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P Kast

Publications and source records attributed to P Kast.

At least 19 recordsLinked to original sources
PubMed2001

Searching sequence space for protein catalysts.

Genetic selection was used to explore the probability of finding enzymes in protein sequence space. Large degenerate libraries were prepared by replacing all secondary structure units in a dimeric, helical bundle chorismate mutase with simple binary-patterned modules based on a limited set of four polar and four nonpolar residues. Two-stage in vivo selection yielded catalytically active variants possessing biophysical and kinetic properties typical of the natural enzyme even though approximately 80% of the protein originates from the simplified modules and >90% of the protein consists of only eight different amino acids. This study provides a quantitative assessment of the number of sequences compatible with a given fold and implicates previously unidentified residues needed to form a functional active site. Given the extremely low incidence of enzymes in completely unbiased libraries, strategies that combine chemical information with genetic selection, like the one used here, may be generally useful in designing novel protein scaffolds with tailored activities.

Amino Acid Sequence↗
PubMed2000

A strategically positioned cation is crucial for efficient catalysis by chorismate mutase.

Combinatorial mutagenesis and in vivo selection experiments previously afforded functional variants of the AroH class Bacillus subtilis chorismate mutase lacking the otherwise highly conserved active site residue Arg(90). Here, we present a detailed kinetic and crystallographic study of several such variants. Removing the arginine side chain (R90G and R90A) reduced catalytic efficiency by more than 5 orders of magnitude. Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand in k(cat). Remarkably, the lysine could be placed at position 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site. Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended conformation that would place its epsilon-ammonium group within hydrogen-bonding distance of the ether oxygen of bound chorismate in the transition state. These results provide support for the hypothesis that developing negative charge in the highly polarized transition state is stabilized electrostatically by a strategically placed cation. The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificial catalysts are discussed.

Binding Sites↗
PubMed2000

Probing the role of the C-terminus of Bacillus subtilis chorismate mutase by a novel random protein-termination strategy.

A novel strategy combining random protein truncation and genetic selection has been developed to identify dispensable C-terminal segments of an enzyme. This approach, which entails the random introduction of premature termination codons, was applied to the last 17 residues of chorismate mutase from Bacillus subtilis (BsCM). Although structurally ill-defined, the C-terminus of BsCM has been proposed to cap the active site upon substrate binding and affect catalysis. However, sequence patterns of 178 selected gene variants show that the final 11 residues of the protein can be mutated and even removed without significantly impairing activity in vivo. In fact, none of the randomized residues is absolutely required, but a preference for wild-type Lys111, Ala112, Leu115, and Arg116 is apparent. These residues are part of a C-terminal 3(10)-helix and provide contacts with the rest of the protein or its ligands. The kinetic parameters of selected enzyme variants show that truncations and mutations do not significantly impair catalytic turnover (k(cat)) but substantially decrease k(cat)/K(m). Thus, while the 17 C-terminal residues of BsCM do not participate directly in the chemical rearrangement, they appear to contribute to enzymatic efficiency via uniform binding of the substrate and transition state.

Amino Acid Sequence↗
PubMed2000

Efficient introduction of aryl bromide functionality into proteins in vivo.

Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amounts of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20-25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addition, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.

Amino Acid Substitution↗
PubMed1999

Bacillus subtilis chorismate mutase is partially diffusion-controlled.

The effect of viscosogens on the enzyme-catalyzed rearrangement of chorismate to prephenate has been studied. The steady-state parameters kcat and kcat/Km for the monofunctional chorismate mutase from Bacillus subtilis (BsCM) decreased significantly with increasing concentrations of glycerol, whereas the 'sluggish' BsCM mutants C75A and C75S were insensitive to changes in microviscosity. The latter results rule out extraneous interactions of the viscosogen as an explanation for the effects observed with the wild-type enzyme. Additional control experiments show that neither viscosogen-induced shifts in the pH-dependence of the enzyme-catalyzed reaction nor small perturbations of the conformational equilibrium of chorismate can account for the observed effects. Instead, BsCM appears to be limited by substrate binding and product release at low and high substrate concentrations, respectively. Analysis of the kinetic data indicates that diffusive transition states are between 30 and 40% rate-determining in these concentration regimes; the chemical step must contribute to the remaining kinetic barrier. The relatively low value of the 'on' rates for chorismate and prephenate (approximately 2 x 106 m-1.s-1) probably reflects the need for a rare conformation of the enzyme, the ligand, or both for successful binding. Interestingly, the chorismate mutase domain of the bifunctional chorismate mutase-prephenate dehydratase from Escherichia coli, which has steady-state kinetic parameters comparable to those of BsCM but has a much less accessible active site, is insensitive to changes in viscosity and the reaction it catalyses is not diffusion-controlled.

Bacillus subtilis↗
PubMed1998

A small, thermostable, and monofunctional chorismate mutase from the archaeon Methanococcus jannaschii.

