Biotransformation of β-amino nitriles: the role of the N-protecting group

N-Tolylsulfonyl- and N-butyloxycarbonyl-protected β-amino nitriles were prepared to study the effect of the N-protecting group on the biotransformation of the β-amino nitriles to the corresponding β-amino amides and acids. The bioconversions were carried out by using whole cells of Rhodococcus sp. R312 and Rhodococcus erythropolis NCIMB 11540. The bioconversion products of five-membered carbocyclic nitriles were mainly the respective acids whereas the carbocyclic six-membered nitriles were accumulated at the stage of the amide. Benefits of the enzymatic compared with the chemical hydrolysis of β-amino nitriles are the mild reaction conditions for the transformation of the nitrile group in the presence of acid or base labile N-protecting groups. In the present work we concentrated on this chemoselectivity of the biotransformation rather than its potential enantioselectivity, which will be subject of future investigations. Thus, some new compounds were prepared: (±)-(2-cyano-cyclohexyl) carbamic acid tert-butyl ester (4a), (±)-(2-carbamoyl-cyclopentyl) carbamic acid tert-butyl ester (3b) and (±)-(2-carbamoyl-cyclohexyl) carbamic acid tert-butyl ester (4b).     

On-line deconjugation of glucuronides using an immobilized enzyme reactor based upon β-glucuronidase

An immobilized enzyme reactor based upon β-glucuronidase (BG–IMER) has been developed for the on-line deconjugation of substrates. The activity of the BG–IMER and its applicability to on-line deconjugation was investigated. The BG–IMER was coupled to a reversed-phase column (C₈ or C₁₈) and the latter column was used to separate substrates and products eluted from the β-glucuronidase reactor. The activity of the BG–IMER was followed by measurement of percent deconjugation and the parameters investigated were: substrate concentration, pH (4 to 6), temperature (r.t., 37°C), enzyme–substrate contact time using flow-rates of 0.1 to 1.0 ml/min (15–1.5 min). The glucuronides used in the evaluation of the BG–IMER were: 4-methylumbelliferyl-β- -glucuronide, p-acetaminophen-β- -glucuronide, 3′-azido-3′-deoxythymidine-β- -glucuronide, phenyl-β- -glucuronide, chloramphenicol-β- -glucuronide, estradiol-17-β- -glucuronide and morphine-β- -glucuronide. The development of on-line HPLC deconjugation of glucuronide substrates using the BG–IMER will facilitate the identification of metabolites and quantification of aglycones in metabolic and pharmacokinetic studies.     

N-Nitroso-β-lactams as β-lactamase inhibitor

N-Nitroso-β-phenyl-β-lactam has been found to be a specific inhibitor of β-lactamase. N-Nitroso--phenyl-β-lactam, by contrast, was virtually ineffective although a transient inhibition of short duration was observed. The acyl enzyme derived from the β-phenyl isomer is presumably involved in a cross-linking reaction, whereas that from the -phenyl isomer was quenched by spontaneous hydrolysis without formation of a covalent bond. No inhibitory effect of the β-phenyl isomer on chymotrypsin has been observed.     

Separative preparation of the four stereoisomers of β-methylphenylalanine with N-carbamoyl amino acid amidohydrolases

The selective preparation of the four stereoisomers of β-methylphenylalanine (Mphe) from mixtures of the four stereoisomers of N-carbamoyl-β-methylphenylalanine (NCMphe) with N-carbamoyl amino acid amidohydrolases (carbamoylases) was developed. -Carbamoylase specifically hydrolyzed threo- -NCMphe with a little side activity toward erythro- -NCMphe, thus threo- -Mphe was produced with high optical purity from a mixture of the four stereoisomers of NCMphe. -Carbamoylase specifically produced threo- -Mphe from a mixture of the four stereoisomers of NCMphe. The erythro- -Mphe was obtained from erythro- -NCMphe which was prepared through diastereomer resolution by separative crystallization of benzoyl Mphe with a little side activity of -carbamoylase toward erythro- -NCMphe and the remaining erythro- -NCMphe was chemically hydrolyzed to erythro- -Mphe.     

