Biomedical subjects

Jan Steyaert

Publications and source records attributed to Jan Steyaert.

13 recordsLinked to original sources

PubMed2006

Multiple transients in the pre-steady-state of nucleoside hydrolase reveal complex substrate binding, product base release, and two apparent rates of chemistry.

We have investigated the transient kinetics of the nucleoside hydrolase from Trypanosoma vivax (TvNH) at low temperatures (5 degrees C). Three novel absorbance transients (termed tau1, tau3, and tau4) were detected during multiple-guanosine turnover stopped-flow absorbance spectroscopy, in addition to a transient (tau2) that had been observed previously at 35 degrees C. At 5 degrees C, TvNH displays full-sites activity and not half-of-the-sites activity as is apparent at 35 degrees C. Both tau1 and tau2 are assigned to chemistry based on rapid-quench results. For tau1, the rate of chemistry is ca. 3000-fold faster than kcat (1-2 orders of magnitude greater than previous estimates). The pH dependencies of the burst amplitudes for tau1 and tau2 indicate that these transients arise from the formation of two different dimeric TvNH.substrate complexes and not from TvNH that contains kinetically asymmetric monomers. The saturating burst rates for tau1 and tau2 are surprisingly pH-independent, given the prominent role of acid-base chemistry in the proposed mechanism for TvNH. tau3 and tau4 are assigned to the substrate binding and base release processes, respectively, and shown to be equivalent to two fluorescence transients (tau3 and tau4, respectively) observed previously by stopped-flow methods at 35 degrees C. The rate of base release is shown to be an apparent rate. Together with steady-state product inhibition results, the data indicate that TvNH follows an ordered uni-bi kinetic mechanism with a TvNH.base dead-end complex, and not the rapid equilibrium random uni-bi mechanism proposed for other NHs. Two applicable kinetic models are presented and their implications for future mechanistic studies discussed.

Animals↗

PubMed2006

New insights into the mechanism of nucleoside hydrolases from the crystal structure of the Escherichia coli YbeK protein bound to the reaction product.

Nucleoside hydrolases (NHs) are enzymes that catalyze the excision of the N-glycosidic bond in nucleosides to allow recycling of the nitrogenous bases. The fine details of the catalytic mechanism and the structural features imposing the substrate specificity of the various members of the NH family are still debated. Here we present the functional characterization of the Escherichia coli YbeK (RihA) protein as a pyrimidine nucleoside-preferring NH and its first crystal structure to 1.8 A resolution. The enzyme active site is occupied by either the alpha or beta anomer of ribose and provides the first structural description of the binding of the NH reaction product. While the amino acid residues involved in ribosyl binding are strictly conserved in pyrimidine-specific NHs, the residues involved in specific interactions with the nitrogenous bases differ considerably. Further comparison of the active site architecture of YbeK with the related NHs establishes structural determinants involved in triggering the conformational transition between the open and closed structures and suggests a mechanism for product release.

Crystallography, X-Ray↗

PubMed2005

Influence of the pi-pi interaction on the hydrogen bonding capacity of stacked DNA/RNA bases.

The interplay between aromatic stacking and hydrogen bonding in nucleobases has been investigated via high-level quantum chemical calculations. The experimentally observed stacking arrangement between consecutive bases in DNA and RNA/DNA double helices is shown to enhance their hydrogen bonding ability as opposed to gas phase optimized complexes. This phenomenon results from more repulsive electrostatic interactions as is demonstrated in a model system of cytosine stacked offset-parallel with substituted benzenes. Therefore, the H-bonding capacity of the N3 and O2 atoms of cytosine increases linearly with the electrostatic repulsion between the stacked rings. The local hardness, a density functional theory-based reactivity descriptor, appears to be a key index associated with the molecular electrostatic potential (MEP) minima around H-bond accepting atoms, and is inversely proportional to the electrostatic interaction between stacked molecules. Finally, the MEP minima on surfaces around the bases in experimental structures of DNA and RNA-DNA double helices show that their hydrogen bonding capacity increases when taking more neighboring (intra-strand) stacking partners into account.

Base Pairing↗

PubMed2005

Substrate-assisted leaving group activation in enzyme-catalyzed N-glycosidic bond cleavage.

In enzymatic depurination of nucleosides, the 5'-OH group of the ribose moiety of the substrate is often shown to contribute substantially to catalysis. The purine-specific nucleoside hydrolase from Trypanosoma vivax (TvNH) fixes the 5'-OH group in a gauche,trans orientation about the C4'-C5' bond, enabling the 5'-oxygen to accept an intramolecular hydrogen bond from the C8-atom of the purine leaving group. High level ab initio quantum chemical calculations indicate that this interaction promotes protonation of the purine at N7. Steady state kinetics comprising engineered substrates confirm that a considerable fraction of the catalytic 5'-OH effect can be attributed to leaving group activation.

