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PubMed2002

Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells.

For application of DNA in gene medicine plasmid or viral DNA is usually used as a vector for the gene of interest. To generate DNA with a minimum of foreign DNA sequences, we used the prokaryotic telomerase, protelomerase TelN, of bacteriophage N15. This is a novel enzyme with cleaving-joining activity, which is required for the formation of linear prophage DNA with closed ends in lysogenic bacteria. Acting on a telomere resolution site telRL, the protelomerase converts circular plasmid DNA into linear covalently closed dumbbell-shaped molecules ("doggybones") in a single-step enzyme reaction. Two such sites were inserted into an expression plasmid flanking a gene of interest. This is cleaved and joined by means of the protelomerase, yielding linear closed mini DNA coding for green fluorescent protein (EGFP) or interleukin-12 (IL-12). Upon transient transfection of human embryonal kidney cells, EGFP was expressed at higher levels from linear closed molecules than from linear open molecules generated by restriction endonucleases for comparison. The level of transcription was comparable to that observed for the parental plasmid DNA. To test whether the linear closed mini DNA molecules are functional in vivo the B16F10/C57BL/6 melanoma metastasis model was applied, where injection of IL-12-expressing DNA inhibits metastasis formation in the lung. The anti-metastatic effect of the IL-12-expressing linear closed DNA was equal or higher than that of the parental plasmid DNA. Therefore, the TelN/ telRL system is well suited to generate linear closed mini DNA with high stability and a minimum of foreign nucleotide sequences.

Animals↗
PubMed2004

Hydroxylation activity of P450 BM-3 mutant F87V towards aromatic compounds and its application to the synthesis of hydroquinone derivatives from phenolic compounds.

Cytochrome P450 BM-3 from Bacillus megaterium is a fatty acid hydroxylase exhibiting selectivity for long-chain substrates (12-20 carbons). Replacement of Phe87 in P450 BM-3 by Val (F87V) greatly increased its activity towards a variety of aromatic and phenolic compounds. The apparent initial reaction rates of F87V as to benzothiophene, indan, 2,6-dichlorophenol, and 2-(benzyloxy)phenol were 227, 204, 129, and 385 nmol min(-1) nmol(-1) P450, which are 220-, 66-, 99-, and 963-fold those of the wild type, respectively. These results indicate that Phe87 plays a critical role in the control of the substrate specificity of P450 BM-3. Furthermore, F87V catalyzed regioselective hydroxylation at the para position of various phenolic compounds. In particular, F87V showed high activity as to the hydroxylation of 2-(benzyloxy)phenol to 2-(benzyloxy)hydroquinone. With F87V as the catalyst, 0.71 mg ml(-1) 2-(benzyloxy)hydroquinone was produced from 1.0 mg ml(-1) 2-(benzyloxy)phenol in 4 h, with a molar yield of 66%.

Bacillus megaterium↗
PubMed1997

Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis.

Artificial cold adaptation of a mesophilic protease, subtilisin BPN' was attempted by means of random mutagenesis of its entire gene coupled with screening of cleared-zone-forming colonies on skim-milk plates at a low temperature. Out of sixty clones screened at 10 degrees C, one mutant enzyme (termed M-15) was found to acquire higher proteolytic activities, specifically dependent on low temperatures ranging from 10 degrees C to 1 degree C, in comparison with those of the wild-type. DNA sequencing analysis revealed that, by this mutation, the 84th amino acid residue, valine, was substituted by isoleucine, which is located 1.5 nm from the center of the catalytic triad in the tertiary structure of subtilisin. By kinetic analysis of the purified enzyme samples, the higher proteolytic activities of M-15 at low temperatures were found to be due to the decrease in the Km value. There was no difference in thermostability between the wild-type and mutant enzymes, when tested by heat treatment. Circular dichroism spectra also showed no difference between them at 10 degrees C, indicating that the mutation of V841 had no effect on the secondary structure of subtilisin.

Adaptation, Biological↗
PubMed1997

Site-directed mutagenesis of a fungal beta-1,4-endoglucanase increases the minimum size required for the substrate.

