Cloning and sequencing of the genes of 2‐hydoxyglutaryl‐CoA dehydratase from <i>Acidaminococcus fermentans</i>

Dutscho, Roland; Wohlfarth, Gert; Buckel, Peter

doi:10.1111/j.1432-1033.1989.tb14786.x

Cited by 22 publications

(17 citation statements)

References 25 publications

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“…The deduced amino acid sequences of the BadD, E, F, and G proteins are notable for their lack of similarity to all characterized proteins in genome databases except one: 2-hydroxyglutaryl-CoA dehydratase, an enzyme responsible for the reversible dehydration of (R)-2-hydroxyglutaryl-CoA to glutaconyl-CoA in the pathway of glutamate fermentation by the bacterium Acidaminococcus fermentans (29,31). The reaction catalyzed is unusual because it involves removal of hydrogen atoms from an unactivated carbon atom.…”

Section: Resultsmentioning

confidence: 99%

A cluster of bacterial genes for anaerobic benzene ring biodegradation

Egland

Pelletier

Dispensa

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

156

226

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A reductive benzoate pathway is the central conduit for the anaerobic biodegradation of aromatic pollutants and lignin monomers. Benzene ring reduction requires a large input of energy and this metabolic capability has, so far, been reported only in bacteria. To determine the molecular basis for this environmentally important process, we cloned and analyzed genes required for the anaerobic degradation of benzoate and related compounds from the phototrophic bacterium, Rhodopseudomonas palustris. A cluster of 24 genes was identified that includes twelve genes likely to be involved in anaerobic benzoate degradation and additional genes that convert the related compounds 4-hydroxybenzoate and cyclohexanecarboxylate to benzoyl-CoA. Genes encoding benzoylCoA reductase, a novel enzyme able to overcome the resonance stability of the aromatic ring, were identified by directed mutagenesis. The gene encoding the ring-cleavage enzyme, 2-ketocyclohexanecarboxyl-CoA hydrolase, was identified by assaying the enzymatic activity of the protein expressed in Escherichia coli. Physiological data and DNA sequence analyses indicate that the benzoate pathway consists of unusual enzymes for ring reduction and cleavage interposed among enzymes homologous to those catalyzing fatty acid degradation. The cloned genes should be useful as probes to identify benzoate degradation genes from other metabolically distinct groups of anaerobic bacteria, such as denitrifying bacteria and sulfate-reducing bacteria.

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Section: Resultsmentioning

confidence: 99%

A cluster of bacterial genes for anaerobic benzene ring biodegradation

Egland

Pelletier

Dispensa

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

156

226

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“…The first inverted repeat is located 10 bases, the other 122 bases behind the stop codon of hgdB, each followed by an oligo(dT) sequence (Dutscho et al, 1989). These inverted repeats resemble p-independent terminators and could act as terminators of transcription for a whole glutamate operon containing different proteins of the hydroxyglutarate pathway (Buckel, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…1 shows the relative location of gcdA, h g W B and the OW. The fact that two adjacent terminator structures are located downstream of hgdB (Dutscho et al, 1989) implies that at least these three genes might form a transcription unit. Since the genomic EMBL12 library was based on partially restricted genomic DNA of A. fermentans, it was necessary to test the isolated EMBL12 clone for undesired ligation events between different genomic fragments during construc- Shine and Dalgarno, 1975) as well as the binding sites of the oligonucleotides (Fig.…”

Section: Sequencing Of the Flanking Regionsmentioning

confidence: 99%

Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin‐dependent Na⁺ pump glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli

Bendrat

1993

European Journal of Biochemistry

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1. The primary sodium-ion pump glutaconyl-CoA decarboxylase (GCD) from Acidaminococcus femzentans is composed of four subunits: GCDA, the carboxytransferase (65 m a ) , GCDB, the carboxylyase (36 m a ) , GCDC, the biotin carrier (24 kDa) and GCDD (14 kDa) of unknown function. A genomic library of A. femzentans was screened with an antiserum raised against whole GCD. A clone giving the strongest reaction in an immunoassay contained a 12-kbp genomic fragment from A. femzentans and was analysed further. An oligonucleotide deduced from the N-terminus of GCDA was used for probing the corresponding gene gcdA. It is 1761 bp in length and encodes for a protein of 64.3 kDa. Both partial amino acid sequences obtained from GCDA, the N-terminus as well as an internal tryptic peptide, were detected in the open reading frame (ORF) of gcdA.2. Sequencing of the flanking regions revealed three adjacent ORF (ORF1-3) which do not code for any of the peptide sequences known of the other GCD subunits. The ORF downstream of gcdA (ORF3) is followed by hgdA and hgdB coding for 2-hydroxyglutaryl-CoA dehydratase, the preceeding enzyme of the pathway of glutamate fermentation. Our results suggest that at least these three genes of the hydroxyglutarate pathway are organised in an operon and that the genes of the other GCD subunits from which peptide sequences are known (GCDB and GCDC) are not located adjacent to gcdA.3. gcdA was amplified from genomic DNA using the polymerase chain reaction and cloned into the expression vector pJFll8HE. Active GCDA subunit (up to 2.8 nkat/mg protein), catalysing the biotin-dependent formation of crotonyl-CoA from glutaconyl-CoA, was obtained in cell-free extracts of Escherichia coli DH5a by moderately inducing the tac promoter of pJFll8HE with 25 -100 pM isopropyl-1-thio-P-D-galactoside. Strong induction (1 mM isopropyl-1-thio-P-D-galactoside) led to the formation of inclusion bodies from which GCDA could not be reactivated. The apparent K, = 51 mM for free biotin of the expressed GCDA subunit with V = 1.9 nkat/mg protein is similar to that of butanol-treated GCD composed of GCDA and GCDC (apparent K, = 40mM). Biocytin was found to be a somewhat better carboxy acceptor for the expressed GCDA subunit (apparent K, = 13 mM; V = l.Onkat/mg protein).4. Native GCD and expressed GCDA were treated with 2 mM N-ethylmaleimide showing different kinetics of inactivation: GCD lost half of its activity within 6 min, whereas expressed GCDA required 21 min.

