BACKGROUND: The incidence of acute coronary syndrome (ACS) in young people (</=65 years) is continuously rising. While prognostic factors in ACS are well-investigated less attention has been paid to their age-dependent prognostic value and their particular relevance in younger patients. The aim of our study was to assess the age-dependent prognostic impact of butyrylcholinesterase (BChE). METHODS: Retrospective cohort study including 624 patients with ACS. Patients were stratified by age into equal groups (n = 208) corresponding to "young patients" (45-64 years), "middle-aged patients" (65-84 years) and "old patients" (85-100 years). Cox regression hazard analysis was used to assess the influence of BChE on survival. RESULTS: After a mean follow-up time of 4.0 (interquartile range [IQR] 2.0-6.4) years, 154 patients (24.7%) died due to a cardiac cause. In the overall cohort, BChE was indirectly associated with cardiac mortality-free survival (adjusted hazard ratio (HR): 0.70 (95% confidence interval [CI] 0.53-0.93, p = 0.01). The primary-analysis of BChE by age strata showed the strongest effect in the age group 45-64 years with an adjusted HR per 1-SD of 0.28 (95% CI 0.12-0.64, p = 0.003), a weaker association with mortality in middle aged (65-84 years: adjusted HR per 1-SD 0.66 [95% CI: 0.41-1.06], p = 0.087), and no association in older patients (85-100 years: adjusted HR per 1-SD 0.89 [95% CI: 0.58-1.38], p = 0.613). CONCLUSION: BChE is a strong predictor for cardiac mortality specifically in younger patients with ACS aged between 45 and 64 years. No significant association of BChE with cardiac-mortality was detected in other age classes.
Several tryptophan128-substituted mutants of the hydroxynitrile lyase from Manihot esculenta (MeHNL) are constructed and applied in the MeHNL-catalyzed addition of HCN to various aromatic and aliphatic aldehydes as well as to methyl and ethyl ketones to yield the corresponding cyanohydrins. The mutants (especially MeHNL-W128A) are in most cases superior to the wild-type (wt) enzyme when diisopropyl ether is used as the solvent. Substitution of tryptophan128 by an alanine residue enlarges the entrance channel to the active site of MeHNL and thus facilitates access of sterically demanding substrates to the active site, as clearly demonstrated for aromatic aldehydes, especially 3-phenoxybenzaldehyde. These experimental results are in accordance with the X-ray crystal structure of MeHNL-W128A. Aliphatic aldehydes, surprisingly, do not demonstrate this reactivity dependence of mutants on substrate bulkiness. Comparative reactions of 3-phenoxybenzaldehyde with wtMeHNL and MeHNL-W128A in both aqueous citrate buffer and a two-phase system of water/methyl tert-butyl ether again reveal the superiority of the mutant enzyme: 3-phenoxybenzaldehyde was converted quantitatively into a cyanohydrin nearly independently of the amount of enzyme present, with a space-time yield of 57 g L(-1) h(-1).
Title: Crystal structure of hydroxynitrile lyase from Sorghum bicolor in complex with the inhibitor benzoic acid: a novel cyanogenic enzyme Lauble H, Miehlich B, Forster S, Wajant H, Effenberger F Ref: Biochemistry, 41:12043, 2002 : PubMed
The crystal structure of the hydroxynitrile lyase from Sorghum bicolor (SbHNL) in complex with the inhibitor benzoic acid has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 16.5%. The SbHNL sequence places the enzyme in the alpha/beta hydrolase family where the active site nucleophile is predicted to be organized in a characteristic pentapeptide motif which is part of the active site strand-turn-helix motif. In SbHNL, however, a unique two-amino acid deletion is next to the putative active site Ser158, removing thereby the putative oxyanion hole-forming Tyr residue. The presented X-ray structure shows that the overall folding pattern of SbHNL is similar to that of the closely related wheat serine carboxypeptidase (CPD-WII); however, the deletion in SbHNL is forcing the putative active site residues away from the expected hydrolase binding site toward a small hydrophobic cleft, which also contains the inhibitor benzoic acid, defining thereby a completely different SbHNL active site architecture where the traditional view of a classic triad is not given any more. Rather, we propose a mechanism involving general base catalysis by the carboxy-terminal Trp270 carboxyl group and proton transfer toward the leaving nitrile group by an active site water molecule. The unexpected interactions of the inhibitor with the new SbHNL active site also reveal the structural basis for the enzyme's limited substrate specificity. The implications of this structure on the evolution of catalysis in the hydroxynitrile lyase superfamily are discussed.
Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL-W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild-type MeHNL and MeHNL-W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL-W128A for the unnatural substrates mandelonitrile and 4-hydroxymandelonitrile are increased 9-fold and approximately 450-fold, respectively, compared with the wild-type MeHNL. The crystal structure of the MeHNL-W128A substrate-free form at 2.1 A resolution indicates that the W128A substitution has significantly enlarged the active-site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL-W128A--4-hydroxybenzaldehyde complex structure at 2.1 A resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.
Title: Structure of hydroxynitrile lyase from Manihot esculenta in complex with substrates acetone and chloroacetone: implications for the mechanism of cyanogenesis Lauble H, Forster S, Miehlich B, Wajant H, Effenberger F Ref: Acta Crystallographica D Biol Crystallogr, 57:194, 2001 : PubMed
The crystal structures of hydroxynitrile lyase from Manihot esculenta (MeHNL) complexed with the native substrate acetone and substrate analogue chloroacetone have been determined and refined at 2.2 A resolution. The substrates are positioned in the active site by hydrogen-bond interactions of the carbonyl O atom with Thr11 OG, Ser80 OG and, to a lesser extent, Cys81 SG. These studies support a mechanism for cyanogenesis as well as for the stereospecific MeHNL-catalyzed formation of (S)-cyanohydrins, which closely resembles the base-catalyzed chemical reaction of HCN with carbonyl compounds.
Title: Mechanistic aspects of cyanogenesis from active-site mutant Ser80Ala of hydroxynitrile lyase from Manihot esculenta in complex with acetone cyanohydrin Lauble H, Miehlich B, Forster S, Wajant H, Effenberger F Ref: Protein Science, 10:1015, 2001 : PubMed
The structure and function of hydroxynitrile lyase from Manihot esculenta (MeHNL) have been analyzed by X-ray crystallography and site-directed mutagenesis. The crystal structure of the MeHNL-S80A mutant enzyme has been refined to an R-factor of 18.0% against diffraction data to 2.1-A resolution. The three-dimensional structure of the MeHNL-S80A-acetone cyanohydrin complex was determined at 2.2-A resolution and refined to an R-factor of 18.7%. Thr11 and Cys81 involved in substrate binding have been substituted by Ala in site-directed mutagenesis. The kinetic measurements of these mutant enzymes are presented. Combined with structural data, the results support a mechanism for cyanogenesis in which His236 as a general base abstracts a proton from Ser80, thereby allowing proton transfer from the hydroxyl group of acetone cyanohydrin to Ser80. The His236 imidazolium cation then facilitates the leaving of the nitrile group by proton donating.
(R)- as well as (S)-cyanohydrins are now easily available as a result of the excellent accessibility, the relatively high stability and the easy handling of hydroxynitrile lyases (HNLs). The optimization of reaction conditions (solvent, temperature, and using site-directed mutagenesis, etc.) has enabled HNL-catalyzed preparations of optically active cyanohydrins on a technical scale. The enantioselectivity of chiral metal-complex-catalyzed additions of trimethylsilyl cyanide to aldehydes has been improved, but is, by far, not yet competitive with the HNL-catalyzed reactions.
