Johann Heider - Academia.edu (original) (raw)

Papers by Johann Heider

function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and... more function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon

Archives of Microbiology, 2021

A novel acyl-CoA dehydrogenase involved in degradation of the auxin indoleacetate by Aromatoleum ... more A novel acyl-CoA dehydrogenase involved in degradation of the auxin indoleacetate by Aromatoleum aromaticum was identified as a decarboxylating benzylmalonyl-CoA dehydrogenase (IaaF). It is encoded within the iaa operon coding for enzymes of indoleacetate catabolism. Using enzymatically produced benzylmalonyl-CoA, the reaction was characterized as simultaneous oxidation and decarboxylation of benzylmalonyl-CoA to cinnamoyl-CoA and CO2. Oxygen served as electron acceptor and was reduced to H2O2, whereas electron transfer flavoprotein or artificial dyes serving as electron acceptors for other acyl-CoA dehydrogenases were not used. The enzyme is homotetrameric, contains an FAD cofactor and is enantiospecific in benzylmalonyl-CoA turnover. It shows high catalytic efficiency and strong substrate inhibition with benzylmalonyl-CoA, but otherwise accepts only a few medium-chain alkylmalonyl-CoA compounds as alternative substrates with low activities. Its reactivity of oxidizing 2-carboxyacy...

This study documents the involvement of ETF in anaerobic toluene metabolism as the physiological ... more This study documents the involvement of ETF in anaerobic toluene metabolism as the physiological electron acceptor for benzylsuccinyl-CoA dehydrogenase. While toluene-degrading denitrifying proteobacteria use a common ETF species, which is also used for other β-oxidation pathways, obligately anaerobic sulfate- or ferric-iron-reducing bacteria use specialized ETF paralogues for toluene degradation. Based on the structure and sequence conservation of these ETFs, they form a new clade that is only remotely related to the previously characterized members of the ETF family. An exhaustive analysis of the available sequences indicated that the protein family consists of several closely related clades of proven or potential electron-bifurcating ETF species and many deeply branching nonbifurcating clades, which either follow the host phylogeny or are affiliated according to functional criteria. ABSTRACT (R)-Benzylsuccinate is the characteristic initial intermediate of anaerobic toluene metab...

Anaerobic Utilization of Hydrocarbons, Oils, and Lipids

The FEBS Journal

Anaerobic toluene degradation proceeds by fumarate addition to produce (R)-benzylsuccinate as fir... more Anaerobic toluene degradation proceeds by fumarate addition to produce (R)-benzylsuccinate as first intermediate, which is further degraded via β-oxidation by five enzymes encoded in the conserved bbs operon. This study characterizes two enzymes of this pathway, (E)-benzylidenesuccinyl-CoA hydratase (BbsH), and (S,R)-2-(α-hydroxybenzyl)succinyl-CoA dehydrogenase (BbsCD) from Thauera aromatica. BbsH, a member of the enoyl-CoA hydratase family, converts (E)-benzylidenesuccinyl-CoA to 2-(α-hydroxybenzyl)succinyl-CoA and was subsequently used in a coupled enzyme assay with BbsCD, which belongs to the short chain dehydrogenases/reductase (SDR) family. The BbsCD crystal structure shows a C2-symmetric heterotetramer consisting of BbsC2 and BbsD2 dimers. BbsD subunits are catalytically active and capable of binding NAD+ and substrate, whereas BbsC subunits represent built-in pseudoenzyme moieties lacking all motifs of the SDR family required for substrate binding or catalysis. Molecular modeling studies predict that the active site of BbsD is specific for conversion of the (S,R)-diastereomer of 2-(α-hydroxybenzyl)succinyl-CoA to (S)-2-benzoylsuccinyl-CoA by hydride transfer to the re-face of NAD+ . Furthermore, BbsC subunits are not engaged in substrate binding and merely serve as scaffold for the BbsD dimer. BbsCD represents a novel clade of related enzymes within the SDR family which adopt a heterotetrameric architecture and catalyse the β-oxidation of aromatic succinate-adducts.

