Biomedical subjects

V Schellenberger

Publications and source records attributed to V Schellenberger.

At least 19 recordsLinked to original sources

PubMed2004

Construction of stabilized proteins by combinatorial consensus mutagenesis.

We constructed stabilized variants of beta-lactamase (BLA) from Enterobacter cloacae by combinatorial recruitment of consensus mutations. By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E.cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions. Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability. The stability of these isolates suggest that all tested mutations make additive contributions to protein stability. A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and eight mutations that destabilized the protein. A second-generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization. The most stable variant had a mid-point of thermal denaturation (Tm) that was 9.1 degrees C higher than the starting molecule and contained eight consensus mutations. Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis. Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability.

Amino Acid Sequence↗

PubMed2001

Rapid evolution of novel traits in microorganisms.

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20- to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.

Dimethylformamide↗

PubMed2000

Rapid in vivo evolution of a beta-lactamase using phagemids.

RNA viruses are capable of undergoing extremely rapid evolution due to their high rates of reproduction, small genome size, and a high frequency of spontaneous mutagenesis. Here we demonstrate that a virus-like, evolutionary state can be created by propagating a phagemid population in a hypermutator strain of Escherichia coli in the presence of a helper phage. This enables one to subject individual phagemid-encoded genes to rapid in vivo evolution. We applied this approach to TEM-1 beta-lactamase which confers resistance to 0.05 mg/L of the antibiotic cefotaxime. After 3 weeks of in vivo evolution we were able to isolate a double mutant, E104K/G238S, of the enzyme which confers a 500-fold increased level of resistance to cefotaxime compared to the starting enzyme. In two independent experiments we obtained a triple mutant, E104K/G238S/T263M, which confers a 1000-fold increase in resistance compared to the wild type enzyme. The same three mutations have been previously observed in TEM-4 beta-lactamase which was discovered in a highly cefotaxime-resistant clinical isolate. The probability of randomly obtaining a beta-lactamase carrying three identical point mutations is less than 10(-10). This indicates that phagemid evolution can rapidly reproduce evolution occurring in nature.

Bacteriophages↗

PubMed1997

Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization.

We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.

Bacillus subtilis↗

PubMed1995

Substrate preferences of Vsr DNA mismatch endonuclease and their consequences for the evolution of the Escherichia coli K-12 genome.

The substrate spectrum of Vsr DNA mismatch endonuclease of Escherichia coli K-12 was investigated using fluorescence-labelled oligonucleotide substrates and a DNA sequencer for detection and quantification of substrates and reaction products. Fourteen substrates were found to be processed by the enzyme, which differ in one or two positions from the canonical pentanucleotide sequence CTA/TGG (T mismatched to G). Relative second-order rate constants of these substrates were determined in groups of four by multiple substrate kinetics and compared to the underresentation of the corresponding pentanucleotides in the E. coli K-12 genome. The high quality of correlation further establishes active mutagenesis by VSP repair as a significant driving force of the evolution of the E. coli K-12 genome and provides clues to its possible selective value.

Base Sequence↗

PubMed1994

The substrate specificity of Uca pugilator collagenolytic serine protease 1 correlates with the bovine type I collagen cleavage sites.

Affinity-based purification and characterization of the collagenolytic serine protease 1 from Uca pugilator (fiddler crab) hepatopancreas shows that the enzyme cleaves the native bovine alpha 1(I) collagen chain carboxyl-terminal to Gln and Arg residues adjacent to the metallocollagenase site. Cleavage carboxyl-terminal to Leu residues is observed in the alpha 2(I) chain and at a secondary site in alpha 1(I). These sites correlate with the preferences observed toward p-nitroanilide substrates varying at the P1 position, for which the specificity (kcat/Km) is Arg > Leu, Phe, Lys > Gln > Ala. Furthermore, collagen cleavage after Gln was found exclusively between two Gln-Arg bonds. The P'1-P'3 specificity of collagenase, as determined by nucleophile acyl transfer, indicated a strong preference for Arg in the P'1 position. Crab collagenase cleaves peptide bonds adjacent to Leu and Gln at the P1 position more efficiently than trypsin, chymotrypsin, or elastase. Moreover, the efficiency of collagenase toward P1-Arg substrates is equivalent to that of trypsin. Crystals of crab collagenase have been grown complexed with the protein inhibitor ecotin. These crystals diffract to better than 2.8 A resolution and belong to the space group P3(2)21 with unit cell dimensions of a = b = 89.0 A, c = 291.7 A.

