PubMed Health⌕ Search

Biomedical subjects

Peter Kast

Publications and source records attributed to Peter Kast.

12 recordsLinked to original sources
PubMed2006

Experimental and computational investigation of the uncatalyzed rearrangement and elimination reactions of isochorismate.

The versatile biosynthetic intermediate isochorismate decomposes in aqueous buffer by two competitive pathways, one leading to isoprephenate by a facile Claisen rearrangement and the other to salicylate via elimination of the enolpyruvyl side chain. Computation suggests that both processes are concerted but asynchronous pericyclic reactions, with considerable C-O cleavage in the transition state but relatively little C-C bond formation (rearrangement) or hydrogen atom transfer to the enolpyruvyl side chain (elimination). Kinetic experiments show that rearrangement is roughly 8-times more favorable than elimination. Moreover, transfer of the C2 hydrogen atom to C9 was verified by monitoring the decomposition of [2-(2)H]isochorismate, which was prepared chemoenzymatically from labeled shikimate, by (2)H NMR spectroscopy and observing the appearance of [3-(2)H]pyruvate. Finally, the isotope effects obtained with the C2 deuterated substrate are in good agreement with calculations assuming pericyclic reaction mechanisms. These results provide a benchmark for mechanistic investigations of isochorismate mutase and isochorismate pyruvate lyase, the enzymes that respectively catalyze the rearrangement and elimination reactions in plants and bacteria.

Chorismate Mutase↗
PubMed2006

1.6 A crystal structure of the secreted chorismate mutase from Mycobacterium tuberculosis: novel fold topology revealed.

The presence of exported chorismate mutases produced by certain organisms such as Mycobacterium tuberculosis has been shown to correlate with their pathogenicity. As such, these proteins comprise a new group of promising selective drug targets. Here, we report the high-resolution crystal structure of the secreted dimeric chorismate mutase from M. tuberculosis (*MtCM; encoded by Rv1885c), which represents the first 3D-structure of a member of this chorismate mutase family, termed the AroQ(gamma) subclass. Structures are presented both for the unliganded enzyme and for a complex with a transition state analog. The protomer fold resembles the structurally characterized (dimeric) Escherichia coli chorismate mutase domain, but exhibits a new topology, with helix H4 of *MtCM carrying the catalytic site residue missing in the shortened helix H1. Furthermore, the structure of each *MtCM protomer is significantly more compact and only harbors one active site pocket, which is formed entirely by one polypeptide chain. Apart from the structural model, we present evidence as to how the substrate may enter the active site.

Amino Acid Sequence↗
PubMed2005

Isochorismate pyruvate lyase: a pericyclic reaction mechanism?

Isochorismate pyruvate lyase (IPL) catalyzes the cleavage of isochorismate to give salicylate and pyruvate, a key step in bacterial siderophore biosynthesis. We investigated the enzyme from Pseudomonas aeruginosa using isochorismate selectively deuterated at C2 as a substrate. Monitoring the reaction by 2H NMR spectroscopy revealed that the label is quantitatively transferred from C2 to C9, producing stoichiometric amounts of [3-2H]pyruvate as product. Moreover, the deuterium kinetic isotope effect of 2.34 +/- 0.08 on kcat indicates that C-H cleavage is significantly rate limiting. Consistent with these data, hybrid density functional theory (HDFT) calculations at the Becke3LYP/DZ+(2d,p) level of theory predict a concerted but highly asynchronous pericyclic transition structure, in which carbon-oxygen bond cleavage is more advanced than hydrogen atom transfer from C2 to C9; the calculated 2H isotope effect of 2.22 at C2 is in excellent accord with the experimental value. Together, these findings indicate that IPL should be added to the small set of proteins that are known to catalyze pericyclic reactions. They also raise the possibility that enzymes, such as chorismate pyruvate lyase, salicylate synthase, 4-amino-4-deoxychorismate lyase, and anthranilate synthase, which accelerate formally similar reaction steps, may also exploit pericyclic mechanisms.

Binding Sites↗
PubMed2005

Mechanistic insights into the isochorismate pyruvate lyase activity of the catalytically promiscuous PchB from combinatorial mutagenesis and selection.

