Mutagenesis

The design of b-glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the b-glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzymetransition state complex. In an attempt to fill this gap, sitedirected mutagenesis and enzyme steady-state kinetic experiments with different substrates were conducted, using as model a digestive b-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbgly50).

10p system191 01-06-2013 41 4   Download

The plant enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138....

11p system191 01-06-2013 65 4   Download

In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epivellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the a/b hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved.

8p system191 01-06-2013 35 4   Download

Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. ...

9p system191 01-06-2013 39 3   Download

Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased Km whereas hydrophobic amino acids in this position tended to decrease Km.

6p system191 01-06-2013 40 4   Download

Cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best-characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution. Experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation. Heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive investigation of the biological process by which the functional structure is formed. ...

7p research12 01-06-2013 32 3   Download

In an effort to explore the role of glycine clusters on the cold adaptation of enzymes, we designed point mutations aiming to alter the distribution of glycine residues close to the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Gly261 and Gly262. The replacement of Gly262 by Ala resulted in an inactive enzyme. Substitution of Gly261 by Ala resulted to an enzyme with lower stability and increased energy of activation.

6p research12 01-06-2013 37 5   Download

The function of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was studied by labelling critical cysteine residues of the enzyme, either native or inserted by sitedirected mutagenesis, with different thiol-reacting molecules. The access of the substrate to the active centre cavity through a nonpolar channel that contains a narrow constriction harbouring a cysteine residue (C435) was probed by labelling experiments on both a C435S mutant, lacking C435 of the channel constriction, and a C25S/C50S/C455S/C537S mutant, bearing C435 as the only cysteine residue.

9p research12 01-06-2013 44 3   Download

Y238, oneof thevery fewconservedresidues in theactive site ofD-amino acid oxidases (DAAO), was mutated to phe-nylalanine and serine in the enzyme from the yeastRhodo-torula gracilis. The mutated proteins are catalytically competent thus eliminating Tyr238 as an active-site acid/ base catalyst. Y238FandY238Smutants exhibit a threefold slower turnover on D-alanine as substrate, which can be attributed to a slower rate of product release relative to the wild-type enzyme (a change of the rate constants for sub-strate binding was also evident). ...

10p research12 29-04-2013 58 5   Download

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehy-drogenase complex (PDC).Analysis of the primary amino-acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-component His-kinases, despite the fact that PDKexclusivelyphosphorylates Ser residues in theE1a subunit of the PDC.This seeming contradiction might be resolved if the PDK-catalyzed reaction employed a phos-pho-His intermediate....

6p research12 29-04-2013 45 4   Download

Osmoregulated periplasmic glucans (OPGs) ofRhodobacter sphaeroidesare anionic cyclic molecules that accumulate in large amounts in the periplasmic space in response to low osmolarity of the medium. Their anionic character is pro-vided by the substitution of the glucosidic backbone by succinyl residues. A wild-type strain was subject to trans-poson mutagenesis, and putative mutant clones were screened for changes in OPGs by thin layer chromatogra-phy.

12p research12 29-04-2013 43 6   Download

Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estro-gen-dependent tumor proliferation. Themolecular structure model is largelyderived fromtheX-ray structure of bacterial cytochromes sharing only 15±20% identities with hP-450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding....

13p research12 29-04-2013 33 3   Download

Random PCR mutagenesis was applied to theThermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylAgenes were cloned and expressed in Escherichia coliHB101 using the vector pGEMÒ-T Easy, and their physicochemical and catalytic properties were determined.

7p research12 29-04-2013 54 3   Download

Theb1,3-glycosyltransferase enzymes identi®ed to date share several conserved regions and conserved cysteine res-idues, all being located in the putative catalytic domain. To investigate the importance of these motifs and cysteines for the enzymatic activity, 14 mutants of the murine b1,3-galactosyltransferase-I gene were constructed and expressed in Sf9 insect cells. Seven mutations abolished the galacto-syltransferase activity.

7p research12 29-04-2013 38 3   Download

A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin.

5p zingzing09 24-04-2013 59 5   Download

The envelope protein gp64 of the baculovirusAutographa californicanuclear polyhedrosis virus is essential for viral entry into insect cells, as the glycoprotein bothmediates pH-dependentmembrane fusionandbinds tohost cell receptors. Surface modification of baculovirus particles by genetic engineering of gp64 has been demonstrated by various strategies and thus has become an important and powerful tool in molecular biology.

10p research12 23-04-2013 27 2   Download

N-Acetylglucosamine is a major component of com-plex carbohydrates. The mammalian salvage pathway of N-acetylglucosamine recruitment from glycoconjugate deg-radation or nutritional sources starts with phosphorylation byN-acetylglucosamine kinase. In this studywe describe the identification of two active site cysteines of the sugar kinase by site-directed mutagenesis and computer-based structure prediction.

7p research12 23-04-2013 33 2   Download

The aimof this workwas to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loopwere generated by PCR-based site-directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods.

10p tumor12 22-04-2013 34 2   Download

Upon mutation of Asp153 by asparagine, the catalytic activity of agmatinase (agmatine ureohydrolase, EC 3.5.3.11)fromEscherichia coliwas reduced to about 5% of wild-type activity. Tryptophan emission fluorescence (kmax ¼340 nm), and CD spectra were nearly identical for wild-type and D153N agmatinases. TheKmvalue for agmatine (1.6 ± 0.1 mM),aswellastheKi for putrescine inhibition (12 ± 2 mM)and the interaction of the enzyme with the requiredmetal ion,werealsonot alteredbymutation.

5p tumor12 22-04-2013 22 3   Download

Genes homologous to hydrogenase accessory genes are scattered over the whole genome in the cyanobacteriumSynechocystissp. PCC 6803. Deletion and insertion mutants of hypA1 (slr1675), hypB1 (sll1432), hypC, hypD, hypE and hypF were constructed and showed no hydrogenase activity. Involvement of the respective genes in maturation of the enzyme was con-firmed by complementation. Deletion of the additional homologueshypA2 (sll1078) and hypB2(sll1079) had no effect on hydrogenase activity. Thus, hypA1andhypB1are specific for hydrogenase maturation....

12p tumor12 20-04-2013 31 4   Download