Amino Acid Sequence↗

PubMed1994

Role of the S' subsites in serine protease catalysis. Active-site mapping of rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni.

The S' subsite specificity of four homologous serine proteases, rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni, was studied by measuring acyl-transfer reactions to 100 pentapeptide nucleophiles. Peptides of the general structures H-Xaa-Ala-Ala-Ala-Ala-NH2, H-Ala-Xaa-Ala-Ala-Ala-NH2, and H-Ala-Ala-Xaa-Ala-Ala-NH2 were synthesized, where Xaa is D-Ala, Cit, and all natural amino acids except Cys. The variable residues of these nucleophiles occupy the P'1, P'2, and P'3 positions in acyl-transfer reactions. The P'1 and P'2 residues were found to influence the efficiency of the nucleophiles by more than 2 orders of magnitude, whereas the S'3 subsite shows a lower specificity in all four enzymes. We synthesized consensus peptides of the general structure H-aa1-aa2-aa3-Ala-Ala-NH2, in which two or three positions were occupied by amino acids that showed the highest specificity in the first series of nucleophiles. Peptides with optimal amino acid residues in the P'2 and P'3 positions show a very high efficiency in chymotrypsin- and trypsin-catalyzed reactions. Otherwise, large specific side chains in the P'1 and P'3 positions of the nucleophiles show less than additive binding contributions due to steric hindrance. Comparison of chymotrypsin-catalyzed acyl-transfer reactions to nucleophiles of the structures H-Xaa-Leu-Arg-Ala-Ala-NH2 and H-Xaa-Ala-Ala-Ala-Ala-NH2 reveals a significantly different P'1 specificity for both series which confirms steric hindrance between large P'1 and P'3 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Analysis of enzyme specificity by multiple substrate kinetics.

Multiple approaches for screening large sets of compounds for a specific function are of growing interest. The use of substrate mixtures to characterize the specificity of enzymes has been limited so far to compounds with similar kinetic parameters, because the data were analyzed by applying the kinetics of two competing substrates. In this study we introduce a statistical method for the analysis of reactions with many competing substrates which makes use of the specific features of multiple substrate kinetics. It is assumed that the relative concentrations of all substrates in a mixture can be monitored by high-performance liquid chromatography or a similar technique. Relative second-order rate constants, i.e., kcat/KM values, can be calculated for all substrates in the mixture from the resulting data set. The calculation uses the fact that there is a relationship between the concentrations of all pairs of substrates in the mixture. As a result, the precision of the calculated parameters is increased and the range of kinetic constants that can be obtained from one experiment is considerably expanded. Simulations demonstrate that the precision in the kinetic parameters increases with the number of substrates in the mixture. In fact, estimation of ratios of rate constants can be improved (or made possible) for substrates with order of magnitude differences in reactivity by adding "dummy" substrates with intermediate reactivities, even though the rate constants for dummy substrates are themselves of no intrinsic interest.

Binding, Competitive↗

PubMed1993

Mapping the S' subsites of serine proteases using acyl transfer to mixtures of peptide nucleophiles.