PchB from Pseudomonas aeruginosa possesses isochorismate pyruvate lyase (IPL) and weak chorismate mutase (CM) activity. Homology modeling based on a structurally characterized CM, coupled with randomization of presumed key active site residues (Arg54, Glu90, Gln91) and in vivo selection for CM activity, was used to derive mechanistic insights into the IPL activity of PchB. Mutation of Arg54 was incompatible with viability, and the CM and IPL activities of an engineered R54K variant were reduced 1,000-fold each. The observation that position 90 was tolerant to substitution but position 91 was essentially confined to Gln or Glu in functional variants rules out involvement of Glu90 in general base catalysis. Counter to the generally accepted mechanistic hypothesis for pyruvate lyases, we propose for PchB a rare [1,5]-sigmatropic reaction mechanism that invokes electrostatic catalysis in analogy to the [3,3]-pericyclic rearrangement of chorismate in CMs. A common catalytic principle for both PchB functions is also supported by the covariance of the catalytic parameters for the CM and IPL activities and the shared functional requirement for a protonated Glu91 in Q91E variants. The experiments demonstrate that focusing directed evolution strategies on the readily accessible surrogate activity of an enzyme can provide valuable insights into the mechanism of the primary reaction.

Amino Acid Sequence↗
PubMed2005

Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis.

The gene encompassing ORF Rv1885c with weak sequence similarity to AroQ chorismate mutases (CMs) was cloned from the genome of Mycobacterium tuberculosis and expressed in Escherichia coli. The gene product (*MtCM) complements a CM-deficient E. coli strain, but only if produced without the predicted N-terminal signal sequence typical of M. tuberculosis. The mature *MtCM, which was purified by exploiting its resistance to irreversible thermal denaturation, possesses high CM activity in vitro. The enzyme follows simple Michaelis-Menten kinetics, having a k(cat) of 50 s(-1) and a K(m) of 180 microM (at 30 degrees C and pH 7.5). *MtCM was shown to be a dimer by analytical ultracentrifugation and size-exclusion chromatography. Secondary-structure prediction and CD spectroscopy confirmed that *MtCM is a member of the all-alpha-helical AroQ class of CMs, but it seems to have a topologically rearranged AroQ fold. Because CMs are normally intracellular metabolic enzymes required for the biosynthesis of phenylalanine and tyrosine, the existence of an exported CM in Gram-positive M. tuberculosis is puzzling. The observation that homologs of *MtCM with a predicted export sequence are generally only present in parasitic or pathogenic organisms suggests that secreted CMs may have evolved to participate in some aspect of parasitism or pathogenesis yet to be unraveled.

Amino Acid Sequence↗
PubMed2005

Strategy for chromosomal gene targeting in RecA-deficient Escherichia coli strains.

Reengineering DNA by homologous recombination in Escherichia coli often depends on helper functions provided on a temporarily introduced replicon that is subsequently cured from the cells. The suicide vector pKSS offers a new curing strategy. pKSS specifies a variant of phenylalanyl-transfer RNA (tRNA) synthetase conferring relaxed substrate specificity towards phenylalanine analogs that results in their lethal incorporation into cellular proteins. Consequently, the presence of p-chlorophenylalanine selects for strains that have lost pKSS. This principle, in conjunction with a plasmid-borne recA gene, was exploited for targeted chromosomal mutagenesis by double homologous recombination in RecA-negative E. coli strains. Gene replacement with a kanamycin-resistance cassette was possible in a single step by plating on kanamycin and p-chlorophenylalanine agar plates and incubating at 37 degrees C. The presence of the correct chromosomal mutation and the absence of the plasmid were established by several control experiments. A simple screen confirmed the desired resistance phenotype in 44% of the initially selected clones, and 75% of these had the correct genotype.

Chromosomes, Bacterial↗
PubMed2004

An enzymatic molten globule: efficient coupling of folding and catalysis.

A highly active, monomeric chorismate mutase, obtained by topological redesign of a dimeric helical bundle enzyme from Methanococcus jannaschii, was investigated by NMR and various other biochemical techniques, including H/D exchange. Although structural disorder is generally considered to be incompatible with efficient catalysis, the monomer, unlike its natural counterpart, unexpectedly possesses all of the characteristics of a molten globule. Global conformational ordering, observed upon binding of a transition state analog, indicates that folding can be coupled to catalysis with minimal energetic penalty. These results support the suggestion that many modern enzymes might have evolved from molten globule precursors. Insofar as their structural plasticity confers relaxed substrate specificity and/or catalytic promiscuity, molten globules may also be attractive starting points for the evolution of new catalysts in the laboratory.

Chorismate Mutase↗
PubMed2004

Probing the proteolytic stability of beta-peptides containing alpha-fluoro- and alpha-hydroxy-beta-amino acids.