Environmental Microbiology, 2016

The plant hormone auxin (indoleacetate) is anaerobically degraded by the Betaproteobacterium Arom... more The plant hormone auxin (indoleacetate) is anaerobically degraded by the Betaproteobacterium Aromatoleum aromaticum. We report here on a CoA ligase (IaaB) and a CoA-transferase (IaaL) which are encoded in the apparent substrate-induced iaa operon containing genes for indoleacetate degradation. IaaB is a highly specific indoleacetate-CoA ligase which activates indoleacetate to the CoA-thioester immediately after uptake into the cytoplasm. This enzyme only activates indoleacetate and some closely related compounds such as naphthylacetate, phenylacetate and indolepropionate, and is inhibited by high concentrations of substrates, and by the synthetic auxin compound 2,4-dichlorophenoxyacetate, which does not serve as substrate. IaaL is a CoA-transferase recognizing several C4-dicarboxylic acids, such as succinate, phenylsuccinate or benzylsuccinate and their CoA-thioesters, but only few monocarboxylic acids and no C3-dicarboxylic acids such as benzylmalonate. The enzyme shows no stereospecific discrimation of the benzylsuccinate enantiomers. Moreover, benzylsuccinate is regiospecifically activated to 2-benzylsuccinyl-CoA, whereas phenylsuccinate is converted to an equal mixture of both regioisomers (2- and 3-phenylsuccinyl-CoA). The identification of these two enzymes allows us to set up a modified version of the metabolic pathway of anaerobic indoleacetate degradation and to investigate the sequences databases for the occurrence and distribution of this pathway in other microorgansisms.

JBIC Journal of Biological Inorganic Chemistry

Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from ... more Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleumaromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe–4S cluster (FS0) in the α-subunit, three 4Fe–4S clusters (FS1 to FS3) and a 3Fe–4S cluster (FS4) in the β-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the MoVI to the MoIV state in two subsequent one-electron steps. The electrons are then transferred via the Fe–S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1–FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 m...

International Journal of Molecular Sciences, 2016

Molecular modeling techniques and density functional theory calculations were performed to study ... more Molecular modeling techniques and density functional theory calculations were performed to study the mechanism of enzymatic radical CC coupling catalyzed by benzylsuccinate synthase (BSS). BSS has been identified as a glycyl radical enzyme that catalyzes the enantiospecific fumarate addition to toluene initiating its anaerobic metabolism in the denitrifying bacterium Thauera aromatica, and this reaction represents the general mechanism of toluene degradation in all known anaerobic degraders. In this work docking calculations, classical molecular dynamics (MD) simulations, and DFT+D2 cluster modeling was employed to address the following questions: (i) What mechanistic details of the BSS reaction yield the most probable molecular model? (ii) What is the molecular basis of enantiospecificity of BSS? (iii) Is the proposed mechanism consistent with experimental observations, such as an inversion of the stereochemistry of the benzylic protons, syn addition of toluene to fumarate, exclusive production of (R)-benzylsuccinate as a product and a kinetic isotope effect (KIE) ranging between 2 and 4? The quantum mechanics (QM) modeling confirms that the previously proposed hypothetical mechanism is the most probable among several variants considered, although C-H activation and not CC coupling turns out to be the rate limiting step. The enantiospecificity of the enzyme seems to be enforced by a thermodynamic preference for binding of fumarate in the pro(R) orientation and reverse preference of benzyl radical attack on fumarate in pro(S) pathway which results with prohibitively high energy barrier of the radical quenching. Finally, the proposed mechanism agrees with most of the experimental observations, although the calculated intrinsic KIE from the model (6.5) is still higher than the experimentally observed values (4.0) which suggests that both C-H activation and radical quenching may jointly be involved in the kinetic control of the reaction.

ACS Catalysis

Anaerobic toluene degradation is initiated by adding toluene to fumarate via the glycyl radical e... more Anaerobic toluene degradation is initiated by adding toluene to fumarate via the glycyl radical enzyme benzylsuccinate synthase (BSS). We developed a recombinant production system producing active BSS and present data on the effects of some active-site mutants. A single substitution of Ile617 to Val inside a hydrophobic pocket of the active site leads to an extended substrate range of the variant, which includes conversion of m-xylene, but not of o-or p-xylene. Moreover, a mutation of the conserved Arg508, which participates in fumarate binding, to Lys or Gln decreases or eliminates BSS activity, proving its catalytic importance. Surprisingly, the Arg508Lys mutant gained the ability to synthesize a small amount of the product 3-benzyl-4ketopentanoate from toluene and the fumarate analogue 3-acetyl acrylate. This study provides insight into substrate recognition by BSS and opens avenues for possible broader biotechnological applications in C−C bond formation.