Conserved regions within a family-5 beta-1,4,-endoglucanase (Eg11), produced by the fungus Macrophomina phaseolina, have been modified through site-directed mutagenesis, resulting in production of an enzyme with novel substrate requirements. The engineered form was generated through mutagenesis of D232A, which lies within the substrate-binding cleft of Eg11. Wild-type Eg11 requires a minimum substrate size of five glucosyl units, while the engineered form requires a minimum of six glucosyl units. Screening was facilitated by the unique ability to obtain functional expression of the fungal endoglucanase in Escherichia coli. Wild-type and mutated Eg11 have equivalent activity on cellohexaose, and both release cellobiose from the reducing end of the cellodextrin. This is the first example of protein engineering of an endoglucanase that results in a novel minimum substrate requirement for cellohexaose. This substrate specificity has not been reported for any native endoglucanases, thus the modified Eg11 may prove useful in applications requiring specific hydrolysis of complex carbohydrates such as beta-glucans.

Amino Acid Sequence↗
PubMed2000

Multidomain and multifunctional glycosyl hydrolases from the extreme thermophile Caldicellulosiruptor isolate Tok7B.1.

DNA sequencing techniques have revealed widespread molecular diversity of the genomic organization of apparently closely related bacteria (as judged from SSU rDNA sequence similarity). We have previously described the extreme thermophile Caldicellulosiruptor saccharolyticus, which is unusual in possessing multi-catalytic, multidomain arrangements for the majority of its glycosyl hydrolases. We report here the sequencing of three gene clusters of glycosyl hydrolases from Caldicellulosiruptor sp. strain Tok7B.1. These clusters are not closely linked, and each is different in its organization from any described for Cs. saccharolyticus. The catalytic domains of the enzymes belong to glycosyl hydrolase families 5, 9, 10, 43, 44, and 48. The cellulose binding domains (CBDs) of these enzymes from Caldicellulosiruptor sp. Tok7B.1 are types IIIb, IIIc, or VI. A number of individual catalytic and binding domains have been expressed in Escherichia coli, and biochemical data are reported on the purified enzymes for cellulose degradation encoded by engineered derivatives of celB and celE.

Amino Acid Sequence↗
PubMed1998

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

Alkaloids↗
PubMed1992

The folding of an enzyme. III. Structure of the transition state for unfolding of barnase analysed by a protein engineering procedure.

The structure of the first significant transition state on the unfolding pathway of barnase has been analysed in detail by protein engineering methods. Over 50 mutations placed strategically over the whole protein have been used as probes to report on the local structure in the transition state. Several different probes for many regions of the protein give consistent results as do multiple probes at the same site. The overall consistency of phi values indicates that the mutations have not produced changes in the protein that significantly alter the transition state for unfolding. A fine-structure analysis of interactions has also been conducted by removing different parts of the same side-chains. Many of the results of simple mutations fall nicely into the two clear-cut cases of phi = 1 or 0, indicating that the local noncovalent bonds are either fully broken or fully made in the transition state. Much of the structure of barnase in the transition state for unfolding is very similar to that in the folded protein. Both major alpha-helices fray at the N terminus. The last two turns in helix1 are certainly intact, as is the C terminus of helix2. The general picture of the beta-sheet is that the three central beta-strands are completely intact while the two edge beta-strands are mainly present but certainly weakened. The first five residues of the protein unwind but the C terminus remains folded. Three of the five loops are unfolded. The edges of the main hydrophobic core (core1) are significantly weakened, however, and their breaking appears partly rate determining. The centre of the small hydrophobic core3 remains intact. Core2 is completely disrupted. The first events in unfolding are thus: the unfolding of several loops, the unwinding of the helices from the N termini, and the weakening and disruption of the hydrophobic cores. The values of phi are found to be substantially the same under conditions that favour folding as under conditions that are highly denaturing, and so the structure of the unfolding transition state is substantially the same in water as in the presence of denaturant. The structure of the final kinetically significant transition state for refolding is identical to that for unfolding. The final events in refolding are, accordingly, the consolidation of the hydrophobic cores, the closing of many loops and the capping of the N termini of the helices.

Amino Acid Sequence↗
PubMed1992

The folding of an enzyme. IV. Structure of an intermediate in the refolding of barnase analysed by a protein engineering procedure.

The pathway of refolding of barnase has been analysed by the protein engineering method using phi plots. The description comprises a folding intermediate, a major transition state (the unfolding transition state) and the fully folded structure. Over 40 mutations have been analysed in the different structural motifs, frequently with several probes in each region. Many of the mutations in this study give phi values for formation of the intermediate of 0, showing that the relevant regions of the structure are as fully unfolded in the intermediate as the unfolded state. Some folding phi values are close to unity, indicating that those regions are fully formed in the intermediate. Even if the data do not report back on a single intermediate but give the averaged properties of a heterogeneous population of sequential or parallel intermediates, then this simplicity of phi data shows that the intermediates tend to have structural features in common. Many phi values are intermediate between those for the unfolded state and the transition state, consistent with either partial structure formation in a single intermediate or a heterogeneous mixture of populations, although the former is more likely. The data are consistent with the intermediate, or collection of intermediates, being on the reaction pathway, rather than side products, because the phi values increase throughout the folding pathway. The main conclusions on the formation of substructure and sequence of folding events from the phi plots are as follows. (1) The major hydrophobic core (core1) begins to form in the intermediate and strengthens in the major transition state. The centre of the core is formed earlier and is stronger in the intermediate and in the transition state than are the edges. (2) Core2 is not formed until after the major transition state. (3) Core3 begins to form in the intermediate and is compact in the transition state. (4) Loop2, loop4 and part of loop1 do not fold until after the major transition state, but the guanosine-binding loop (loop3) is formed in the intermediate and loop5 is partially formed in the intermediate and the transition state. (5) The centre of the beta-sheet is substantially formed in the intermediate, and is fully present in the transition state, but the edges, and associated turns, are definitely weakened.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1986

A simple and rapid nucleotide sequencing strategy and its application in analyzing a rice histone 3 gene.

An improved rapid method for sequencing a target DNA is described. A new plasmid, pAA-PZ1, which contains the origin of replication from phage M13 and a portion of the Tn9 transposon was constructed. A long fragment of target DNA cloned into this vector is progressively shortened in vivo from one end by transposon-mediated deletions. The plasmids carrying different lengths of target DNA are then made into single-stranded DNA in the same host upon infection with an M13 phage and their sequence is determined using the dideoxynucleotide chain-termination method. This method bypasses the in vitro enzymatic manipulations for progressive deletions and requires no subcloning. Using this strategy, we sequenced 1.3 kb of rice DNA containing a histone 3 gene within three weeks.

Amino Acid Sequence↗
PubMed2004

XIP-I, a xylanase inhibitor protein from wheat: a novel protein function.

Endo-(1,4)-beta-xylanases of plant and fungal origin play an important role in the degradation of arabinoxylans. Two distinct classes of proteinaceous endoxylanase inhibitors, the Triticum aestivum xylanase inhibitor (TAXI) and the xylanase inhibitor protein (XIP), have been identified in cereals. Engineering of proteins in conjunction with enzyme kinetics, thermodynamic, real-time interaction, and X-ray crystallographic studies has provided knowledge on the mechanism of inhibition of XIP-I towards endoxylanases. XIP-I is a 30 kDa protein which belongs to glycoside hydrolase family 18, and folds as a typical (beta/alpha)8 barrel. Although the inhibitor shows highest homology with plant chitinases, XIP-I does not hydrolyse chitin; probably due to structural differences in the XIP-I binding cleft. The inhibitor is specific for fungal xylanases from glycoside hydrolases families 10 and 11, but does not inhibit bacterial enzymes. The inhibition is competitive and, depending on the xylanase, the Ki value can be as low as 3.4 nM. Site-directed mutagenesis of a xylanase from Aspergillus niger suggested that the XIP-I binding site was the conserved hairpin loop "thumb" region of family 11 xylanases. Furthermore, XIP-I shows the ability to inhibit barley alpha-amylases of glycoside hydrolase family 13, providing the first example of a protein able to inhibit members of different glycoside hydrolase families (10, 11, and 13), and additionally a novel function for a protein of glycoside hydrolase family 18.

Amino Acid Sequence↗
PubMed2006

Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3.

Recently, we described a triple mutant of the bacterial cytochrome P450 BM3 as the first mutant with affinity for drug-like compounds. In this paper, we show that this mutant, but not wild-type BM3, is able to metabolise testosterone and several drug-like molecules such as amodiaquine, dextromethorphan, acetaminophen, and 3,4-methylenedioxymethylamphetamine that are known substrates of human P450s. Interestingly, the metabolism of 3,4-methylenedioxymethylamphetamine and acetaminophen could be stimulated up to 70-fold by the addition of caffeine, a known activator of rat P450 3A2. With testosterone metabolism, homotropic cooperativity was observed. This shows that heterotropic and homotropic cooperativity, known to occur in the P450 3A family, can also take place in BM3. BM3 therefore can be used as a model system to study atypical kinetics in mammalian P450s. Second, this study shows that BM3 can be engineered to a drug-metabolising enzyme, making it a promising candidate to use as biocatalyst in drug discovery and synthesis.