The gene for chorismate mutase (CM) from the archaeon Methanococcus jannaschii, an extreme thermophile, was subcloned and expressed in Escherichia coli. This gene, which belongs to the aroQ class of CMs, encodes a monofunctional enzyme (AroQf) able to complement the CM deficiency of an E. coli mutant strain. The purified protein follows Michaelis-Menten kinetics (kcat = 5.7 s-1 and Km = 41 microM at 30 degreesC) and displays pH-independent activity in the range of pH 5-9. Its activation parameters [Delta H = 16.2 kcal/mol, Delta S = -1. 7 cal/(mol.K)] are similar to those of another well characterized AroQ class CM, the mesophilic AroQp domain from E. coli. Like AroQp, the thermophilic CM is an alpha-helical dimer, but approximately 5 kcal/mol more stable than its mesophilic counterpart as judged from equilibrium denaturation studies. The possible origins of the thermostability of M. jannaschii AroQf, the smallest natural CM characterized to date, are discussed in light of available sequence and tertiary structural information.

Amino Acid Sequence↗
PubMed1998

Redesigning enzyme topology by directed evolution.

Genetic selection was exploited in combination with structure-based design to transform an intimately entwined, dimeric chorismate mutase into a monomeric, four-helix-bundle protein with near native activity. Successful reengineering depended on choosing a thermostable starting protein, introducing point mutations that preferentially destabilize the wild-type dimer, and using directed evolution to optimize an inserted interhelical turn. Contrary to expectations based on studies of other four-helix-bundle proteins, only a small fraction of possible turn sequences (fewer than 0.05 percent) yielded well-behaved, monomeric, and highly active enzymes. Selection for catalytic function thus provides an efficient yet stringent method for rapidly assessing correctly folded polypeptides and may prove generally useful for protein design.

Amino Acid Sequence↗
PubMed1998

Exploring sequence constraints on an interhelical turn using in vivo selection for catalytic activity.

The role of interhelical turns in determining protein structure has been investigated previously in relatively simple four-helix-bundle proteins using combinatorial mutagenesis coupled with screening for functional variants. To assess the tolerance to sequence substitution of a short, interhelical turn in a larger, more complicated protein, we have exploited a more sensitive in vivo selection for catalytic activity. Randomization of three solvent-exposed turn residues in Escherichia coli chorismate mutase (Ala65, His66, and His67), followed by selection, indicated that >63% of tripeptides, including some with significantly altered backbone conformations, can functionally replace the native sequence. The increased sensitivity of the catalytic assay allowed optimal sequences to be distinguished from less appropriate ones, revealing a statistically significant preference for hydrophilic residues in solvent-exposed positions. It also enabled investigation of the extent to which either secondary structure or tertiary interactions influence substitution patterns. Randomization of an alpha-helical residue (Lys64), together with the adjacent solvent-exposed tripeptide, Ala65-His66-His67, showed that the secondary structure at position 64 does not limit the range of side chains allowed at this site. In contrast, randomization of a buried turn residue (Leu68), together with the same tripeptide, revealed an extremely strict requirement for hydrophobic aliphatic amino acids at this position. The strong constraint imposed by the tertiary interaction, in contrast to the weak influence of secondary structure, has important implications for protein design.

Catalysis↗
PubMed1998

Probing enzyme quaternary structure by combinatorial mutagenesis and selection.

Genetic selection provides an effective way to obtain active catalysts from a diverse population of protein variants. We have used this tool to investigate the role of loop sequences in determining the quaternary structure of a domain-swapped enzyme. By inserting random loops of four to seven residues into a dimeric chorismate mutase and selecting for functional variants by genetic complementation, we have obtained and characterized both monomeric and hexameric enzymes that retain considerable catalytic activity. The low percentage of active proteins recovered from these selection experiments indicates that relatively few loop sequences permit a change in quaternary structure without affecting active site structure. The results of our experiments suggest further that protein stability can be an important driving force in the evolution of oligomeric proteins.

Chorismate Mutase↗
PubMed1998

UGA read-through artifacts--when popular gene expression systems need a pATCH.

pET and similar vectors are widely used for efficient gene expression in Escherichia coli and subsequent protein purification, often by means of a C-terminal histidine (His) tag. We found that the TGA translation termination signal following the His-tag sequence in pET constructs gives rise to a significant fraction of read-through protein extended by 21 amino acids. Mass spectrometry indicated that tryptophan is inserted at the UGA (opal) stop codon in the examined non-opal suppressor strains; no evidence for translational frameshifting was detected. We have shown that the problem of obtaining heterogeneous protein preparations can easily be corrected. Plasmid pATCH1 provides a replacement sequence for the inefficient stop signal and can be used to repair both pET vectors and existing pET-based expression constructs. Our observation illustrates the largely ignored fact that a UGA codon is the worst choice for proper translation termination in efficient overexpression vectors.

Amino Acid Sequence↗
PubMed1997

3D structural information as a guide to protein engineering using genetic selection.

A great variety of protein systems have been investigated in the past year using structure-guided evolutionary strategies. On the basis of available 3D structural information, critical regions of proteins have been targeted for randomizing mutagenesis and active variants of the corresponding genes have been selected. These approaches help characterize structural and mechanistic features of proteins and have important implications for design.