We have developed a rapid and convenient procedure for the characterization of the S' subsite specificity of serine proteases. A mixture of peptide nucleophiles is incubated with the enzyme in the presence of excess of a specific ester substrate. The decrease in each nucleophile concentration is monitored by high-performance liquid chromatography analysis of the dansylated mixture. Relative kinetic parameters for each nucleophile in the mixture are then calculated using a new statistical algorithm that relates all pairs of nucleophiles. As a first application, we investigated the S'1 subsite specificity of chymotrypsin, trypsin, and a recently described trypsin mutant, Tr-->Ch[S1 + L1 + L2] with chymotrypsin-like primary specificity [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255, 1249-1253]. For this purpose 21 peptide nucleophiles of the general structure H-Xaa-Ala-Ala-Ala-Ala-NH2 were prepared by multiple solid-phase synthesis, where Xaa represents D-alanine, citrulline, and all natural amino acids except cysteine. Relative second-order rate constants for the enzyme-catalyzed acyl transfer to these nucleophiles were determined over a range of 10(2). Chymotrypsin and trypsin have markedly different S'1 specificities. The order of preference in chymotrypsin-catalyzed acyl transfer reactions is positively charged > aliphatic > aromatic >> negatively charged, D-Ala, Pro P'1 side chain. Trypsin prefers hydrophobic residues, but like chymotrypsin aliphatic residues are better than aromatic residues in P'1 position. The S'1 specificity of the mutant Tr-->Ch[S1 + L1 + L2] is similar to the specificity of trypsin; however, P'1 aromatic residues have low reactivity characteristic of chymotrypsin.

Acyltransferases↗

PubMed1993

Peptide production by a combination of gene expression, chemical synthesis, and protease-catalyzed conversion.

We describe a new approach for the production of peptides using a combination of recombinant DNA technology, chemical synthesis, and proteinase-catalyzed processing. An artificial substance P-precursor is produced as a beta-galactosidase (1-459) fusion protein containing nine copies of the decapeptide sequence Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe. The fusion protein accumulates in E. coli as insoluble inclusion bodies which are easily isolated and purified. The decapeptide blocks are selectively cleaved from the insoluble fusion protein by alpha-chymotrypsin. Alternatively, a dodecapeptide ester is produced when a dipeptide ester is included in the chymotrypsin reaction mixture. This peptide ester is converted converted to substance P by papain-catalyzed acyl transfer and subsequent tryptic cleavage. These results demonstrate that peptides can be readily produced by a combination of recombinant DNA technology and proteinase-catalyzed conversion. The approach allows incorporation of groups other than natural amino acids into oligo- and polypeptides.

Amino Acid Sequence↗

PubMed1992

Proteinase-catalyzed conversion of a substance P-precursor peptide.

The protease-catalyzed conversion of peptides and proteins produced by recombinant DNA technology is a promising method for large-scale production of peptides including those with non-proteinogenic structural elements. As a model system we have investigated the proteinase-catalyzed modification of a chemically synthesized substance P-precursor. In the precursor peptide the residues of substance P(1-8) were flanked by tripeptide linkers on both sides. In the first step the C-terminal tripeptide amide was replaced by the authentic C-terminal tripeptide amide of substance P via alpha-chymotrypsin-catalyzed transpeptidation. The enzyme simultaneously attacks two peptide bonds in the precursor molecule leading to the formation of several side-products. The desired peptide was obtained with 25% yield. In the second step the other tripeptide linker was selectively and almost quantitatively removed from the N-terminus of the precursor via trypsin-catalyzed hydrolysis. This study demonstrates that substance P can be obtained from an engineered protein by proteinase-catalyzed processing.

Amino Acid Sequence↗

PubMed1991

Penicillin acylase-catalyzed protection and deprotection of amino groups as a promising approach in enzymatic peptide synthesis.

Penicillin acylase from E. coli is able to catalyze both the introduction and the removal of the phenylacetyl group. We have established that phenylacetyl derivatives of amino acids and peptides can be used in protease-catalyzed peptide synthesis. Here the synthesis of leucine-enkephalin using enzymes for N-terminal amino group protection, peptide bond formation and deprotection is described.

Amino Acid Sequence↗

PubMed1991

The specificity of chymotrypsin. A statistical analysis of hydrolysis data.

From the literature we collected all available quantitative data on the chymotrypsin-catalyzed hydrolysis of series of amino acid and peptide substrates. Utilizing this data base, we performed calculations on their quantitative structure/activity relationship (QSAR). The substrates were considered to be composed of fragments; log(kcat/Km) values for the substrates resulted from additive contributions of their fragments. Despite the fact that the kinetic constants in the data base were determined by different authors under various reaction conditions, the data are well described by the simple additivity model. Obviously, the intrinsic specificity of chymotrypsin dominates the influence of varying reaction conditions.