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“…In fact, the deduced N-terminal amino acid sequences from the R. palustris genes show a high degree of identity to the N-termini of the benzoyl-CoA reductase subunits (Harwood, C. S., personal communication). In addition, the overall deduced amino acid sequences show considerable similarity to those of the subunits of 2-hydroxyglutaryl-CoA dehydratase and the activating enzyme from A. fermentam (Dutscho et al, 1989;Bendrat et al, 1993;Egland and Harwood, 1996).…”

Section: -Fluorobenzoate For Azoarcus Evansii) Direct Reduction Of Thementioning

confidence: 91%

Anaerobic Metabolism of Aromatic Compounds

Heider

Fuchs

1997

European Journal of Biochemistry

285

214

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Aromatic compounds comprise a wide variety of low-molecular-mass natural compounds (amino acids, quinones, flavonoids, etc.) and biopolymers (lignin, melanin). They are almost exclusively degraded by microorganisms. Aerobic aromatic metabolism is characterised by the extensive use of molecular oxygen. Monoxygenases and dioxygenases are essential for the hydroxylation and cleavage of aromatic ring structures. Accordingly, the characteristic central intermediates of the aerobic pathways (e.g. catechol) are readily attacked oxidatively. Anaerobic aromatic catabolism requires, of necessity, a quite different strategy. The basic features of this metabolism have emerged from studies on bacteria that degrade soluble aromatic substrates to CO, in the complete absence of molecular oxygen.Essential to anaerobic aromatic metabolism is the replacement of all the oxygen-dependent steps by an alternative set of novel reactions and the formation of different central intermediates (e.g. benzoylCoA) for breaking the aromaticity and cleaving the ring; notably, in anaerobic pathways, the aromatic ring is reduced rather than oxidised. The two-electron reduction of benzoyl-CoA to a cyclic diene requires the cleavage of two molecules of ATP to ADP and P, and is catalysed by benzoyl-CoA reductase. After nitrogenase, this is the second enzyme known which overcomes the high activation energy required for reduction of a chemically stable bond by coupling electron transfer to the hydrolysis of ATP. The alicyclic product cyclohex-I ,5-diene-l-carboxyl-CoA is oxidised to acetyl-CoA via a modified P-oxidation pathway ; the ring structure is opened hydrolytically. Some phenolic compounds are anaerobically transformed to resorcinol (1,3-dihydroxybenzene) or phloroglucinol (1,3,5-trihydroxybenzene). These intermediates are also first reduced and then as alicyclic products oxidised to acetyl-CoA.This review gives an outline of the anaerobic pathways which allow bacteria to utilize aromatics even in the absence of oxygen. We focus on previously unknown reactions and on the enzymes characteristic for such novel metabolism.Keywords ; aromatic metabolism ; benzoyl-CoA ; anaerobic bacteria ; aromatic-ring reduction ; phloroglucinol; resorcinol.This review deals with the art of degrading aromatic compounds to CO, in the complete absence of molecular oxygen. This ability allows bacteria to make use of natural compounds of this widespread class as substrates for growth under anoxic conditions. In nature, anoxic conditions are created when oxygen consumption exceeds its supply. This situation prevails in many environments, e.g. in the intestinal tract of animals and man, in the sediments of all natural bodies of water, in ground water and in part in soil. Aromatic compounds are produced or transformed by all organisms, plants being the most prolific producers. There are numerous low-molecular-mass aromatic compounds includ-

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Cloning and sequencing of the genes of 2‐hydoxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans

Cited by 22 publications

References 25 publications

A cluster of bacterial genes for anaerobic benzene ring biodegradation

A cluster of bacterial genes for anaerobic benzene ring biodegradation

Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin‐dependent Na⁺ pump glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli

Anaerobic Metabolism of Aromatic Compounds

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Cloning and sequencing of the genes of 2‐hydoxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans

Cited by 22 publications

References 25 publications

A cluster of bacterial genes for anaerobic benzene ring biodegradation

A cluster of bacterial genes for anaerobic benzene ring biodegradation

Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin‐dependent Na+ pump glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli

Anaerobic Metabolism of Aromatic Compounds

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Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin‐dependent Na⁺ pump glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli