Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli

Skinner, Deborah D.; Morgenstern, Margaret R.; Fedechko, Ronald; Denoya, Claudio D.

doi:10.1128/jb.177.1.183-190.1995

Cited by 26 publications

(18 citation statements)

References 69 publications

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“…mdeB was not clustered with structural genes for other components of the complex. This is a remarkable result, because all other known genes encoding E1 components of ␣-keto acid dehydrogenase complexes which have been identified so far occur together with the respective genes for the E2 components as operons or gene clusters (10,34,37,38). The only exception to this is the pdhE gene from A. eutrophus, which was recently reported by Hein and Steinbüchel (9).…”

Section: Discussionmentioning

confidence: 86%

Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida

et al. 1997

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A 15-kb region of Pseudomonas putida chromosomal DNA containing the mde operon and an upstream regulatory gene (mdeR) has been cloned and sequenced. The mde operon contains two structural genes involved in L-methionine degradative metabolism: the already-identified mdeA, which encodes L-methionine ␥-lyase (H. Inoue, K. Inagaki, M. Sugimoto, N. Esaki, K. Soda, and H. Tanaka. J. Biochem. (Tokyo) 117:1120-1125, 1995), and mdeB, which encodes a homologous protein to the homodimeric-type E1 component of pyruvate dehydrogenase complex. A rho-independent terminator was present just downstream of mdeB, and open reading frames corresponding to other components of ␣-keto acid dehydrogenase complex were not found. When MdeB was overproduced in Escherichia coli, the cell extract showed the E1 activity with high specificity for ␣-ketobutyrate rather than pyruvate. These results suggest that MdeB plays an important role in the metabolism of ␣-ketobutyrate produced by MdeA from L-methionine. Accordingly, mdeB encodes a novel E1 component, ␣-ketobutyrate dehydrogenase E1 component, of an unknown ␣-keto acid dehydrogenase complex in P. putida. In addition, we found that the mdeR gene was located on the opposite strand and began at 127 bp from the translational start site of mdeA. The mdeR gene product has been identified as a member of the leucineresponsive regulatory protein (Lrp) family and revealed to act as an essential positive regulator allowing the expression of the mdeAB operon.Methionine plays a central role in the metabolism of sulfur amino acids. Many bacteria and eukaryotes catabolize L-methionine through ␣-ketobutyrate by three main pathways (36): (i) conversion of methionine to cystathionine through S-adenosylmethionine and homocysteine and then to cysteine, ␣-ketobutyrate, and ammonia; (ii) deamination to ␣-keto-␥-methylthiobutyrate and the subsequent dethiomethylation to ␣-ketobutyrate; and (iii) simultaneous deamination and dethiomethylation to ␣-ketobutyrate by L-methionine ␥-lyase.L-Methionine ␥-lyase (EC 4.4.1.11), a pyridoxal 5Ј-phosphate-dependent enzyme, catalyzes the direct conversion of L-methionine into ␣-ketobutyrate, methanethiol, and ammonia. This enzyme has been demonstrated to be present in various bacteria, such as Pseudomonas putida (27,40), Aeromonas sp. (26), and Clostridium sporogenes (16), and in the parasite Trichomonas vaginalis (20). L-Methionine ␥-lyase is induced by the addition of L-methionine to the medium and is regarded as a key enzyme in methionine catabolism. ␣-Ketobutyrate, a main product of L-methionine catabolism, is converted to propionyl-coenzyme A by pyruvate dehydrogenase complex (2, 19) or to ␣-aceto-␣-hydroxybutyrate with pyruvate by acetolactate synthase, which is the isoleucine biosynthetic enzyme (6, 41). It has been proposed that high ␣-ketobutyrate levels interfere with a number of metabolic pathways by several mechanisms (5, 41). Therefore, a study of the L-methionine catabolism pathway (iii above) should be considered along with a study of the metabolism of ␣-ketobu...

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Section: Discussionmentioning

confidence: 86%

Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida

et al. 1997

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“…The sequence obtained was compared to the GenBank database. These data revealed the insertion of Tn1409C␤ 59 bp upstream of a gene homologous to branched-chain ␣-keto acid dehydrogenases (5,28) in mutant A27. From another mutant Arthrobacter strain, SU A1, the hybridizing 4.8-kb SphI fragment was cloned in an analogous procedure (pKGT1000).…”

Section: Resultsmentioning

confidence: 99%

Isolation and Characterization of IS 1409 , an Insertion Element of 4-Chlorobenzoate-Degrading Arthrobacter sp. Strain TM1, and Development of a System for Transposon Mutagenesis

