ or Mn2+. However, many of the mutations within the suspected DNA-binding motif saw significant differences depending on which metal was used. Only some of the improvements in DNase activity could be attributed to improved folding of the mutants compared to the unmodified CRM197. This study should provide a basis for further mutagenesis studies to remove the DNase activity of CRM197.
Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.
Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.
As an indispensable enzyme for the hydrolysis of dextran, dextranase has been widely used in the fields of food and medicine. It should be noted that the weak thermostability of dextranase has become a restricted factor for industrial applications. This study aims to improve the thermostability of dextranase AoDex in glycoside hydrolase (GH) family 49 that derived from Arthrobacter oxydans KQ11. Some mutants were predicted and constructed based on B-factor analysis, PoPMuSiC and HotMuSiC algorithms, and four mutants exhibited higher heat resistance. Compared with the wild-type, mutant S357P showed the best improved thermostability with a 5.4-fold increase of half-life at 60 °C, and a 2.1-fold increase of half-life at 65 °C. Furthermore, S357V displayed the most obvious increase in enzymatic activity and thermostability simultaneously. Structural modeling analysis indicated that the improved thermostability of mutants might be attributed to the introduction of proline and hydrophobic effects, which generated the rigid optimization of the structural conformation. These results illustrated that it was effective to improve the thermostability of dextranase AoDex by rational design and site-directed mutagenesis. The thermostable mutant of dextranase AoDex has potential application value, and it can also provide references for engineering other thermostable dextranases of the GH49 family.
Haemoglobins are found ubiquitously in eukaryotes and many bacteria. In plants, haemoglobins were first identified in species, which can fix nitrogen via symbiosis with bacteria. Recent findings suggest that another class of haemoglobins termed as nonsymbiotic haemoglobins are present through out the plant kingdom and are expressed differentially during plant development. Limited data available suggests that non-symbiotic haemoglobins are involved in hypoxic stress and oversupply of nutrients. Due to lack of information on structurally conserved, functionally important residues in non-symbiotic haemoglobins, further studies to elucidate the molecular mechanisms underlying the biological role are hampered. To determine functionally important residues in non-symbiotic haemoglobins, I have analyzed a number of sequences from plant haemoglobin family, in the context of the known crystal structures of plant by evolutionary trace method. Results indicate that the, evolutionary trace method like conventional phylogentic analysis, could resolve phylogentic relationships between plant haemoglobin family. Evolutionary trace analysis has identified candidate functional (trace) residues that uniquely characterize the heme-binding pocket, dimer interface and possible novel functional surfaces. Such residues from specific three-dimensional clusters might be of functional importance in nonsymbiotic haemoglobins. These data, together with our improved knowledge of possible functional residues, can be used in future structure-function analysis experiments.
Members of the genus Bifidobacterium are notoriously recalcitrant to genetic manipulation due to their extensive and variable repertoire of Restriction-Modification (R-M) systems. Non-replicating plasmids are currently employed to achieve insertional mutagenesis in Bifidobacterium. One of the limitations of using such insertion vectors is the presence within their sequence of various restriction sites, making them sensitive to the activity of endogenous restriction endonucleases encoded by the target strain. For this reason, vectors have been developed with the aim of methylating and protecting the vector using a methylase-positive Escherichia coli strain, in some cases containing a cloned bifidobacterial methylase. Here, we present a mutagenesis approach based on a modified and synthetically produced version of the suicide vector pORI28 (named pFREM28), where all known restriction sites targeted by Bifidobacterium breve R-M systems were removed by base substitution (thus preserving the codon usage). After validating the integrity of the erythromycin marker, the vector was successfully employed to target an α-galactosidase gene responsible for raffinose metabolism, an alcohol dehydrogenase gene responsible for mannitol utilization and a gene encoding a priming glycosyltransferase responsible for exopolysaccharides (EPS) production in B. breve. The advantage of using this modified approach is the reduction of the amount of time, effort and resources required to generate site-directed mutants in B. breve and a similar approach may be employed to target other (bifido)bacterial species.
N-acetyl-L-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K(m) for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.
Naturally occurring core-Streptavidin (c-Strep) would serve as a more useful agent in vivo if not for its high kidney retention. This retention is mediated by an integrin-binding motif-RYDS-that shares homology to the more common RGDS. We generated a c-Strep molecule constituting amino acids 13-139 of streptavidin and by site-directed mutagenesis altered the RYDS motif to RYES. RYDS-c-Streptavidin and RYES-c-Streptavidin were expressed in E. coli and purified on a 2-imminobiotin matrix. Each demonstrated an affinity for biotin similar to that of native post-secretory streptavidin while maintaining their ability to form dimers and tetramers. The mutant RYES-c-Streptavidin was no longer able to mediate normal rat kidney cell attachment in an in vitro assay. RYDS-c-Streptavidin-mediated kidney cell attachment was inhibited by competition with c-Streptavidin, RYDS-c-Streptavidin and RGDS-containing peptides but not with an irrelevant peptide or RYES-c-Streptavidin. Therefore, the point mutation D49E generates a molecule, which may not display the in vivo kidney retention observed for RYDS-c-Streptavidin, potentially finding more widespread clinical application.
