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At least 433 records · Page 24Linked to original sources
PubMed1991

The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli.

We have recently cloned a full-length cDNA encoding the rat hepatic cholesterol 7 alpha-hydroxylase cytochrome P450 (P450c7) (Li, Y. C., Wang, D. P., and Chiang, J. Y. L. (1990) J. Biol. Chem. 265, 12012-12019), which catalyzes the rate-limiting reaction of bile acid synthesis in the liver. By using the polymerase chain reaction, we have designed two P450c7 cDNAs. One has the second Met codon deleted and the third Thr codon replaced with an Ala. The other lacks codons for the NH2-terminal hydrophobic sequence of amino acids 2-24 (P450c7 delta 2-24). The cDNAs were separately cloned into the expression vector pKK233-2 and transformed into Escherichia coli. After induction with isopropyl-beta-D-thiogalactopyranoside, bacteria harboring recombinant plasmids expressed a polypeptide which reacted with the antibody against cholesterol 7 alpha-hydroxylase in immunoblots. The slightly modified full-length enzyme was expressed to 0.2% of the total bacterial lysate and was located in the membrane fraction, whereas P450c7 delta 2-24 was expressed at a 10-fold higher level (2%), of which 85% was in the cytosol and the remaining associated with the membranes. We have purified P450c7 delta 2-24 which showed a typical reduced-CO difference spectrum of cytochrome P450 and reconstituted cholesterol 7 alpha-hydroxylase activity in the presence of NADPH-cytochrome P450 reductase. P450c7 delta 2-24 has a similar Km for cholesterol (24.6 microM) but a lower Vmax (0.10 nmol/min) and a lower turnover number (1.93 min-1) as compared with the enzyme isolated from rat liver microsomes. The purified P450c7 delta 2-24 has an unique hydrophilic NH2 terminus and contains monomers and dimers in equal amounts. This is the first report demonstrating that a genetically engineered cytochrome P450 enzyme lacking a typical NH2-terminal hydrophobic sequence is mainly cytosolic and catalytically active.

Amino Acid Sequence↗
PubMed2007

Phospholipases and their industrial applications.

Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A1, A2, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions--white biotechnology is a strong alternative.

Animals↗
PubMed2003

Crystal structure of D-Hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability.

D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYD(Bp)) at a 2.7-A resolution. The structure of HYD(Bp) consists of a core (alpha/beta)(8) triose phosphate isomerase barrel fold and a beta-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYD(Bp) shares only moderate sequence similarity with D-HYDs from Thermus sp. (HYD(Tsp)) and Bacillus stearothermophilus (HYD(Bst)), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs. The more thermostable HYD(Tsp) contains more aromatic residues in the interior of the structure than HYD(Bp) and HYD(Bst). Changes of large aromatic residues in HYD(Tsp) to smaller residues in HYD(Bp) or HYD(Bst) decrease the hydrophobicity and create cavities inside the structure. HYD(Tsp) has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYD(Bp) and HYD(Bst). Besides, HYD(Tsp) also contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.

Amidohydrolases↗
PubMed2004

Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis.

AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine. METHODS: By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the P(BF) -aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively. Then, they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays. RESULTS: Engineering strains of C.glutamicum (Tyr(-)) were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively. CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1998

[Evaluation of penicilloyl proteins of allergic impurity in gene engineering drugs].

It is well known that penicillins are necessary for prevention of contamination during cell culture or fermentation in the production of gene engineering drugs. As penicillins are easily combined with proteins forming allergic impurities, the benzyl penicilloyl proteins in aqueous solution, an evaluation system should be established for control of the gene engineering drugs. An enzyme linked immunosorbent assay (ELISA) with high specificity and high sensitivity in vitro, by which the conjugated penicillins of 0.3 ppm in a sample could be detected and a passive cutaneous anaphylaxis (PCA) test, a classical method for evaluation of allergic reaction in vivo are integral parts of this system. Some domestic gene engineering drugs such as erythropoietin (EPO), G-CSF, GM-CSF and 125SerIL-2 are evaluated with this system. No matter whether the expression products was produced in secretion manner or in inclusion body, it was possible that some residues of allergic impurities may remain in the finished products if an unsuitable process of isolation and purification was used. The maximum absolute content of the conjugated penicillins in a sample was up to about one per thousand.