Gartemann

Eichenlaub

2001

J Bacteriol

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Insertion elements (IS elements) are small mobile non-selfreplicating DNA regions specifying only the gene(s) required for their transposition (16). According to the phylogenetic relationship of the transposases and some features involved in the transposition (e.g., the inverted repeats which constitute the ends of most elements, as well as the recognition sites of the transposase and the target duplication caused by most elements), they can be grouped in different families (16). At least for some IS elements, a target-sequence-dependent site preference has been demonstrated, while members of other families apparently transpose in a random fashion. They are widespread in bacteria and contribute to the plasticity of bacterial genomes due to their transposition ability and to their role as target sites for homologous recombination, which can give rise to deletions, inversions, or more complex rearrangements (20). Some composite transposons derived from IS elements, in addition to the genes required for transposition, harbor genes encoding resistances to antibiotics or degradative pathways.Arthrobacter, a very common soil bacterium, and related genera like Micrococcus form one of the major branches of the actinomycetes, the Micrococcaceae (30). Some Arthrobacter strains are known to degrade aromatic compounds as well as chlorinated aromatic compounds such as 4-chlorobenzoate (4-CBA). Hydrolytic dehalogenation of 4-CBA by Arthrobacter sp. strain SU DSM20407 requires three genes organized in an operon which maps on plasmid pASU1 (25). The same set of genes is also present in Arthrobacter sp. strain TM1 (17). In the course of cloning and sequencing of these genes (unpublished data), we identified an IS element upstream of the dehalogenase operon in Arthrobacter sp. strain TM1. So far, only one other IS element in Arthrobacter and the Micrococcaceae, IS1473 from Arthrobacter nicotinovorans (which belongs to the IS3 family), has been described (19). A growing number of genes specifying degradation of aromatic compounds are known to be located on or associated with transposable elements (for recent reviews see references 31 and 41), with the best known examples being the very large catabolic transposons Tn4651 and Tn4653 on the TOL plasmids of pseudomonads. Thus, it seems possible that the genes for the hydrolytic dehalogenation of 4-CBA are located on a transposon.In order to demonstrate that IS1409 is a functional transposable element, a series of transposons were constructed based on a slightly modified IS1409 and carrying antibiotic resistance genes. These transposons are the first which can be used for transposon mutagenesis in Arthrobacter. MATERIALS AND METHODSBacterial strains, plasmids, culture media, and antibiotics. Bacterial strains and plasmids used in this study are listed in Table 1. 4-CBA-degrading strains were grown at 28°C in a minimal medium as described by Seiler (27) at pH 8.0, supplemented with 1 g of 4-CBA/liter as the only carbon and energy source and 0.2 ml of trace element solution/liter (9). Al...

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“…show significant similarity (28 to 42% identity) to the known heterotetrameric E1 ␣-subunits of the BCOADHC from B. subtilis (69), S. avermitilis (60), P. putida (6), T. thermophilus (36), and M. musculus (10). All these BCOADHC E1 ␣-proteins cluster with the PDHC E1 ␣-proteins of the low-GC-content gram-positive bacteria (Fig.…”

Section: Resultsmentioning

confidence: 99%

E1 Enzyme of the Pyruvate Dehydrogenase Complex in Corynebacterium glutamicum : Molecular Analysis of the Gene and Phylogenetic Aspects

et al. 2005

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The E1p enzyme is an essential part of the pyruvate dehydrogenase complex (PDHC) and catalyzes the oxidative decarboxylation of pyruvate with concomitant acetylation of the E2p enzyme within the complex. We analyzed the Corynebacterium glutamicum aceE gene, encoding the E1p enzyme, and constructed and characterized an E1p-deficient mutant. Sequence analysis of the C. glutamicum aceE gene and adjacent regions revealed that aceE is not flanked by genes encoding other enzymes of the PDHC. Transcriptional analysis revealed that aceE from C. glutamicum is monocistronic and that its transcription is initiated 121 nucleotides upstream of the translational start site. Inactivation of the chromosomal aceE gene led to the inability to grow on glucose and to the absence of PDHC and E1p activities, indicating that only a single E1p enzyme is present in C. glutamicum and that the PDHC is essential for the growth of this organism on carbohydrate substrates. Surprisingly, the E1p enzyme of C. glutamicum showed up to 51% identity to homodimeric E1p proteins from gram-negative bacteria but no similarity to E1 ␣-or ␤-subunits of heterotetrameric E1p enzymes which are generally assumed to be typical for gram-positives. To investigate the distribution of E1p enzymes in bacteria, we compiled and analyzed the phylogeny of 46 homodimeric E1p proteins and of 58 ␣-subunits of heterotetrameric E1p proteins deposited in public databases. The results revealed that the distribution of homodimeric and heterotetrameric E1p subunits in bacteria is not in accordance with the rRNA-based phylogeny of bacteria and is more heterogeneous than previously assumed.The pyruvate dehydrogenase complex (PDHC) represents a member of a multienzyme complex family that also comprises the 2-oxoglutarate dehydrogenase complex (OGDHC) and the branched-chain 2-oxoacid dehydrogenase complex (BCOADHC). These enzymes catalyze the oxidative decarboxylation of pyruvate, 2-oxoglutarate, and the 2-oxo acids of the branched-chain amino acids L-leucine, L-valine, and L-isoleucine, respectively. In general, the multienzyme complexes are composed of multiple copies of three different enzymes, a thiamine pyrophosphate (TPP) containing 2-oxoacid decarboxylase (E1), a lipoic acid-containing dihydrolipoamide acyltransferase (E2), and the flavin-containing lipoamide dehydrogenase (LPD). The E1 enzyme catalyzes the irreversible, TPP-dependent oxidative decarboxylation of the 2-oxoacid, followed by the acylation of the lipoyl prosthetic group covalently attached to the E2 chain. The E2 component catalyzes the transfer of the acyl group from the lipoyl group to coenzyme A (CoA). The resulting dihydrolipoyl group is reoxidized by LPD, generating NADH and H ϩ from NAD ϩ (for a recent review, see reference 11). The E1 and E2 enzymes are specific for each of the three multienzyme complexes and therefore specified as E1p and E2p in the PDHC, E1o and E2o in the OGDHC, and E1b, and E2b in the BCOADHC. In contrast, the LPD component is common in the three multienzyme complexes in most organ...

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Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli

Cited by 26 publications

References 69 publications

Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida

Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida

Isolation and Characterization of IS 1409 , an Insertion Element of 4-Chlorobenzoate-Degrading Arthrobacter sp. Strain TM1, and Development of a System for Transposon Mutagenesis

E1 Enzyme of the Pyruvate Dehydrogenase Complex in Corynebacterium glutamicum : Molecular Analysis of the Gene and Phylogenetic Aspects

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