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H Griengl

Publications and source records attributed to H Griengl.

At least 37 records · Page 2Linked to original sources
PubMed1997

2-Naphthoate catabolic pathway in Burkholderia strain JT 1500.

Burkholderia strain (JT 1500), able to use 2-naphthoate as the sole source of carbon, was isolated from soil. On the basis of growth characteristics, oxygen uptake experiments, enzyme assays, and detection of intermediates, a degradation pathway of 2-naphthoate is proposed. The features of this pathway are convergent with those for phenanthrene. We propose a pathway for the conversion of 2-naphthoate to 1 mol (each) of pyruvate, succinate, and acetyl coenzyme A and 2 mol of CO2. During growth in the presence of 2-naphthoate, six metabolites were detected by thin-layer chromatography, high-performance liquid chromatography, and spectroscopy. 1-Hydroxy-2-naphthoate accumulated in the culture broth during growth on 2-naphthoate. Also, the formation of 2'-carboxybenzalpyruvate, phthalaldehydate, phthalate, protocatechuate, and beta-carboxy-cis,cis-muconic acid was demonstrated. (1R,2S)-cis-1,2-Dihydro-1,2-dihydroxy-2-naphthoate was thus considered an intermediate between 2-naphthoate and 1-hydroxy-2-naphthoate, but it was not transformed by whole cells or their extracts. We conclude that this diol is not responsible for the formation of 1-hydroxy-2-naphthoate from 2-naphthoate but that one of the other three diastereomers is not eliminated as a potential intermediate for a dehydration reaction.

Biodegradation, Environmental↗
PubMed1997

Stereochemical course of two arene-cis-diol dehydrogenases specifically induced in Pseudomonas putida.

Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. There are two stereochemical questions to answer. (i) To which face of NAD is hydride transferred giving NADH? (ii) Which hydrogen of the arene-cis-diols is donated to NAD? We report the results of 1H nuclear magnetic resonance [1H NMR] experiments for two diol dehydrogenases induced during growth of Pseudomonas putida PaW1(TOL) and JT105 with p-xylene and p-toluate, respectively. per-[2H5]benzoate-1,2-dihydrodiol and per-[2H7]- and specifically [2H]p-toluate-2,3-dihydrodiols were the substrates used to examine this by 1H NMR, as the two protons of the prochiral center (C-4 of the nicotinamide ring) are easily distinguished in the region of 2.6 to 2.7 ppm. We found that with the partially purified dehydrogenases (i) 2H from the (2R) center of per-(1S,2R)-benzoate-1,2-dihydrodiol was donated to the Si-face of NAD to give (4S)-NAD2H; (ii) p-toluate-2,3-diol dehydrogenase also provided exclusively (4S)-NAD2H, but the 2H was transferred from both the 2- and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo- and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies.

Benzoates↗
PubMed1997

Hydroxynitrile lyase from Hevea brasiliensis: molecular characterization and mechanism of enzyme catalysis.

(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis is a 29 kDa single chain protein that catalyses the breakdown or formation of a C--C bond by reversible addition of hydrocyanic acid to aldehydes or ketones. The primary sequence of Hnl has no significant homology to known proteins. Detailed homology investigations employing PROFILESEARCH and secondary structure prediction algorithms suggest that Hnl is a member of the alpha/beta hydrolase fold protein family and contains a catalytic triad as functional residues for catalysis. The significance of predicted catalytic residues was tested and confirmed by site-directed mutagenesis and expression of mutant and wild-type proteins in the yeast, Saccharomyces cerevisiae. Based on these data we suggest a mechanistic model for the (S)-cyanohydrin synthesis catalyzed by hydroxynitrile lyase from Hevea brasiliensis.

Aldehyde-Lyases↗
PubMed1996

Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis.

BACKGROUND: Over three thousand species of plants, including important food crops such as cassava, use cyanogenesis, the liberation of HCN upon tissue damage, as a defense against predation. Detoxification of cyanogenic food crops requires disruption of the cyanogenic pathway. Hydroxynitrile lyase is one of the key enzymes in cyanogenesis, catalyzing the decomposition of an alpha-cyanohydrin to form HCN plus the corresponding aldehyde or ketone. These enzymes are also of potential utility for industrial syntheses of optically pure chiral cyanohydrins, being used to catalyze the reverse reaction. We set out to gain insight into the catalytic mechanism of this important class of enzymes by determining the three-dimensional structure of hydroxynitrile lyase from the rubber tree, Hevea brasiliensis. RESULTS: The crystal structure of the enzyme has been determined to 1.9 A resolution. It belongs to the alpha/beta hydrolase superfamily, with an active site that is deeply buried within the protein and connected to the outside by a narrow tunnel. The catalytic triad is made up of Ser80, His235 and Asp207. By analogy with known mechanisms of other members of this superfamily, catalysis should involve an oxyanion hole formed by the main chain NH of Cys81 and the side chains of Cys81 and Thr11. Density attributed to a histidine molecule or ion is found in the active site. CONCLUSIONS: By analogy with other alpha/beta hydrolases, the reaction catalyzed by hydroxynitrile lyase involves a tetrahedral hemiketal or hemiacetal intermediate formed by nucleophilic attack of Ser80 on the substrate, stabilized by the oxyanion hole. The SH group of Cys81 is probably involved in proton transfer between the HCN and the hydroxynitrile OH. This mechanism is significantly different from the corresponding uncatalyzed solution reaction.