Animals↗
PubMed2002

Purification, crystallization and preliminary structural studies of dTDP-4-keto-6-deoxy-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, the fourth enzyme in the dTDP-L-epivancosamine biosynthetic pathway.

The vancomycin class of antibiotics is regarded as the last line of defence against Gram-positive bacteria. The compounds used clinically are very complex organic molecules and are made by fermentation. The biosynthesis of these is complex and fascinating. Its study holds out the prospect of utilizing genetic engineering of the enzymes in the pathway in order to produce novel vancomycin analogues. In part, this requires detailed structural insight into substrate specificity as well as the enzyme mechanism. The crystallization of one of the enzymes in the chloroeremomycin biosynthetic pathway (a member of the vancomycin family), dTDP-3-amino-4-keto 2,3,6-trideoxy-3-C-methyl-glucose-5-epimerase (EvaD) from Amycolatopsis orientalis, is reported here. The protein is fourth in the pathway which makes a carbohydrate essential for the activity of chloroeremomycin. The crystals of EvaD diffract to 1.5 A and have unit-cell parameters a = 98.6, b = 72.0, c = 57.1 A with space group P2(1)2(1)2. Data to this resolution were collected at the European Synchrotron Radiation Facility.

Actinomycetales↗
PubMed2001

Engineering the properties of a cold active enzyme through rational redesign of the active site.

In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.

Alkaline Phosphatase↗
PubMed1979

Molecular engineering of the L-tryptophan-depleting enzyme indolyl-3-alkane alpha-hydroxylase.

Polymaleic acid and polyacrylic acid polymers have been covalently linked to indolyl-3-alkane alpha-hydroxylase with a shift of the pH optimum from 3.5 to between 5.0 and 5.5. In addition, the specific activity of the modified enzyme was increased approximately threefold at pH 7.0. The Km values of the polymer-bound enzyme preparations are essentially the same as that of the native enzyme. The plasma half-life was shortened from 6-7 hours with the native enzyme to 2-4 hours with the modified enzymes. The degree of L-tryptophan depletion in the circulation of mice, however, was the same during a 24-hour period after injection of native or modified enzymes.

Animals↗
PubMed2002

The 1.9 A structure of alpha-N-acetylgalactosaminidase: molecular basis of glycosidase deficiency diseases.

In the lysosome, glycosidases degrade glycolipids, glycoproteins, and oligosaccharides. Mutations in glycosidases cause disorders characterized by the deposition of undegraded carbohydrates. Schindler and Fabry diseases are caused by the incomplete degradation of carbohydrates with terminal alpha-N-acetylgalactosamine and alpha-galactose, respectively. Here we present the X-ray structure of alpha-N-acetylgalactosaminidase (alpha-NAGAL), the glycosidase that removes alpha-N-acetylgalactosamine, and the structure with bound ligand. The active site residues of alpha-NAGAL are conserved in the closely related enzyme a-galactosidase A (alpha-GAL). The structure demonstrates the catalytic mechanisms of both enzymes and reveals the structural basis of mutations causing Schindler and Fabry diseases. As alpha-NAGAL and alpha-GAL produce type O "universal donor" blood from type A and type B blood, the alpha-NAGAL structure will aid in the engineering of improved enzymes for blood conversion.

Acetylgalactosamine↗
PubMed2000

Processing and trafficking of cysteine proteases in Leishmania mexicana.

Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPB(C25G)). When CPB(C25G) was expressed in a L. mexicana mutant lacking all CPB genes, the inactive pro-enzyme was processed to the mature protein and trafficked to the lysosome. These results show that auto-activation is not required for correct processing of CPB in vivo. When CPB(C25G) was expressed in a L. mexicana mutant lacking both CPA and CPB genes, the majority of the pro-enzyme remained unprocessed and accumulated in the flagellar pocket. These data reveal that CPA can directly or indirectly process CPB(C25G) and suggest that cysteine proteases are targeted to lysosomes via the flagellar pocket. Moreover, they show that another protease can process CPB in the absence of either CPA or CPB, albeit less efficiently. Abolition of the glycosylation site in the mature domain of CPB did not affect enzyme processing, targeting or in vitro activity towards gelatin. This indicates that glycosylation is not required for trafficking. Together these findings provide evidence that the major route of trafficking of Leishmania cysteine proteases to lysosomes is via the flagellar pocket and therefore differs significantly from cysteine protease trafficking in mammalian cells.

Animals↗
PubMedDate not supplied

Enzyme-carrying polymeric nanofibers prepared via electrospinning for use as unique biocatalysts.

Improvement of catalytic efficiency of immobilized enzymes via materials engineering was demonstrated through the preparation of bioactive nanofibers. Bioactive polystyrene (PS) nanofibers with a typical diameter of 120 nm were prepared and examined for catalytic efficiency for biotransformations. The nanofibers were produced by electrospinning functionalized PS, followed by the chemical attachment of a model enzyme, alpha-chymotrypsin. The observed enzyme loading as determined by active site titration was up to 1.4% (wt/wt), corresponding to over 27.4% monolayer coverage of the external surface of nanofibers. The apparent hydrolytic activity of the nanofibrous enzyme in aqueous solutions was over 65% of that of the native enzyme, indicating a high catalytic efficiency as compared to other forms of immobilized enzymes. Furthermore, nanofibrous alpha-chymotrypsin exhibited a much-improved nonaqueous activity that was over 3 orders of magnitude higher than that of its native counterpart suspended in organic solvents including hexane and isooctane. It appeared that the covalent binding also improved the enzyme's stability against structural denaturation, such that the half-life of the nanofibrous enzyme in methanol was 18-fold longer than that of the native enzyme.

Animals↗
PubMed1998

Engineering of solvent-exposed loops in Escherichia coli beta-galactosidase.

The Escherichia coli beta-galactosidase is a high molecular mass tetrameric enzyme extensively used as a molecular marker. Despite its proven utility as a partner in fusion proteins, previous attempts to generate insertional mutants rendered inactive or poorly active enzymes, hampering its further engineering for the construction of multifunctional enzymes. We have explored several solvent-exposed loops on the tetramer, namely those spanning residues 246-254, 271-287, 578-584, 770-773, and 793-806, as acceptor sites to accommodate functional protein segments on the surface of active beta-galactosidase enzymes. An RGD-containing antigenic peptide positioned in these sites interacts efficiently with specific monoclonal antibodies as well as target integrins on the surface of mammalian cells. The resulting chimeric enzymes are soluble, stable, produced in high yields and enzymatically active. Moreover, the identified insertion sites could be appropriated for the design of promising beta-galactosidase-based molecular sensors.

Amino Acid Sequence↗
PubMed2004

Controlling the regiospecific oxidation of aromatics via active site engineering of toluene para-monooxygenase of Ralstonia pickettii PKO1.

A primary goal of protein engineering is to control catalytic activity. Here we show that through mutagenesis of three active site residues, the catalytic activity of a multicomponent monooxygenase is altered so that it hydroxylates all three positions of toluene as well as both positions of naphthalene. Hence, for the first time, an enzyme has been engineered so that its regiospecific oxidation of a substrate can be controlled. Through the A107G mutation in the alpha-subunit of toluene para-monooxygenase, a variant was formed that hydroxylated toluene primarily at the ortho-position while converting naphthalene to 1-naphthol. Conversely, the A107T variant produced >98% p-cresol and p-nitrophenol from toluene and nitrobenzene, respectively, as well as produced 2-naphthol from naphthalene. The mutation I100S/G103S produced a toluene para-monooxygenase variant that formed 75% m-cresol from toluene and 100% m-nitrophenol from nitrobenzene; thus, for the first time a true meta-hydroxylating toluene monooxygenase was created.