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products

Epidemiological studies testing the effect of β-carotene in humans have found a relative risk for lung cancer in smokers supplemented with β-carotene. We investigated the reactions of retinal and β-apo-8′-carotenal, two β-carotene oxidation products, with 2′-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N²-etheno-2′-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with β-carotene or β-carotene oxidation products, significantly increased levels of 1,N²-etheno-2′-deoxyguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of β-carotene oxidation products.     

Inhibition stereochemistry of hydroxamate inhibitors for thermolysin

N-Acyl-N-hydroxy-β-amino acid derivatives were prepared and tested as inhibitors for thermolysin to find that these inhibitors show the -stereospecificity in contrast to the corresponding hydroxamates prepared from -amino acid, which exhibit the -stereochemistry. N-Formyl-N-hydroxy-β- -Phe-NHMe is the most potent inhibitor having the K_i value of 1.66 μM.     

Fungus β-glycosidases: immobilization and use in alkyl-β-glycoside synthesis

Production of β-glycosidases: β-xylosidase and β-glucosidase by the fungus Sclerotinia sclerotiorum was optimized in the presence of different carbon sources. Immobilization supports with different physico-chemical characteristics were evaluated for use in continuous reactors. Immobilization and activity yields were calculated. Among the adsorption on Duolite, Amberlite, Celite and DEAE-sepharose, and entrapment in polyacrylamide gel or reticulation using glutaraldehyde, highest yields were obtained when β-xylosidase was adsorbed on Duolite A 7 and when β-glucosidase was adsorbed on DEAE-sepharose.

Enzyme preparations from S. sclerotiorum cultures were used in a biphasic (alcohol/aqueous) medium for the synthesis of alkyl-glycosides by trans-glycosylation of sugars and long-chain alcohols. The synthesis was studied under different conditions with primary and secondary alcohols as substrates, in the presence of free or immobilized enzyme. Xylan and cellobiose were used for the synthesis of alkyl-xylosides and alkyl-glucosides, respectively. The majority of the immobilized preparations were unable to catalyze the synthesis of alkyl-glycosides.

Highest yields were obtained when using xylan and C₄–C₆-alcohols. The reaction produced alkyl-β-xyloside and alkyl-β-xylobioside, as confirmed by MS/MS. Up to 22 mM iso-amyl-xyloside and 14 mM iso-amyl-xylobioside were produced from iso-amyl alcohol and xylan.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

The posttranslational modification of β-endorphin in the intermediate pituitary of the toad, Bufo marinus, includes processing at a monobasic cleavage site

Fractionation of an acid extract of 15 B. marinus intermediate pituitaries by a combination of gel filtration chromatography and cation exchange chromatography revealed one major and five minor forms of β-endorphin in this tissue. Based on reversed-phase HPLC and immunological properties, as well as amino acid composition and primary sequence analysis, it was deduced that the sequence of the major form of B. marinus β-endorphin is N-acetyl-YGGFMTPE. Overall, the steady-state analyses of the minor forms of β-endorphin indicated that the posttranslational processing of β-endorphin in the toad intermediate pituitary includes endoproteolytic cleavage at both paired basic and monobasic cleavage sites.     

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for β-galactosidase activity, were used for the synthesis of 1-hexyl-β- -galactoside and 1-octyl-β- -galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6–36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50 mM lactose for at least a week.     