Aldehyde-Lyases↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase from Hevea brasiliensis.

Crystals of the hydroxynitrile lyase from Hevea brasiliensis overexpressed in Pichia pastoris have been obtained by the hanging-drop technique at 294 K with ammonium sulfate and PEG 400 as precipitants. The crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 A. The crystals diffract to about 2.5 A resolution on a rotating-anode X-ray source.

Journal Article↗
PubMed1996

Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue.

The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1. 1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.

Aldehyde-Lyases↗
PubMed1996

Stereospecific Biohydroxylations of Protected Carboxylic Acids with Cunninghamella blakesleeana.

Cunninghamella blakesleeana DSM 1906 was found to be an efficient biocatalyst for the biotransformation of cycloalkylcarboxylic acids into hydroxy and oxo derivatives. When cultivated in submerged culture, the fungus grew in pellets. In comparison with malt extract-glucose-peptone-yeast extract medium (medium E), Czapek-Dox medium was found to reduce pellet size. Cycloalkylcarboxylic acids were protected against microbial degradation by chemical transformation into 2-cycloalkyl-1,3-benzoxazoles. The transformations of protected cyclopentyl-, cyclohexyl-, cycloheptyl-, and cyclooctylcarboxylic acids by C. blakesleeana were investigated. The biotransformations were performed in medium E by using an aerated, stirred-tank bioreactor. The transformation of 2-cyclopentyl-1,3-benzoxazole yielded (1S,3S)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol as the main product. The main by-product was (1R)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-one, and 2-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol was also obtained in small amounts. During the experiment, the enantiomeric excess of the main product increased up to 64%. 2-Cyclohexyl-1,3-benzoxazole was hydroxylated to 4-(benz-1,3-oxazol-2-yl)cyclohexan-1-ol. 2-Cycloheptyl-1,3-benzoxazole and 2-cyclooctyl-1,3-benzoxazole were transformed into several alcohols and ketones, all in low yields (2 to 19%).

Journal Article↗
PubMed1994

Microbial Baeyer-Villiger reaction of bicyclo[3.2.0]heptan-6-ones--a novel approach to sarkomycin A.

Racemic (1 alpha, 2 alpha, 5 alpha)- and (1 beta, 2 alpha, 5 beta)-2- bromobicyclo[3.2.0]heptan-6-one (rac-7, rac-10, respectively), (1 alpha, 2 alpha, 5 beta)- and (1 beta, 2 alpha, 5 beta)-2- benzyloxybicyclo[3.2.0]heptan-6-one (rac-15, rac-13, respectively), (1 beta, 2 alpha, 5 beta)-2-hydroxybicyclo[3.2.0]heptan-6-one (rac-17) and cis-bicyclo[3.2.0]hept-2-en-7-one (rac-18) were subjected to a microbial Baeyer-Villiger reaction by Acinetobacter calcoaceticus NCIB 9871. In each case both regioisomeric lactones were formed (67-93% yield) having always the opposite configuration (20 to > 99 % e.e.). Both the ratio of the regioisomers and the enantiomeric excess proved to be dependent on the type of substitution. Analogously cis-bicyclo[3.2.0]heptan-2,6-dione (rac-1) gave besides other products cyclosarkomycin (1b) (7 % yield, 97 % e.e.). Compound 1b was also obtained from the Baeyer-Villiger product of rac-17 by Swern oxidation (total yield starting from rac-17 9 %, > 98 % e.e.).

Acinetobacter calcoaceticus↗
PubMed1990

Strategies for the chemoenzymatic preparation of optically active 1-alkyn-3-ols.

A series of (R)- and (S)-1-alkyn-3-ols, chiral building units for the synthesis of leukotrienes and pheromones, were prepared via enantioselective hydrolysis of their racemic esters. While the majority of biocatalysts employed (lipases, fermenting or freeze-dried microorganisms) failed in discriminating between enantiomers, lyophilized cells of baker's yeast (Saccharomyces cerevisiae Hansen) gave (S)-1-alkyn-3-ols and their corresponding (R)-esters with greater than 90% e.e.

Alcohols↗
PubMed1990

Carbocyclic 5-iodo-2'-deoxyuridine (C-IDU) and carbocyclic (E)-5-(2-bromovinyl)-2'-deoxyuridine (C-BVDU) as unique examples of chiral molecules where the two enantiomeric forms are biologically active: interaction of the (+)- and (-)-enantiomers of C-IDU and C-BVDU with the thymidine kinase of herpes simplex virus type 1.

The (+)- and (-)-enantiomers of the carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (C-BVDU) and 5-iodo-2'-deoxyuridine (C-IDU) were synthesized by separate routes. Both the (+)- and (-)-enantiomers of C-BVDU and C-IDU were markedly inhibitory to herpes simplex virus type 1 (HSV-1) replication. (+)-C-BVDU and (+)-C-IDU were as inhibitory to HSV-1 as the racemic (+/-)-C-BVDU and (+/-)-C-IDU, respectively, whereas the (-)-enantiomers were only 10-fold less active. Also, the (+)- and (-)-enantiomers of C-BVDU were equally inhibitory to the growth of murine mammary carcinoma cells transformed by the HSV-1 or HSV-2 thymidine kinase (TK) gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+). The (+)- and (-)-enantiomers of C-BVDU and the (+)- and (-)-enantiomers of C-IDU had a remarkably similar affinity for HSV-1 TK [Ki, 0.09 and 0.19 microM for (+)-C-BVDU and (+)-C-IDU and 0.16 and 0.19 microM for (-)-C-BVDU and (-)-C-IDU, respectively]. The inhibition of HSV-1 TK by BVDU, IDU, (+)-C-BVDU, and (+)-C-IDU was purely competitive with regard to the natural substrate (thymidine), whereas (-)-C-BVDU, (-)-C-IDU, (+/-)-C-BVDU, and (+/-)C-IDU showed a linear mixed-type inhibition of HSV-1 TK. C-BVDU and C-IDU are examples of chiral molecules of which both isomeric forms are markedly active at both the cellular and enzymatic level.

Animals↗
PubMed1989

Sugar modified oligonucleotides. I. Carbo-oligodeoxynucleotides as potential antisense agents.

For the first time, carbo-oligodeoxynucleotides, namely c-dT4 and c-dT12, have been synthesized. As compared to the natural oligomers these carbo-oligodeoxynucleotides are at least 5 times more stable toward enzymatic degradation and bind more strongly to complementary DNA. These preliminary data indicate that such oligomers fulfill the requirements to be considered as potential antisense agents.

Base Composition↗
PubMed1989

Synthesis and antiviral activity of the enantiomeric forms of carba-5-iodo-2'-deoxyuridine and carba-(E)-5-(2-bromovinyl)-2'-deoxyuridine.

Both enantiomers of the carbocyclic analogues of 5-iodo-2'-deoxyuridine (14 and ent-14) and of (E)-5-(2-bromo-vinyl)-2'-deoxyuridine (16 and ent-16) were synthesized by using (+)- or (-)-endo-norborn-5-en-2-yl acetate or butyrate, respectively, as starting materials. Against herpes simplex virus type 1 (+)-C-BVDU (16) was only slightly less active than BVDU itself, whereas (-)-C-BVDU (ent-16) proved to be 10-400-fold less effective, depending on the strain investigated. Against HSV-2 both (+)- and (-)-C-BVDU as well as (+)- and (-)-C-IDU showed minor activity. All carbocyclic analogues were inactive against TK-HSV-1 strains, pointing to the prerequisite of phosphorylation (activation) by the viral thymidine kinase (TK).

Antiviral Agents↗
PubMed1988

Phosphonoformate and phosphonoacetate derivatives of 5-substituted 2'-deoxyuridines: synthesis and antiviral activity.

The synthesis of potential "combined prodrugs" wherein phosphonoformate or phosphonoacetate was attached to the 5'-position of 2'-deoxyuridine, 2'-deoxythymidine, 5-iodo-2'-deoxyuridine (IDU), 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), or 5-(2-bromovinyl)-2'-deoxyuridine (BVDU) or to the 3'-position of CEDU is described. The antiviral activities of these derivatives and of reference compounds were compared in Vero, HEp-2, and primary rabbit kidney cells against herpes simplex virus types 1 and 2 (HSV-1 and -2). The CEDU and BVDU analogues were also evaluated against systemic and intracutaneous HSV-1 infection in mice. The nature of the 5-substituent proved critical for antiviral activity, since only the 5-iodo-, 5-(2-bromovinyl)-, and 5-(2-chloroethyl)-substituted derivatives were inhibitory to the herpesviruses. Furthermore, the type specificity is determined by the nature of the 5-substituent: the IDU analogues were similarly inhibitory to HSV-1 and -2 whereas the CEDU and BVDU analogues inhibited HSV-2 replication only at considerably higher concentrations than HSV-1. In vivo, several derivatives were shown to possess significant antiviral activity; however, none surpassed its respective parent compound, CEDU or BVDU, in potency. It seems improbable, therefore, that a synergistic effect between PFA or PAA and the nucleoside analogue occurred. The extent of in vitro and in vivo activity of the CEDU and BVDU 5'-phosphonoformates and 5'-phosphonoacetates is most plausibly explained by the ease by which the "combined prodrugs" are hydrolyzed and the parent compound, CEDU and BVDU, respectively, is released.