Amino Acid Sequence↗
PubMed2000

Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst.

The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.

Bacillus megaterium↗
PubMed1991

Antibody engineering: an overview.

We can now isolate and manipulate antibody genes. Mouse antibodies can be humanized, resulting in chimaeric or reshaped antibodies. Antibody engineering is useful in large scale production of antibodies, in production of active antibody fragments, bifunctional, single-domain and catalytic antibodies, and has lead to the production of novel expression systems useful in many other areas. It allows production of new antibody conjugates, e.g. antibody-toxin or antibody-enzyme linked proteins. Engineered antibodies have many potential applications e.g. imaging, therapy and biosensors.

Animals↗
PubMed1991

Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase.

We have cloned and sequenced the gene for DNA ligase from Thermus thermophilus. A comparison of this sequence and those of other ligases reveals significant homology only with that of Escherichia coli. The overall amino acid composition of the thermophilic ligase and the pattern of amino acid substitutions between the two proteins are consistent with compositional biases in other thermophilic enzymes. We have engineered the expression of the T. thermophilus gene in Escherichia coli, and we show that E. coli proteins may be substantially removed from the thermostable ligase by a simple heat precipitation step.

Amino Acid Sequence↗
PubMed2004

Site-directed mutagenesis of human soluble calcium-activated nucleotidase 1 (hSCAN-1): identification of residues essential for enzyme activity and the Ca(2+)-induced conformational change.

Human soluble calcium-activated nucleotidase 1 (hSCAN-1) is the human homologue of soluble apyrases found in blood-sucking insects. This family of nucleotidases is unrelated in sequence to more well-studied nucleotidases, and very little is known about the enzymatic mechanism. By multiple sequence alignment, eight regions that are highly conserved in the hSCAN-1 family were identified and named. To identify amino acids important for catalytic activity and enzyme specificity, seven point mutations were constructed, expressed in bacteria, refolded, purified, and characterized. Substitution of glutamic acid 130 with tyrosine resulted in dramatically increased nucleotidase activities, while mutagenesis of aspartic acid 151 to alanine and aspartic acid 84 to alanine completely abolished activity. Mutagenesis of arginine 133 and arginine 271 resulted in enzymes with very little nucleotidase activity. Mutagenesis of aspartic acid 175 to alanine and glycine 122 to glutamic acid had smaller negative effects on enzyme activities. Previously, our laboratory showed that calcium triggers a conformational change in hSCAN-1 necessary for nucleotidase activity. Here we show that several mutants (D84A, R133A, and D151A) that lost most of their activity were unable to undergo the conformational change induced by Ca(2+), as shown by Cibacron blue binding, limited proteolysis, and tryptophan fluorescence. We conclude that aspartic acid residues 84 and 151, as well as arginine residue 133, are essential for the Ca(2+)-induced conformational change that is necessary for enzyme activity. Aspartic acid 175 and glutamic acid 130 are important for determining substrate specificity. In addition, we show that Sr(2+), unlike Mg(2+) and other divalent cations, can substitute for Ca(2+) to induce the conformational change necessary for enzyme activity. However, Sr(2+) cannot substitute for Ca(2+) to support nucleotide hydrolysis, presumably because Sr(2+) cannot substitute for Ca(2+) in its second role as a nucleotide cosubstrate. The ramifications of our results on the interpretation of a recently published crystal structure are discussed. This information will facilitate future engineering of this enzyme designed to enhance its ability to hydrolyze ADP and thus increase its potential for therapeutic use in the treatment of pathological ischemic events triggered via activation of platelets by ADP.

Amino Acid Sequence↗