Amino Acid Sequence↗

PubMed1991

Electrostatic effects in the alpha-chymotrypsin-catalyzed acyl transfer. I. Influence of different inorganic salts.

We investigated the deacylation of two acyl-alpha-chymotrypsins by added nucleophiles. The nucleophile binding site of the enzyme shows a strong preference for positively charged compounds. Most of our data can be explained by direct electrostatic interaction between the ionic nucleophiles and two negatively charged residues which are located close to the active site of the enzyme molecule. The influence of inorganic salts on the acyl transfer includes the following effects: (1) reduction of electrostatic interactions between the acyl-enzyme and the nucleophile by addition of salts; (2) binding of divalent cations to the nucleophile binding site of the acyl-enzyme leading to a significantly changed specificity; and (3) linear dependence of the activity coefficients of the added nucleophiles on salt concentration.

Acylation↗

PubMed1991

Electrostatic effects in the alpha-chymotrypsin-catalyzed acyl transfer. II. Efficiency of nucleophiles bearing charged groups in various locations.

We investigated the alpha-chymotrypsin-catalyzed acyl transfer to a series of glycine oligomers. It could be established that the electrostatic interactions between the carboxylate group of the nucleophiles and the S'-subsites of the enzyme fall off with the length of the nucleophile molecule. Additional negatively charged residues in the nucleophile lead to a considerable reduction of the acyl transfer efficiency. An arginine residue in P'1- or P'3-position, but not in P'2-position, makes favourable interactions with the appropriate S'-subsites of the enzyme.

Acylation↗

PubMed1991

The alkaline phosphatase from bone: transphosphorylating activity and kinetic mechanism.

For the purified alkaline phosphatase from bone, the ability to catalyze a phosphate transfer reaction from p-nitrophenyl phosphate to two different hydroxy acceptor compounds, ethanolamine and glycerol, was established by identification of the formed phosphorylated products, phosphoethanolamine and glycerol 3-phosphate, respectively. In addition, a steady-state kinetic analysis of the hydrolysis of p-nitrophenyl phosphate in the presence of an added nucleophile, diethanolamine, gave rise to the proposal of a simple model for the kinetic mechanism of the enzyme. This mechanism includes a covalent phosphoryl enzyme intermediate, the dephosphorylation of which by water (k3) or a nucleophile (k4) is rate-determining. According to this model, in the presence of diethanolamine, k3 and k4 were determined to be 4.44 s-1 M-1 and 1000 s-1 M-1, respectively. Therefore, in vitro a suitable nucleophile, such as diethanolamine, seems to be a better phosphate acceptor than water. These results may suggest that alkaline phosphatase from bone could be well suited for catalyzing phosphate transfer reactions in vivo as well.

Alkaline Phosphatase↗

PubMed1991

Chymotrypsin-catalyzed fragment coupling synthesis of GnRH-analogs.

D-Xaa6-GnRH analogs (Xaa: Ala, Nal1), Phe, Ser(tBu), Trp) were prepared by chymotrypsin catalyzed 3 + 7 fragment coupling synthesis with conversion rates of the amino component in the range from 90.3 to 97.4%. For D-Phe6-GnRH the method was scaled up to production level.

Catalysis↗

PubMed1990

Characterization of the S'-subsite specificity of V8 proteinase via acyl transfer to added nucleophiles.

The S'-subsite specificity of endoproteinase Glu-C (V8 proteinase) was studied by acyl transfer reactions using Z-Glu-OMe as acyl donor and a series of amino acid- and peptide-derived nucleophiles. The partition constant, which characterizes specificity, was determined by a method based on the integrated rate equation. V8 proteinase prefers amino acid residues with hydrophobic side chains in the P'1 position. Di- and tripeptide amides are more efficient nucleophilic amino components than amino acid amides.

Binding Sites↗