Acetaminophen↗
PubMed2005

Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals.

The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and epsilon-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the beta2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 degrees C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 degrees C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.

Amino Acid Sequence↗
PubMed2005

Predicting enzyme function from protein sequence.

There are two main reasons to try to predict an enzyme's function from its sequence. The first is to identify the components and thus the functional capabilities of an organism, the second is to create enzymes with specific properties. Genomics, expression analysis, proteomics and metabonomics are largely directed towards understanding how information flows from DNA sequence to protein functions within an organism. This review focuses on information flow in the opposite direction: the applicability of what is being learned from natural enzymes to improve methods for catalyst design.

Animals↗
PubMed2004

Endograft technology: a delivery vehicle for intravascular gene therapy.

PURPOSE: The purpose of this study was to determine whether vascular smooth muscle cells (SMCs) suffused into a bilayered stent graft retain and express a retrovirally transduced gene for 7 months in vivo. METHODS: SMCs harvested from dog jugular vein were retrovirally transduced to introduce genes for tissue plasminogen activator (t-PA) and beta-galactosidase. These cells were then suffused into a novel dual-layered Dacron graft and cultured for 36 to 48 hours. The grafts were mounted on a Palmaz stent and balloon- expanded in the infrarenal aorta of the SMC donor dogs (n = 6). Grafts were recovered at 1, 2, 3, 4, 5, and 7 months. A control endograft suffused with SMCs transduced with only the beta-galactosidase gene was placed in the dogs with grafts recovered at 2, 3, and 4 months. t-PA antigen concentration and expression were analyzed with an enzyme-linked immunosorbent assay. RESULTS: Retained engineered SMCs (blue nuclei) were identified in the explanted grafts, neointima, and underlying aorta with X-gal staining. The t-PA antigen concentration and t-PA activity from the SMCs recovered from the grafts remained elevated for the duration of the experiment (7 months) at levels significantly higher (3.7 +/- 0.2 ng/mL per 10(5) cells per 24 hours and 1.4 +/- 0.1 IU/mL per 10(5) cells per 24 hours) than in control endografts (0.5 +/- 0.03 ng/mL per 10(5) cells per 24 hours and 0.07 +/- 0.00 IU/mL per 10(5) cells per 24 hours; P <.001). No graft stenosis was observed. CONCLUSION: Retrovirally engineered vascular SMCs survived the implantation trauma, repopulated each graft, migrated into the underlying aorta, and expressed the transduced genes for the 7-month duration of the experiment. This bilayered Dacron endograft model provides a platform to study direct intravascular gene therapy.

Animals↗
PubMed1997

Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide.

Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N2O reductase (N2OR) (EC 1.7.99.6) of Pseudomonas stutzeri was studied. The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm. The signal peptide of N2OR shares a conserved twin-arginine sequence motif with the signal peptides of other N2O reductases and a sizeable group of periplasmic or membrane-bound enzymes, requiring cofactor insertion or processing. A catalytically inactive reductase, N2ORR20D, that lacked Cu, accumulated in the cytoplasm on mutation of the first arginine of this motif. The CuA site of N2ORR20D could be reconstituted in vitro indicating that the lack of metal was not due to a serious conformational restraint. Our findings locate the event of in vivo Cu insertion into N2OR in the periplasm or allow it to take place concomitant with protein translocation.

Amino Acid Sequence↗
PubMed1999

High level expression of Thermococcus litoralis 4-alpha-glucanotransferase in a soluble form in Escherichia coli with a novel expression system involving minor arginine tRNAs and GroELS.

The Thermococcus litoralis 4-alpha-glucanotransferase (GTase) gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the GTase gene in E. coli resulted in low protein production and the accumulation of inclusion bodies. However, simultaneous expression of GTase with tRNA(AGA), tRNA(AGG) and GroELS affected both the production and solubility of GTase, and production of soluble GTase increasing about 5-fold. This new E. coli expression system should be applicable to the expression of not only archaeal but also eukaryotic genes, which usually contain a large number of AGA and AGG codons.

Chaperonin 60↗