Animals↗
PubMed1987

In vitro and in vivo antiviral activity of 2'-fluorinated arabinosides of 5-(2-haloalkyl)uracil.

5-(2-Fluoroethyl)-2'-deoxyuridine (FEDU), its 2'-fluoroarabinofuranosyl analog (FEFAU) and the 2'-fluoroarabinofuranosyl analog (CEFAU) of the potent anti-herpesvirus compound 5-(2-chloroethyl)-2'-deoxyuridine (CEDU) were evaluated for activity against herpes simplex virus type 1 (HSV-1) and HSV-2 in vitro and in vivo. FEDU, FEFAU and CEFAU proved to be potent and selective anti-herpesvirus agents in vitro. Their potency is evident from their low minimum inhibitory concentrations for HSV-1 and HSV-2, and their selectivity is attested by the marginal inhibition of cell proliferation at relatively high concentrations, and by the high concentrations at which DNA-, RNA- or protein synthesis in normal uninfected host cells is inhibited. Their activity spectrum is broader than that of CEDU: in addition to being highly effective against HSV-1 replication, these derivatives, in particular FEFAU, inhibit HSV-2 replication at concentrations comparable to acyclovir (ACV). In the systemic and cutaneous HSV-1 infection models in mice, FEDU, FEFAU and CEFAU were markedly less potent than CEDU in suppressing the development of lesions and in reducing the mortality rate. In HSV-2 infections in mice and in guinea pigs FEDU, FEFAU and CEFAU were virtually ineffective. CEDU, however, exerted a protective effect in these animal models, albeit at relatively high concentrations.

Acyclovir↗
PubMed1987

2'-Fluorinated arabinonucleosides of 5-(2-haloalkyl)uracil: synthesis and antiviral activity.

The synthesis of 5-(2-fluoroethyl)-2'-deoxyuridine (FEDU, 4b), its 2'-fluoro analogue 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-(2-fluoroethyl)-1H,3H- pyrimidine-2,4-dione (FEFAU, 4k), and the 2'-fluoro analogue of the potent antiherpes virus compound 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), 5-(2-chloroethyl)-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1H,3H-pyr imidine - 2,4-dione (CEFAU, 4i), is described. The antiviral activities of these compounds were determined in cell culture against herpes simplex virus (HSV) types 1 and 2 and varicella zoster virus (VZV). All compounds were shown to possess significant and selective antiviral activity. FEDU proved less potent than CEDU against VZV replication; however, it was more active against HSV-2. CEFAU showed marked activity against HSV-1, HSV-2, and VZV. The compound containing fluorine at both positions, FEFAU, exhibited the strongest antiviral potency against HSV-1, HSV-2, and VZV. It inhibited HSV-1 at a concentration of 0.03-0.2 microgram/mL, HSV-2 at 0.1-0.3 microgram/mL, and VZV at 0.03 microgram/mL. Neither FEDU nor CEFAU or FEFAU exerted a significant inhibitory effect on cell proliferation at a concentration of 100 micrograms/mL. Thus, the cytotoxicity of these compounds is as low as that of CEDU and compares favorably to that of previously described 2'-fluoroarabinosyl nucleoside analogues.

Antiviral Agents↗
PubMed1985

5-(Haloalkyl)-2'-deoxyuridines: a novel type of potent antiviral nucleoside analogue.

Syntheses of 5-(2-haloethyl)-2'-deoxyuridines, 5-(3-chloropropyl)-2'-deoxyuridines, and 5-(2-chloroethyl)-2'-deoxycytidine are described. The antiviral activities of these compounds were determined in cell culture against herpes simplex virus types 1 and 2. All compounds were shown to possess significant and selective antiviral activity. The most potent derivative, 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), inhibited HSV-1 at concentrations below 0.1 microgram/mL. It exerted measurable inhibitory effects on cell proliferation only at concentrations higher than 100 micrograms/mL. In vivo CEDU reduced the mortality rate of HSV-1-infected mice at concentrations lower than 5 mg/kg per day when given intraperitoneally and orally. Thus, it proved to be more effective in this in vivo model than the reference compounds (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-[(2-hydroxyethoxy)methyl]guanine (ACV).

Animals↗