Phenylethanolamine N-methyltransferase has beta-carboline 2N-methyltransferase activity: hypothetical relevance to Parkinson's disease

Mammalian brain has a β-carboline 2N-methyltransferase activity that converts β-carbolines, such as norharman and harman, into 2N-methylated β-carbolinium cations, which are structural and functional analogs of the Parkinsonian-inducing toxin 1-methyl-4-phenylpyridinium cation (MPP⁺). The identity and physiological function of this β-carboline 2N-methylation activity was previously unknown. We report pharmacological and biochemical evidence that phenylethanolamine N-methyltransferase (EC 2.1.1.28) has β-carboline 2N-methyltransferase activity. Specifically, purified phenylethanolamine N-methyltransferase (PNMT) catalyzes the 2N-methylation (21.1 pmol/h per unit PNMT) of 9-methylnorharman, but not the 9N-methylation of 2-methylnorharmanium cation. LY134046, a selective inhibitor of phenylethanolamine N-methyltransferase, inhibits (IC₅₀ 1.9 μM) the 2N-methylation of 9-methylnorharman, a substrate for β-carboline 2N-methyltransferase. Substrates of phenylethanolamine N-methyltransferase also inhibit β-carboline 2N-methyltransferase activity in a concentration-dependent manner. β-Carboline 2N-methyltransferase activity (43.7 pmol/h/mg protein) is present in human adrenal medulla, a tissue with high phenylethanolamine N-methyltransferase activity.

We are investigating the potential role of N-methylated β-carbolinium cations in the pathogenesis of idiopathic Parkinson’s disease. Presuming that phenylethanolamine N-methyltransferase activity forms toxic 2N-methylated β-carbolinium cations, we propose a novel hypothesis regarding Parkinson’s disease—a hypothesis that includes a role for phenylethanolamine N-methyltransferase-catalyzed formation of MPP⁺-like 2N-methylated β-carbolinium cations.     

Kinetically controlled synthesis of ampicillin with immobilized penicillin acylase in the presence of organic cosolvents

Penicillin acylase (PA) is used in the industrial production of 6-amino penicillanic acid (6-APA). However, by proper control of reaction medium, the enzyme can be used in the reverse synthesis of β-lactam antibiotics from the corresponding β-lactam nuclei and suitable acyl donors. Under thermodynamically controlled strategy, the use of organic cosolvents can favor synthesis over hydrolysis by lowering water activity and favoring the non-ionic reactive species. Under kinetically controlled strategy using activated acyl donors, organic solvents can favor synthesis by depressing hydrolytic reactions. Results are presented on the synthesis of ampicillin from phenylglycine methyl ester and 6-APA with immobilized Escherichia coli PA in the presence of organic cosolvents. Several solvents were tested in terms of enzyme stability and solubility of substrates. Ethylene glycol, glycerol, 1–2 propanediol and 1–3 butanediol were selected accordingly and ampicillin synthesis was performed in all of them. Best results in terms of yield and productivity were obtained with ethylene glycol, with which further studies were conducted. Variables studied were enzyme to limiting substrate ratio, acyl acceptor to acyl donor ratio, organic solvent concentration, pH and temperature. Experimental design based on a two-level fractional factorial design was conducted. pH was determined as the most sensitive variable and was further optimized. The best conditions for ampicillin synthesis in terms of productivity, within the range of values studied for those variables, were pH 7.4, 28°C, 36 U_S PA/mmol 6-APA, 3 mol PGME/mol 6-APA and 45 % (v/v) ethylene glycol concentration. Productivity was 7.66 mM ampicillin/h, which corresponds to a specific productivity of 7.02 μmol ampicillin/h U_S at 55 % yield. Productivity was lower than in buffer but product yield was higher because of the much lower relative hydrolysis rates.     

