Lothar Brecker

Acetylacetone-cleaving enzyme Dke1: a novel C-C-bond-cleaving enzyme from Acinetobacter johnsonii.

The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe(2+) is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a K (m) of 9.1 microM and a k(cat) of 8.5 s(-1). A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.

The Obligate PredatoryBdellovibrio bacteriovorusPossesses a Neutral Lipid A Containing α-D-Mannoses That Replace Phosphate Residues: SIMILARITIES AND DIFFERENCES BETWEEN THE LIPID As AND THE LIPOPOLYSACCHARIDES OF THE WILD TYPE STRAINB. BACTERIOVORUSHD100 AND ITS HOST-INDEPENDENT DERIVATIVE HI100

Bdellovibrio bacteriovorus are predatory bacteria that penetrate Gram-negative bacteria and grow intraperiplasmically at the expense of the prey. It was suggested that B. bacteriovorus partially degrade and reutilize lipopolysaccharide (LPS) of the host, thus synthesizing an outer membrane containing structural elements of the prey. According to this hypothesis a host-independent mutant should possess a chemically different LPS. Therefore, the lipopolysaccharides of B. bacteriovorus HD100 and its host-independent derivative B. bacteriovorus HI100 were isolated and characterized by SDS-polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometry. LPS of both strains were identified as smooth-form LPS with different repeating units. The lipid As were isolated after mild acid hydrolysis and their structures were determined by chemical analysis, by mass spectrometric methods, and by NMR spectroscopy. Both lipid As were characterized by an unusual chemical structure, consisting of a β-(1→6)-linked 2,3-diamino-2,3-dideoxy-d-glucopyranose disaccharide carrying six fatty acids that were all hydroxylated. Instead of phosphate groups substituting position O-1 of the reducing and O-4′ of the nonreducing end α-d-mannopyranose residues were found in these lipid As. Thus, they represent the first lipid As completely missing negatively charged groups. A reduced endotoxic activity as determined by cytokine induction from human macrophages was shown for this novel structure. Only minor differences with respect to fatty acids were detected between the lipid As of the host-dependent wild type strain HD100 and for its host-independent derivative HI100. From the results of the detailed analysis it can be concluded that the wild type strain HD100 synthesizes an innate LPS.

Characterization and Scope of S-layer Protein O-Glycosylation in Tannerella forsythia

Background: Bacterial cell surface glycosylation impacts virulence. Result: Surface layer glycans from T. forsythia are O-linked oligosaccharides that modify also multiple other T. forsythia proteins. Conclusion: A general protein O-glycosylation system is present in T. forsythia sharing identical sequon requirements as other Bacteroides species. Significance: Systematic protein O-glycosylation may affect the biology of T. forsythia. Cell surface glycosylation is an important element in defining the life of pathogenic bacteria. Tannerella forsythia is a Gram-negative, anaerobic periodontal pathogen inhabiting the subgingival plaque biofilms. It is completely covered by a two-dimensional crystalline surface layer (S-layer) composed of two glycoproteins. Although the S-layer has previously been shown to delay the bacteriums recognition by the innate immune system, we characterize here the S-layer protein O-glycosylation as a potential virulence factor. The T. forsythia S-layer glycan was elucidated by a combination of electrospray ionization-tandem mass spectrometry and nuclear magnetic resonance spectroscopy as an oligosaccharide with the structure 4-Me-β-ManpNAcCONH2-(1→3)-[Pse5Am7Gc-(2→4)-]-β-ManpNAcA-(1→4)-[4-Me-α-Galp-(1→2)-]-α-Fucp-(1→4)-[-α-Xylp-(1→3)-]-β-GlcpA-(1→3)-[-β-Digp-(1→2)-]-α-Galp, which is O-glycosidically linked to distinct serine and threonine residues within the three-amino acid motif (D)(S/T)(A/I/L/M/T/V) on either S-layer protein. This S-layer glycan obviously impacts the life style of T. forsythia because increased biofilm formation of an UDP-N-acetylmannosaminuronic acid dehydrogenase mutant can be correlated with the presence of truncated S-layer glycans. We found that several other proteins of T. forsythia are modified with that specific oligosaccharide. Proteomics identified two of them as being among previously classified antigenic outer membrane proteins that are up-regulated under biofilm conditions, in addition to two predicted antigenic lipoproteins. Theoretical analysis of the S-layer O-glycosylation of T. forsythia indicates the involvement of a 6.8-kb gene locus that is conserved among different bacteria from the Bacteroidetes phylum. Together, these findings reveal the presence of a protein O-glycosylation system in T. forsythia that is essential for creating a rich glycoproteome pinpointing a possible relevance for the virulence of this bacterium.