Animals↗

PubMed2005

Encapsulation of therapeutic nucleoside hydrolase in functionalised nanocapsules.

Liposomes are introduced as encapsulating carrier for prodrug activating enzymes. Inosineã-adenosineã-guanosine preferring nucleoside hydrolase of Trypanosoma vivax, a potential prodrug activating enzyme, was encapsulated in porin functionalized dioleyl-phosphatidylglycerol/egg-phosphatidylglycerol (DOPC/EPG) liposomes. Reactors had radiuses in the nanometer scale. First, transport of nucleosides through general diffusion porins OmpF and PhoE was measured in swelling assays, after which fully functional nanoreactors were developed. Enzyme catalysis of p-nitrophenylriboside, a substrate analogue for nucleoside hydrolases, was significantly higher in permeabilized vesicles than in control vesicles without porins. Residual activity of control vesicles possibly resides in an interaction between the enzyme and the liposomes. This interaction was not of electrostatic nature, since it remained unaffected after the addition of high salt or after perturbation of liposome surface charge and charge density. With these vesicles, we have introduced a new strategy for prodrug therapy, combining the benefits of ADEPT and liposome targeting strategies.

Drug Compounding↗

PubMed2005

Therapeutic nanoreactors: combining chemistry and biology in a novel triblock copolymer drug delivery system.

Triblock copolymeric nanoreactors are introduced as an alternative for liposomes as encapsulating carrier for prodrug activating enzymes. Inosine-adenosine-guanosine preferring nucleoside hydrolase of Trypanosoma vivax, a potential prodrug activating enzyme, was encapsulated in nanometer-sized vesicles constructed of poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-(2-methyloxazoline) triblock copolymers. The nanoreactor is functionalized by incorporation of bacterial porins, OmpF or Tsx, in the reactor wall. Efficient cleavage of three natural substrates and one prodrug, 2-fluoroadenosine, by the nanoreactors was demonstrated.

Animals↗

PubMed2004

Leaving group activation by aromatic stacking: an alternative to general acid catalysis.

General acid catalysis is a powerful and widely used strategy in enzymatic nucleophilic displacement reactions. For example, hydrolysis/phosphorolysis of the N-glycosidic bond in nucleosides and nucleotides commonly involves the protonation of the leaving nucleobase concomitant with nucleophilic attack. However, in the nucleoside hydrolase of the parasite Trypanosoma vivax, crystallographic and mutagenesis studies failed to identify a general acid. This enzyme binds the purine base of the substrate between the aromatic side-chains of Trp83 and Trp260. Here, we show via quantum chemical calculations that face-to-face stacking can raise the pKa of a heterocyclic aromatic compound by several units. Site-directed mutagenesis combined with substrate engineering demonstrates that Trp260 catalyzes the cleavage of the glycosidic bond by promoting the protonation of the purine base at N-7, hence functioning as an alternative to general acid catalysis.

Acids↗

PubMed2003

Pre-steady-state analysis of the nucleoside hydrolase of Trypanosoma vivax. Evidence for half-of-the-sites reactivity and rate-limiting product release.

The nucleoside hydrolase (NH) of the Trypanosoma vivax parasite catalyzes the hydrolysis of the N-glycosidic bond in ribonucleosides according to the following reaction: beta-purine (or pyrimidine) nucleoside + H(2)O --> purine (pyrimidine) base + ribose. The reaction follows a highly dissociative nucleophilic displacement reaction mechanism with a ribosyl oxocarbenium-like transition state. This paper describes the first pre-steady-state analysis of the conversion of a number of purine nucleosides. The NH exhibits burst kinetics and behaves with half-of-the-sites reactivity. The analysis suggests that the NH of T. vivax follows a complex multistep mechanism in which a common slow step different from the chemical hydrolysis is rate limiting. Stopped-flow fluorescence binding experiments with ribose indicate that a tightly bound enzyme-ribose complex accumulates during the enzymatic hydrolysis of the common purine nucleosides. This is caused by a slow isomerization between a tight and a loose enzyme-ribose complex forming the rate-limiting step on the reaction coordinate.

Animals↗

PubMed2003

Cloning, preliminary characterization and crystallization of nucleoside hydrolases from Caenorhabditis elegans and Campylobacter jejuni.

The nucleoside hydrolases (NHs) are a family of nucleoside-modifying enzymes. They play an important role in the purine-salvage pathway of many pathogenic organisms which are unable to synthesize purines de novo. Although well characterized in protozoan parasites, their precise function and mechanism remain unclear in other species. For the first time, NHs from Caenorhabditis elegans and Campylobacter jejuni, which are representatives of mesozoa and bacteria, respectively, have been cloned and purified. Steady-state kinetics indicate a different substrate-specificity profile to previously described hydrolases. Native diffraction data sets were collected from crystals of NH from each organism. The hexagonal crystals (space group P6(2)22 or P6(4)22) of NH from C. elegans diffracted to a resolution of 2.8 A, while the data set from the orthorhombic crystals (space group I222 or I2(1)2(1)2(1)) of NH from C. jejuni could be processed to 1.7 A resolution. The unit-cell parameters were a = b = 102.23, c = 117.27 A in the former case and a = 101.13, b = 100.13, c = 81.37 A in the latter.