The orphan insulin receptor-related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR) can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains, and shows positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR in, we now address the role of the fibronectin type III (FnIII) repeats in the IRR pH-sensing. The first activation site includes the intrinsically disordered subdomain ID (646-716) within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, and K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, and D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity, suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain.
Biotic stresses caused by microbial pathogens impair crop yield and quality if not restricted by expensive and often ecologically problematic pesticides. For a sustainable agriculture of tomorrow, breeding or engineering of pathogen-resistant crop varieties is therefore a major cornerstone. Maize is one of the four most important cereal crops in the world. The biotrophic fungal pathogen Ustilago maydis causes galls on all aerial parts of the maize plant. Biotrophic pathogens like U. maydis co-evolved with their host plant and depend during their life cycle on successful manipulation of the host's cellular machinery. Therefore, removing or altering plant susceptibility genes is an effective and usually durable way to obtain resistance in plants. Transcriptional time course experiments in U. maydis-infected maize revealed numerous maize genes being upregulated upon establishment of biotrophy. Among these genes is the maize LIPOXYGENASE 3 (LOX3) previously shown to be a susceptibility factor for other fungal genera as well. Aiming to engineer durable resistance in maize against U. maydis and possibly other pathogens, we took a Cas endonuclease technology approach to generate loss of function mutations in LOX3. lox3 maize mutant plants react with an enhanced PAMP-triggered ROS burst implicating an enhanced defense response. Based on visual assessment of disease symptoms and quantification of relative fungal biomass, homozygous lox3 mutant plants exposed to U. maydis show significantly decreased susceptibility. U. maydis infection assays using a transposon mutant lox3 maize line further substantiated that LOX3 is a susceptibility factor for this important maize pathogen.
Redox active cysteine residues including βCys93 are part of hemoglobin's "oxidation hotspot". Irreversible oxidation of βCys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active γCys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of γCys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at α19 on the exposed surface of the α-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.
The condensation step of fatty acid elongation is the addition of a C2 unit from malonyl-CoA to an acyl primer catalyzed by one of two families of enzymes, the 3-ketoacyl-CoA synthases and the ELO-like condensing enzymes. 3-Ketoacyl-CoA synthases use a Claisen-like reaction mechanism while the mechanism of the ELO-catalyzed condensation reaction is unknown. We have used site-directed mutagenesis of Dictyostelium discoideum EloA to identify residues important to catalytic activity and/or structure. Mutation of highly conserved polar residues to alanine resulted in an inactive enzyme strongly suggesting that these residues play a role in the condensation reaction.
The preparation of oligosaccharides via xylan hydrolysis is an effective way to add value to hemicellulosic material of agricultural waste. The bacterial strain Streptomyces L10608, isolated from soil, contains genes encoding xylanases of glucoside hydrolase family 10/11 (GH10/11), and these have been cloned to catalyze the production of xylooligosaccharide (XOS). To improve the XOS proportion of hydrolysates produced by xylanase, four amino acid residues were substituted by site-directed mutagenesis, and the mutant genes were overexpressed in Escherichia coli. Mutations replaced the codons encoding Asn214 (+2) and Asn86 (-2) by Ala and removed the Ricin B-lectin domain in GH10-xyn, and mutants Y115A (-2) and Y123A (-2) were produced for GH11-xyn. Interestingly, GH10-N86Q had significantly increased hydrolysis of XOS and almost eliminated xylose (X1) to <2.5%, indicating that the -2 binding site of GH10-xyn of L10608 is required for binding with xylotriose (X3). The hydrolytic activity of GH10-N86Q was increased approximately 1.25-fold using beechwood xylan as a substrate and had high affinity for the substrate with a low Km of about 1.85 mg·mL-1. Otherwise, there were no significant differences in enzymatic properties between GH10-N86Q and GH10-xyn. These mutants offer great potential for modification of xylanase with desired XOS hydrolysis.
The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones.
Polyphenol oxidases (PPOs) comprise tyrosinases (TYRs) and catechol oxidases (COs), which catalyse the initial reactions in the biosynthesis of melanin. TYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas COs react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono- versus diphenolase activity in the type-III copper enzyme jrPPO1.
Angiotensin-converting enzyme-2 (ACE2) may play an important role in cardiorenal disease and it has also been implicated as a cellular receptor for the severe acute respiratory syndrome (SARS) virus. The ACE2 active-site model and its crystal structure, which was solved recently, highlighted key differences between ACE2 and its counterpart angiotensin-converting enzyme (ACE), which are responsible for their differing substrate and inhibitor sensitivities. In this study the role of ACE2 active-site residues was explored by site-directed mutagenesis. Arg273 was found to be critical for substrate binding such that its replacement causes enzyme activity to be abolished. Although both His505 and His345 are involved in catalysis, it is His345 and not His505 that acts as the hydrogen bond donor/acceptor in the formation of the tetrahedral peptide intermediate. The difference in chloride sensitivity between ACE2 and ACE was investigated, and the absence of a second chloride-binding site (CL2) in ACE2 confirmed. Thus ACE2 has only one chloride-binding site (CL1) whereas ACE has two sites. This is the first study to address the differences that exist between ACE2 and ACE at the molecular level. The results can be applied to future studies aimed at unravelling the role of ACE2, relative to ACE, in vivo.
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