Inorganics

Tungsten is the heaviest element used in biological systems. It occurs in the active sites of sev... more Tungsten is the heaviest element used in biological systems. It occurs in the active sites of several bacterial or archaeal enzymes and is ligated to an organic cofactor (metallopterin or metal binding pterin; MPT) which is referred to as tungsten cofactor (Wco). Wco-containing enzymes are found in the dimethyl sulfoxide reductase (DMSOR) and the aldehyde:ferredoxin oxidoreductase (AOR) families of MPT-containing enzymes. Some depend on Wco, such as aldehyde oxidoreductases (AORs), class II benzoyl-CoA reductases (BCRs) and acetylene hydratases (AHs), whereas others may incorporate either Wco or molybdenum cofactor (Moco), such as formate dehydrogenases, formylmethanofuran dehydrogenases or nitrate reductases. The obligately tungsten-dependent enzymes catalyze rather unusual reactions such as ones with extremely low-potential electron transfers (AOR, BCR) or an unusual hydration reaction (AH). In recent years, insights into the structure and function of many tungstoenzymes have been...

Journal of Bacteriology

Aromatic amines like 2-phenylethylamine (2-PEA) or benzylamine (BAm) have been identified as nove... more Aromatic amines like 2-phenylethylamine (2-PEA) or benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacterium Aromatoleum aromaticum EbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways, respectively. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized. Both enzymes consist of three subunits, contain two heme c cofactors in their α-subunits and exhibit extensive processing of their γ-subunits, generating four intramolecular thioether bonds and a cysteine tryptophylquinone (CTQ) cofactor. One of the enzymes was present in cells grown with 2-PEA or other substrates, showed a...

Frontiers in Microbiology

$Research paper thumbnail of {"__content__"=>"Compatible Solute Synthesis and Import by the Moderate Halophile : Physiology and Genomics.", "i"=>{"__content__"=>"Spiribacter salinus"}}$

Frontiers in microbiology, 2018

Members of the genus are found worldwide and are abundant in ecosystems possessing intermediate s... more Members of the genus are found worldwide and are abundant in ecosystems possessing intermediate salinities between seawater and saturated salt concentrations. M19-40 is the type species of this genus and its first cultivated representative. In the habitats of M19-40, high salinity is a key determinant for growth and we therefore focused on the cellular adjustment strategy to this persistent environmental challenge. We coupled these experimental studies to the mining of the genome sequence of this moderate halophile with respect to systems allowing this bacterium to control its potassium and sodium pools, and its ability to import and synthesize compatible solutes. M19-40 produces enhanced levels of the compatible solute ectoine, both under optimal and growth-challenging salt concentrations, but the genes encoding the corresponding biosynthetic enzymes are not organized in a canonical operon. Instead, they are scrambled (; ) and are physically separated from each other on the M19-40 ...

Genes, Jan 22, 2018

Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats o... more Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyectoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, a...

Environmental Microbiology

Ectoine and hydroxyectoine are effective microbial osmostress protectants, but can also serve as ... more Ectoine and hydroxyectoine are effective microbial osmostress protectants, but can also serve as versatile nutrients for bacteria. We have studied the genetic regulation of ectoine and hydroxyectoine import and catabolism in the marine Roseobacter species Ruegeria pomeroyi and identified three transcriptional regulators involved in these processes: the GabR/MocR-type repressor EnuR, the feast and famine-type regulator AsnC and the two-component system NtrYX. The corresponding genes are widely associated with ectoine and hydroxyectoine uptake and catabolic gene clusters (enuR, asnC), and with microorganisms predicted to consume ectoines (ntrYX). EnuR contains a covalently bound pyridoxal-5&amp;#39;-phosphate as a co-factor and the chemistry underlying the functioning of MocR/GabR-type regulators typically requires a system-specific low molecular mass effector molecule. Through ligand binding studies with purified EnuR, we identified N-(alpha)-L-acetyl-2,4-diaminobutyric acid and L-2,4-diaminobutyric acid as inducers for EnuR that are generated through ectoine catabolism. AsnC/Lrp-type proteins can wrap DNA into nucleosome-like structures, and we found that the asnC gene was essential for use of ectoines as nutrients. Furthermore, we discovered through transposon mutagenesis that the NtrYX two-component system is required for their catabolism. Database searches suggest that our findings have important ramifications for an understanding of the molecular biology of most microbial consumers of ectoines.