Animals↗

PubMed2003

Catalysis by nucleoside hydrolases.

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Because of their vital role in the protozoan purine salvage pathway, nucleoside hydrolases from parasitic protozoa in particular have been studied extensively by X-ray crystallography, kinetic methods and site-directed mutagenesis. An elaborate network of conserved interactions between the metalloenzyme and the ribose enables steric and electrostatic stabilisation of the oxocarbenium-ion-like transition state. Activation of the leaving group by protonation before the formation of the transition state is a recurring catalytic strategy of enzymes that cleave N-glycosidic bonds. However, the mechanisms underlying leaving group activation are still the subject of debate for the nucleoside hydrolases.

Binding Sites↗

PubMed2003

Ribonucleases: from prototypes to therapeutic targets?

Ribonucleases (RNases) have proven to be excellent model systems for the study of protein structure, folding and stability, and enzyme catalysis, resulting in four Nobel Prize lectures in chemistry. Beside this 'academic' success, RNases are also relevant from a medical point of view. The RNA population in cells is controlled post-transcriptionally by ribonucleases (RNases) of varying specificity. Other therapeutic proteins like angiogenin, neurotoxins, and plant allergens have RNase activity or significant structural homology to known RNases. Also, RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases. To date, no clinical drugs are available that target this important class of enzymes. Small-molecule RNase inhibitors derived from mono- or dinucleotides, as well as pentavalent oxyvanadate transition state analogs are found to be rather marginal inhibitors. These compounds bind their target RNase with dissociation constants in the micromolar range, whereas transition state theory predicts picomolar values for genuine transition states. The rational design for new transition state analog inhibitors requires knowledge of the precise nature of the transition state and of the occurring intermolecular enzyme-substrate interactions. This review focuses on these chemical and structural features of RNase A and RNase T(1), the best characterized members of two separate classes of ribonucleases.

Animals↗

PubMed2002

A nucleophile activation dyad in ribonucleases. A combined X-ray crystallographic/ab initio quantum chemical study.

Ribonucleases (RNases) catalyze the cleavage of the phosphodiester bond in RNA up to 10(15)-fold, as compared with the uncatalyzed reaction. High resolution crystal structures of these enzymes in complex with 3'-mononucleotide substrates demonstrate the accommodation of the nucleophilic 2'-OH group in a binding pocket comprising the catalytic base (glutamate or histidine) and a charged hydrogen bond donor (lysine or histidine). Ab initio quantum chemical calculations performed on such Michaelis complexes of the mammalian RNase A (EC ) and the microbial RNase T(1) (EC ) show negative charge build up on the 2'-oxygen upon substrate binding. The increased nucleophilicity results from stronger hydrogen bonding to the catalytic base, which is mediated by a hydrogen bond from the charged donor. This hitherto unrecognized catalytic dyad in ribonucleases constitutes a general mechanism for nucleophile activation in both enzymic and RNA-catalyzed phosphoryl transfer reactions.

Binding Sites↗

PubMed2002

Enzyme-substrate interactions in the purine-specific nucleoside hydrolase from Trypanosoma vivax.

Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae and are considered as targets for drug design. We previously reported the first x-ray structure of an inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-NH) from Trypanosoma vivax (). Here we report the 2.0-A crystal structure of the slow D10A mutant in complex with the inhibitor 3-deaza-adenosine and the 1.6-A crystal structure of the same enzyme in complex with a genuine substrate inosine. The enzyme-substrate complex shows the substrate bound to the enzyme in a different conformation from 3-deaza-adenosine and provides a snapshot along the reaction coordinate of the enzyme-catalyzed reaction. The chemical groups on the substrate important for binding and catalysis are mapped. The 2'-OH, 3'-OH, and 5'-OH contribute 4.6, 7.5, and 5.4 kcal/mol to k(cat)/K(m), respectively. Specific interactions with the exocyclic groups on the purine ring are not required for catalysis. Site-directed mutagenesis indicates that the purine specificity of the IAG-NHs is imposed by a parallel aromatic stacking interaction involving Trp(83) and Trp(260). The pH profiles of k(cat) and k(cat)/K(m) indicate the existence of one or more proton donors, possibly involved in leaving group activation. However, mutagenesis of the active site residues around the nucleoside base and an alanine scan of a flexible loop near the active site fail to identify this general acid. The parallel aromatic stacking seems to provide the most likely alternative mechanism for leaving group activation.

Amino Acid Sequence↗