Title: Crystallization and preliminary x-ray diffraction analysis of hydroxynitrile lyase from cassava (Manihot esculenta) Lauble H, Decanniere K, Wajant H, Forster S, Effenberger F Ref: Acta Crystallographica D Biol Crystallogr, 55:904, 1999 : PubMed
Hydroxynitrile lyase from M. esculenta (cassava) was crystallized in two different crystal forms by the hanging-drop vapour-diffusion method. Crystals of form I were obtained from a mixture of polyethylene glycol 8000 and 2-methyl-2,4-pentanediol, and belong to the tetragonal space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 105.9, c = 188.9 A and with two molecules in the asymmetric unit. These crystals diffract to 2.9 A with conventional X-ray sources and beyond 2.1 A resolution with synchrotron radiation. The crystals are relatively sensitive to radiation damage and conditions for flash-cooling the crystals have been established. A complete native data set has been collected up to 2.2 A resolution. Crystal form II has been obtained at pH 5.6 using lithium sulfate as a precipitant. The crystals apparently belong to the orthorhombic space group P21212, with unit-cell parameters a = 117.52, b = 127.09 and c = 78.08 A, have two molecules in the asymmetric unit and diffract to beyond 2.0 A resolution. A complete native data set has been collected to 2.2 A resolution.
Title: Crystallographic studies and preliminary X-ray investigation of (S)-p-hydroxy-mandelonitrile lyase from Sorghum bicolor (L.) Lauble H, Knodler S, Schindelin H, Forster S, Wajant H, Effenberger F Ref: Acta Crystallographica D Biol Crystallogr, 52:887, 1996 : PubMed
(S)-p-Hydroxy-mandelonitrile lyase from Sorghum bicolor has been crystallized in three different forms using the hanging-drop vapor-diffusion technique. Crystal form I is obtained from 1.4 M (NH(4))(2)SO(4) in 100 mM Na-acetate, pH 4.6, and belongs to the orthorhombic space group P2(1)2(1)2(1). The cell dimensions are a = 71.4, b = 95.8, c = 149.1 A. A complete set of diffraction data has been collected to 2.6 A resolution. Form II crystals are grown from 500 mM Li(2)SO(4) in 13% polyethylene glycol 8000. These crystals appear as hexagonal plates and diffract to 2.98 A resolution but apparently are twinned. Cocrystallizing hydroxynitrile lyase with the inhibitor benzoic acid using 1.4 M (NH(4))(2)SO(4) in 100 mM Na citrate, pH 5.4 as precipitant yields crystal form III, which belongs to the monoclinic space group C2 with a = 150.7, b = 103.7, c = 90.6 A, beta = 101.3. X-ray diffraction data were collected to 2.3 A resolution.
Title: Enantioselective synthesis of aliphatic (S)-cyanohydrins in organic solvents using hydroxynitrile lyase from Manihot esculenta Wajant H, Forster S, Sprauer A, Effenberger F, Pfizenmaier K Ref: Annals of the New York Academy of Sciences, 799:771, 1996 : PubMed
Using high-performance liquid chromatography and nuclear magnetic resonance we identified vicianin as the cyanogenic compound of Phlebodium aureum. The (R)-hydroxynitrile lyase involved during cyanogenesis in the catabolism of the aglycon ([R]-mandelonitrile) was purified to apparent homogeneity. The purified holoenzyme is a homomultimer with subunits of Mr = 20,000. At least three isoforms of the enzyme exist. In contrast to other hydroxynitrile lyases, mandelonitrile lyase (MDL) from P. aureum was not inhibited by sulfhydryl- or hydroxyl-modifying reagents, suggesting a different catalytic mechanism. The enzyme is active over a broad temperature range, with maximum activity between 35 and 50[deg]C, and a pH optimum at 6.5. In contrast to (R)-MDLs isolated from several species of the Rosaceae family, (R)-MDL from P. aureum is not a flavoprotein. The substrate specificity was investigated using immobilized enzyme and diisopropyl ether as solvent. The addition of cyanide to aromatic and heterocyclic carbonyls is catalyzed by this (R)-MDL, whereas aliphatic carbonyls are poorly converted.
Single crystals of three different isoenzymes of (R)-(+) mandelonitrile lyase (hydroxynitrile lyase) from almonds (Prunus amygdalus) have been obtained by hanging drop vapor diffusion using polyethylene glycol 4000 and isopropanol as co-precipitants. The crystals belong to the monoclinic space group P2(1) with unit cell parameters a = 69.9, b = 95.1, c = 95.6 A, and beta = 118.5 degrees. A complete set of diffraction data has been collected to 2.6 A resolution on native crystals of isoenzyme III.