One of the benefits of beta-peptides as potential candidates for biological applications is their stability against common peptidases. Attempts have been made to rationalize this stability by altering the electron availability of a given amide carbonyl bond through the introduction of polar substituents at the alpha-position of a single beta-amino acid. Such beta-amino acids (beta-homoglycine, beta-homoalanine), containing one or two fluorine atoms or a hydroxy group in the alpha-position, were prepared in enantiopure form. A versatile method for preparing these alpha-fluoro-beta-amino acids by the homologation of appropriate alpha-amino acids and C-OH->C-F or C=O-->CF(2) substitution with DAST, is described. Consequently, a series of beta-peptides possessing an electronically modified residue at the N terminus or embedded within the chain was synthesized, and their proteolytic stability was investigated against a selection of enzymes. All ten beta-peptides tested were resilient to proteolysis. Introducing a polar, sterically undemanding group, into the alpha-position of beta-amino acids in a beta-peptide chain does not appear to facilitate localized or general enzymatic degradation.

Amino Acids↗
PubMed2003

Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor.

Citrulline was incorporated via chemical semisynthesis at position 90 in the active site of the AroH chorismate mutase from Bacillus subtilis. The wild-type arginine at this position makes hydrogen-bonding interactions with the ether oxygen of chorismate. Replacement of the positively charged guanidinium group with the isosteric but neutral urea has a dramatic effect on the ability of the enzyme to convert chorismate into prephenate. The Arg90Cit variant exhibits a >104-fold decrease in the catalytic rate constant kcat with a 2.7-fold increase in the Michaelis constant Km. In contrast, its affinity for a conformationally constrained inhibitor molecule that effectively mimics the geometry but not the dissociative character of the transition state is only reduced by a factor of approximately 6. These results show that an active site merely complementary to the reactive conformation of chorismate is insufficient for catalysis of the mutase reaction. Instead, electrostatic stabilization of the polarized transition state by provision of a cationic hydrogen bond donor proximal to the oxygen in the breaking C-O bond is essential for high catalytic efficiency.

Alanine↗
PubMed2002

Salicylate biosynthesis in Pseudomonas aeruginosa. Purification and characterization of PchB, a novel bifunctional enzyme displaying isochorismate pyruvate-lyase and chorismate mutase activities.

Isochorismate pyruvate-lyase (IPL), the second enzyme of pyochelin biosynthesis and the product of the pchB gene, was purified to homogeneity from Pseudomonas aeruginosa. In the reaction catalyzed by this enzyme, isochorismate --> salicylate + pyruvate, no cofactors appear to be required. At the pH optimum (pH 6.8), the enzyme displayed Michaelis-Menten kinetics, with an apparent K(m) of 12.5 microm for isochorismate and a kcat of 106 min(-1), calculated per monomer. The native enzyme behaved as a homodimer, as judged by molecular sieving chromatography, electrophoresis under nondenaturing conditions, and cross-linking experiments. PchB has approximately 20% amino acid sequence identity with AroQ-class chorismate mutases (CMs). Chorismate was shown to be converted to prephenate by purified PchB in vitro, with an apparent K(m) of 150 microm and a kcat of 7.8 min(-1). An oxabicyclic diacid transition state analog and well characterized inhibitor of CMs competitively inhibited both IPL and CM activities of PchB. Moreover, a CM-deficient Escherichia coli mutant, which is auxotrophic for phenylalanine and tyrosine, was functionally complemented by the cloned P. aeruginosa pchB gene for growth in minimal medium. A mutant form of PchB, in which isoleucine 88 was changed to threonine, had no detectable IPL activity, but retained wild-type CM activity. In conclusion, the 11.5-kDa subunit of PchB appears to contain a single active site involved in both IPL and CM activity.

Amino Acid Sequence↗
PubMed2001

Investigating and Engineering Enzymes by Genetic Selection.

Natural enzymes have arisen over millions of years by the gradual process of Darwinian evolution. The fundamental steps of evolution-mutation, selection, and amplification-can also be exploited in the laboratory to create and characterize protein catalysts on a human timescale. In vivo genetic selection strategies enable the exhaustive analysis of protein libraries with 10(10) different members, and even larger ensembles can be studied with in vitro methods. Evolutionary approaches can consequently yield statistically meaningful insight into the complex and often subtle interactions that influence protein folding, structure, and catalytic mechanism. Such methods are also being used increasingly as an adjunct to design, thus providing access to novel proteins with tailored catalytic activities and selectivities.

Journal Article↗