Fibronectin promotes calcium signaling by interferon-gamma in human neutrophils via G-protein and sphingosine kinase-dependent mechanisms

A common intracellular signal activating polymorphonuclear leukocytes (PMN) in inflammation is a change in cytosolic calcium concentration. Previously, we have shown that interferon-γ (IFN-γ) induces transient calcium signals in PMN, but only after intracellular calcium store depletion. Using a digital imaging system, we show that adhesion of PMN is critical for IFN-γ-induced calcium signals, and with PMN attached to the optimal coating, the calcium signals are evoked even in presence of extracellular calcium, that is, non-depleted calcium stores. Adhesion to fibronectin, pure or extracted from plasma by gelatin, improved the IFN-γ responses compared with serum, plasma, or vitronectin coats. In accordance with previous observations, IFN-γ-induced calcium signals in fibronectin adherent cells were totally abolished by the G-protein inhibitor pertussis toxin and were also inhibited by the sphingosine kinase inhibitors dimethylsphingosine (DMS) and N-acetylsphingosine (N-Ac-Sp). PMN contact with fibronectin alone, measured in cells sedimenting onto a fibronectin-coated surface or by addition of fibronectin to glass-adherent cells, evoked transient calcium signals. However, PMN in suspension did not respond to the addition of fibronectin or arginine-glycine-aspartate (RGD). The fibronectin-induced calcium signals were also clearly depressed by pertussis toxin and by the sphingosine kinase inhibitors DMS, dihydrosphingosine (DHS), and N-Ac-Sp. When the product of sphingosine kinase activity, sphingosine I-phosphate (S1-P), was added to the cells, similar calcium signals were induced, which were dependent on a pertussis toxin-sensitive G-protein activity. Finally, addition of S1-P to the cells prior to stimulation with IFN-γ partly mimicked the priming effect of fibronectin. In conclusion, fibronectin contact evokes by itself a calcium signal in PMN and further promotes calcium signaling by IFN-γ. We suggest that fibronectin might activate sphingosine kinase, and that the sphingosine 1-phosphate thereby generated induces a calcium signal via a G-protein-dependent mechanism. Apparently, sphingosine kinase activity is also involved in IFN-γ induced calcium signals.     

Beta-ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.     

Characterization of recombinant fusion constructs of human β1,4-galactosyltransferase 1 and the lipase pre-propeptide from Staphylococcus hyicus

The question whether proteins fused to β1,4galactosyltransferase (β4GalT-1) influence the biocatalytic properties of the glycosyltransferase has not been addressed so far. In the present study we have chosen a novel approach to express the gene encoding for human β4GalT-1 from placenta in an N-terminal fusion with the pre-propeptide of the lipase from Staphylococcus hyicus. The pre-propeptide provides a secretion signal in Escherichia coli and was reported to protect fused proteins against proteolytic degradation. Expression of the fusion protein was challenged with an almost full-length version of human β4GalT-1 including parts of the signal anchor and the stem region (propeptide-natβ4GalT-1) and the full catalytic domain (His₆propeptide-catβ4GalT-1), respectively. We demonstrate that the fusion protein in propeptide-natβ4GalT-1 is cleaved off during purification using immobilized metal ion chromatography IMAC, most probably catalyzed by the immobilized Zn²⁺ ions. Cleavage can be avoided by deletion of five C-terminal amino acids of the propeptide and the stem region yielding His₆propeptide-catβ4GalT-1. Kinetic data reveal that both enzyme constructs possess specific biocatalytic characteristics when compared to a recombinant luminal β4GalT-1 construct. The catalytic efficiency towards more hydrophobic acceptor glycosides, e.g. benzyl 2-acetamido-2-deoxy-β-d-glucopyranoside and p-nitro phenyl 2-acetamido-2-deoxy-β-d-glucopyranoside, and the N-glycans of IgG from rat is significantly increased. In summary, the His₆propeptide-catβ4GalT-1 is very useful for biocatalytic applications involving hydrophobic acceptor glycosides and the propeptide-natβ4GalT-1 is able to glycosylate glycoproteins in an efficient way.     

Volume-regulated Cl- channels in human pleural mesothelioma cells

A novel xyloglucan-specific endo-β-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-β-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (−2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained.     

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.     

The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism

Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp²³⁰ residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.