Selectivity issues in targeted metabolomics: Separation of phosphorylated carbohydrate isomers by mixed‐mode hydrophilic interaction/weak anion exchange chromatography

Phosphorylated carbohydrates are important intracellular metabolites and thus of prime interest in metabolomics research. Complications in their analysis arise from the existence of structural isomers that do have similar fragmentation patterns in MS/MS and are hard to resolve chromatographically. Herein, we present selective methods for the liquid chromatographic separation of sugar phosphates, such as hexose and pentose phosphates, 2- and 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, as well as glucosamine 1- and 6-phosphate utilizing mixed-mode chromatography with reversed-phase/weak anion-exchangers and a charged aerosol detector. The best results were obtained when the reversed-phase/weak anion-exchanger column was operated under hydrophilic interaction liquid chromatography elution conditions. The effects of various chromatographic parameters were examined and are discussed on the basis of a simple stoichiometric displacement model for explaining ion-exchange processes. Employed acidic conditions have led to the complete separation of α- and β-anomers of glucose 6-phosphate at low temperature. The anomers coeluted in a single peak at elevated temperatures (>40°C) (peak coalescence), while at intermediate temperatures on-column interconversion with a plateau in-between resolved anomer peaks was observed with apparent reaction rate constants between 0.1 and 27.8×10(-4) s(-1). Dynamic HPLC under specified conditions enabled to investigate mutarotation of phosphorylated carbohydrates, their interconversion kinetics, and energy barriers for interconversion. A complex mixture of six hexose phosphate structural isomers could be resolved almost completely.

Biotransformations monitored in situ by proton nuclear magnetic resonance spectroscopy

One-dimensional Fourier-transform proton nuclear magnetic resonance (1H-NMR) spectroscopy can be used to study biotransformations in situ, in vivo and in aqua (1H2O). Although an insensitive method, it rapidly provides solution-structural information of mixtures of diverse compounds that are used and formed during enzymic reactions and culture fermentations; the samples do not require any physical or chemical processing for analysis. The absolute stereochemistry of some reactions can also be determined, and assessments of metabolic fluxes made. This technique, with appropriate modifications, is of obvious value for on-line assessments of industrial fermentation processes.

Acid–base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237→Gln mutant enzymes

The role of acid-base catalysis in the two-step enzymatic mechanism of alpha-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237-->Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Brønsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 10(5)-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding alpha-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed alpha-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/K(m), the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pK(a) values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pK(a) approximately 5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pK(a) approximately 6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid-base of sucrose phosphorylase. They support a mechanism in which the pK(a) of Glu237 cycles between approximately 7.2 in free enzyme and approximately 5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.

A novel concept for detoxification: Complexation between aconitine and liquiritin in a Chinese herbal formula (‘Sini Tang’)

ETHNOPHARMACOLOGICAL RELEVANCE Sini Tang (SNT) is a traditional Chinese herbal formulation consisting of three different herbs: Aconitum carmichaelii (Fuzi), Zingiber officinale (Ganjiang), and Glycyrrhiza uralensis (Gancao). For this study, we modified this mixture by adding the bark of Cinnamomum cassia (Rougui, ). Aconitum carmichaelii contains aconitine and its derivatives, all of which are highly toxic alkaloids. These compounds are commonly detoxified with pyrolytic and hydrolytic pretreatments, such as Heishunpian, which requires repeated soaking in salt water, boiling until the roots turn black, and drying in the oven. We now demonstrate that Glycyrrhiza uralensis, which is often used in Traditional Chinese Medicine for detoxification, reduces the concentration of free aconitine in decoctions by forming a complex between liquiritin and aconitine. MATERIALS AND METHODS Aqueous extracts of SNT, each individual herb or herbal mixture, and methanolic extracts of individual herbs were tested for free aconitine by HPLC coupled with a diode array detector. A detected complex was investigated by NMR and UV/vis spectroscopy. The continuous variations method and (1)H-NMR titrations provided the complex stoichiometry and binding constant. A 2D-ROESY experiment was performed to obtain the structural details of the formed complex. RESULTS A fast and simple HPLC method was developed to determine the amounts of aconitine and its derivatives found in herbal extracts. The Heishunpian pretreatment led to nearly complete pyrolysis and hydrolysis of the toxic compounds. However, in some batches, considerable amounts of aconitine remained. The addition of Glycyrrhiza uralensis to Aconitum carmichaelii, or liquiritin to free aconitine, led to a complexation with aconitine. The complex possessed a 1:1 stoichiometry and a binding constant of ca. 3000 L/mol to 4000 L/mol in mixtures of aqueous methanol. CONCLUSIONS A new HPLC based method allows the concentration of toxic aconitine and other diester diterpene alkaloids in herbal extracts to be rapidly determined. This method provides a starting point for the development of routine quality control procedures. The complexation of free aconitine by adding an excess of Glycyrrhiza uralensis or free liquiritin to SNT formulations will make these formulations safer.