Binding Sites↗
PubMed1996

Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential. Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.

Amino Acid Sequence↗
PubMed1994

Substrate specificity is determined by amino acid binding pocket size in Escherichia coli phenylalanyl-tRNA synthetase.

Alanine at position 294 (Ala294) within the motif 3 consensus of Escherichia coli phenylalanyl-tRNA synthetase alpha subunit has previously been implicated as a determinant of amino acid specificity. To characterize the role of Ala294, the catalytic effects of amino acid replacements at this position were tested with purified wild-type and mutant phenylalanyl-tRNA synthetases. We show that Ala294 is involved in amino acid binding and that it influences specificity as a determinant of binding pocket size. Replacement of Ala294 by either glycine or serine, thereby increasing or decreasing the size of the binding pocket, respectively, reduces affinity for phenylalanine. The Gly294 mutant shows a relaxed specificity toward synthetic para-halogenated phenylalanine analogues, the apparent dissociation constant Km increasing in direct relation to an increase of the van der Waals radius of the para group, thus confirming the role of position 294 in determining amino acid binding pocket size. For the substrate analogue p-chlorophenylalanine, attachment to tRNA and in vivo incorporation into cellular protein by the Gly294 mutant were demonstrated. Tyrosine activation was also improved with this mutant, but the resulting enzyme-Tyr-adenylate complex was rapidly hydrolyzed, indicating the presence of a proofreading mechanism in E. coli phenylalanyl-tRNA synthetase.

Adenosine Triphosphate↗
PubMed1994

pKSS--a second-generation general purpose cloning vector for efficient positive selection of recombinant clones.

A new small plasmid vector (pKSS) for the direct selection of insert-containing plasmid clones is presented. The selection strategy is based on the acquired sensitivity of Escherichia coli cells to p-chloro-phenylalanine (p-Cl-Phe) if they carry a pheS allele encoding a phenylalanyl-tRNA synthetase alpha subunit with relaxed substrate specificity. This pheS allele is present on pKSS. Insertion into, or replacement of, the plasmidial pheS gene by a cloned fragment enables transformed pheS wild-type cells to survive on agar plates containing p-Cl-Phe plus ampicillin. This host strain-independent positive selection of recombinant clones proved to be highly efficient (> 99%) and did not require purification of the vector fragment prior to cloning. The high-copy-number vector pKSS offers a multitude of restriction sites and all of the features for analysis of cloned fragments that stem from the cloning vector pBluescript (Stratagene, La Jolla, CA, USA). Thus, pKSS represents a valuable alternative to previously reported positive-selection vectors; it should prove particularly useful for cloning when expecting a high fraction of cells transformed with non-recombinant vector, and for construction of DNA libraries.

Alleles↗
PubMed1992

Cloning and sequence analysis of the phenylalanyl-tRNA synthetase genes (pheST) from Thermus thermophilus.

While crystals suitable for X-ray diffraction analyses are available of phenylalanyl-tRNA synthetase (PheRS) from the thermophilic bacterium Thermus thermophilus, neither the primary structure of its constituent alpha and beta subunits nor the nucleotide sequence of the corresponding pheS and pheT genes were known. Using specific oligonucleotides of conserved pheS regions that were adapted to the T. thermophilus codon usage, we identified, cloned and subsequently sequenced the pheST genes of this bacterium. The sequences reported here will greatly aid in the three-dimensional structure determination of T. thermophilus PheRS, a heterotetrameric (alpha 2 beta 2), class II aminoacyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1992

Identification of the pheS5 mutation, which causes thermosensitivity of Escherichia coli mutant NP37.

The pheS5 mutation responsible for the thermosensitive phenylalanyl-tRNA synthetase of the classical Escherichia coli NP37 was cloned by a recombination event and identified by DNA sequence analysis. The mutation was subsequently verified by direct sequencing of amplified NP37 DNA generated by an asymmetric polymerase chain reaction. The resulting amino acid exchange, Gly-98 to Asp-98 in the phenylalanyl-tRNA synthetase alpha subunit, might cause subunit disaggregation due to electrostatic repulsion.

Amino Acid Sequence↗
PubMed1991

Impaired affinity for phenylalanine in Escherichia coli phenylalanyl-tRNA synthetase mutant caused by Gly-to-Asp exchange in motif 2 of class II tRNA synthetases.

Phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 subunit structure) is a member of class II of tRNA synthetases. We report here the genetic analysis of an Escherichia coli mutant strain which is auxotrophic for phenylalanine because it has a PheRS with a decreased affinity for phenylalanine. The mutant pheS gene encoding the PheRS alpha subunit was cloned and sequenced, and the deviation from the wild-type gene was found to result in a Gly191-to-Asp191 exchange. This alteration is located within motif 2, one of 3 conserved sequence motifs characteristic for class II aminoacyl-tRNA synthetases. Motif 2 may thus participate in the formation of the phenylalanine binding site in PheRS.

Amino Acid Sequence↗