Online NMR for monitoring biocatalysed reactions.

Monitoring biocatalysed reactions and metabolic pathways using NMR spectroscopy is of growing interest. As a non-invasive analytical method providing simultaneous information about intracellular and extracellular constituents, it is superior to other analytical techniques and has a wide range of applications: kinetics and stoichiometrics of metabolic events, metabolic fluxes and enzyme activities can be detected in situ or after taking a sample from the biotransformation mixture. New NMR pulse sequences provide even more valuable experiments in these fields. Research topics range from the monitoring of polymer formation to fermentations producing beverages or antibiotics. Routine monitoring of industrial fermentations by NMR seems to be imminent.

Enzyme identification and development of a whole-cell biotransformation for asymmetric reduction of o-chloroacetophenone

Chiral 1‐(o‐chlorophenyl)‐ethanols are key intermediates in the synthesis of chemotherapeutic substances. Enantioselective reduction of o‐chloroacetophenone is a preferred method of production but well investigated chemo‐ and biocatalysts for this transformation are currently lacking. Based on the discovery that Candida tenuis xylose reductase converts o‐chloroacetophenone with useful specificity (kcat/Km = 340 M−1 s−1) and perfect S‐stereoselectivity, we developed whole‐cell catalysts from Escherichia coli and Saccharomyces cerevisiae co‐expressing recombinant reductase and a suitable system for recycling of NADH. E. coli surpassed S. cerevisiae sixfold concerning catalytic productivity (3 mmol/g dry cells/h) and total turnover number (1.5 mmol substrate/g dry cells). o‐Chloroacetophenone was unexpectedly “toxic,” and catalyst half‐life times of only 20 min (E. coli) and 30 min (S. cerevisiae) in the presence of 100 mM substrate restricted the time of batch processing to maximally ∼5 h. Systematic reaction optimization was used to enhance the product yield (≤60%) of E. coli catalyzed conversion of 100 mM o‐chloroacetophenone which was clearly limited by catalyst instability. Supplementation of external NAD+ (0.5 mM) to cells permeabilized with polymyxin B sulfate (0.14 mM) resulted in complete conversion providing 98 mM S‐1‐(o‐chlorophenyl)‐ethanol. The strategies considered for optimization of reduction rate should be generally useful, however, especially under process conditions that promote fast loss of catalyst activity. Biotechnol. Bioeng. 2011; 108:797–803.

Mechanistic differences among retaining disaccharide phosphorylases : insights from kinetic analysis of active site mutants of sucrose phosphorylase and α,α-trehalose phosphorylase

Sucrose phosphorylase utilizes a glycoside hydrolase-like double displacement mechanism to convert its disaccharide substrate and phosphate into alpha-d-glucose 1-phosphate and fructose. Site-directed mutagenesis was employed to characterize the proposed roles of Asp(196) and Glu(237) as catalytic nucleophile and acid-base, respectively, in the reaction of sucrose phosphorylase from Leuconostoc mesenteroides. The side chain of Asp(295) is suggested to facilitate the catalytic steps of glucosylation and deglucosylation of Asp(196) through a strong hydrogen bond (23 kJ/mol) with the 2-hydroxyl of the glucosyl oxocarbenium ion-like species believed to be formed in the transition states flanking the beta-glucosyl enzyme intermediate. An assortment of biochemical techniques used to examine the mechanism of alpha-retaining glucosyl transfer by Schizophyllum commune alpha,alpha-trehalose phosphorylase failed to provide evidence in support of a similar two-step catalytic reaction via a covalent intermediate. Mutagenesis studies suggested a putative active-site structure for this trehalose phosphorylase that is typical of retaining glycosyltransferases of fold family GT-B and markedly different from that of sucrose phosphorylase. While ambiguity remains regarding the chemical mechanism by which the trehalose phosphorylase functions, the two disaccharide phosphorylases have evolved strikingly different reaction coordinates to achieve catalytic efficiency and stereochemical control in their highly analogous substrate transformations.