Novel aerobic 2-aminobenzoate metabolism. Nucleotide sequence of the plasmid carrying the gene for the flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase in a denitrifying Pseudomonas sp.

Pseudomonas KB 740 degrades 2-aminobenzoate aerobically via a chimeric pathway which combines characteristics of anaerobic and aerobic aromatic metabolism. Atypically, 2-aminobenzoyl-CoA is an intermediate, and the activated aromatic acid is not only hydroxylated but also reduced to an alicyclic compound in a single step. The bacterial strain possesses a small plasmid, pKB 740, which carries all essential information of this new pathway. Its total nucleotide sequence was determined. It consists of 8280 bp and contains the genes for the two initial enzymes of the pathway; 2-aminobenzoate-CoA ligase catalyzes the activation of the aromatic acid, and the flavoenzyme 2-aminobenzoyl-CoA monooxygenase/reductase catalyzes the hydroxylation (monooxygenase activity) and subsequent reduction (reductase activity) of the aromatic ring of 2-aminobenzoyl-CoA. Furthermore, five open reading frames (ORF) possibly coding for polypeptides are on the plasmid. Putative promoter sequences were found for two of the ORF. A nucleotide sequence able to form a possible termination loop was located downstream of the gene for 2-aminobenzoyl-CoA monooxygenase/reductase. This gene consists of 2190 bases. The deduced amino acid sequence of the protein (730 residues; calculated molecular mass of the native 729-residue protein, 83,559 Da) contains a consensus sequence for an FAD-binding site at the N-terminus and a possible NAD(P)H-binding site approximately 150 amino acid residues apart from the N-terminus. The monooxygenase/reductase shows low sequence similarity to the flavoprotein salicylate hydroxylase. Functional and evolutionary aspects of this work are discussed.

Cloning and nucleotide sequence of the psrA gene of Wolinella succinogenes polysulphide reductase.

The polysulphide reductase (formerly sulphur reductase) of Wolinella succinogenes is a component of the phosphorylative electron transport system with polysulphide as the terminal acceptor. Using an antiserum raised against the major subunit (PsrA, 85 kDa) of the enzyme, the corresponding gene (psrA) was cloned from a lambda-gene bank. The N-terminal amino acid sequence of PsrA mapped within the psrA gene product, which also contained an apparent signal peptide. Downstream of the psrA gene two more open reading frames (psrB and psrC) were found. The three genes may form a transcriptional unit with the transcription start site in front of psrA. The three genes were present only once on the genome. PsrA is a hydrophilic protein homologous to the largest subunits of six prokaryotic molybdoenzymes. PsrB is predicted to be hydrophilic, to contain ferredoxin-like cysteine clusters and to be homologous to the smaller hydrophilic subunits of four molybdoenzymes. PsrC is predicted to be a hydrophobic protein that could possibly serve as the membrane anchor of the enzyme.

Cloning and nucleotide sequence of the structural genes encoding the formate dehydrogenase of Wolinella succinogenes.

The formate dehydrogenase of Wolinella succinogenes is a membraneous molybdo-enzyme which is involved in phosphorylative electron transport. The gene (fdhA) encoding the largest subunit was isolated from a gene bank by immunological screening. The fdhA gene was located in an apparent transcriptional unit (fdhA,B,C,D) together with three more structural genes. The N-terminal sequences of three polypeptides present in the isolated enzyme were found to map within the fdhA, B and C structural genes. A polypeptide corresponding to fdhD was not detected in the enzyme preparation. This suggested that the functional formate dehydrogenase was made up of three or four different subunits. The genes fdhA and C encode larger preproteins which differ from the corresponding mature proteins by N-terminal signal peptides. The N-terminal half of the mature FdhA is homologous to the larger subunits of the formate dehydrogenases of E. coli (formate-hydrogenlyase linked) and Methanobacterium formicicum as well as to three bacterial reductases containing molybdenum. It harbours a conserved cysteine cluster and two more domains which may be involved in binding the molybdenum cofactor. FdhB may represent an iron-sulphur protein, twelve cysteine residues of which are arranged in two clusters which are typical of ligands of the iron-sulfur centers in ferredoxins. FdhC is a hydrophobic protein with four predicted transmembrane segments, which appears to be identical with the cytochrome b present in the isolated formate dehydrogenase. It may form the membrane anchor of the enzyme and react with the bacterial menaquinone.

Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg). Purity, activity and novel inhibitors.

Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg) was purified to a stage where, besides the alpha, beta and gamma subunits, no additional polypeptides were detectable in the preparation. Under appropriate conditions the enzyme was found to catalyze the reduction of methyl-CoM with 7-mercaptoheptanoylthreonine phosphate (H-S-HTP) to CH4 at a specific rate of 2.5 mumol.min-1.mg protein-1. This finding contradicts a recent report that methyl-CoM reductase is only active when some contaminating proteins are present. The two polypeptides encoded by the open reading frames ORF1 and ORF2 of the methyl-CoM reductase transcription unit did not co-purify with the alpha, beta and gamma subunits. They were neither required nor did they stimulate the activity under the assay conditions. 3-Bromopropanesulfonate (apparent Ki = 0.05 microM) and 2-azidoethanesulfonate (apparent Ki = 1 microM) were found to be two new competitive inhibitors of methyl-CoM reductase. Both inhibitors were considerably more effective than the "classical" 2-bromoethanesulfonate (apparent Ki = 4 microM).

Conserved gene structures and expression signals in methanogenic archaebacteria.

A comparative analysis of cotranscribed gene clusters comprising the structural genes mcrA, mcrB, mcrC, mcrD, and mcrG was carried out in three species of methanogens. mcrA, mcrB, and mcrG are the structural genes for the three subunits of methyl coenzyme M reductase, while the two other genes encode polypeptides of unknown functions. The degree of conservation of the mcr gene products among different species of methanogens varies. No correlation was found between the conservation of the G+C contents of the homologous genes and of the amino acid sequences of their products among the different bacteria. The comparison of RNA polymerase core subunit genes of Methanobacterium thermoautotrophicum as evolutionary markers with their equivalents in Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster showed that homologous polypeptide domains are encoded by different numbers of genes suggesting gene fusion of adjacent genes in the course of evolution. The archaebacterial subunits exhibit much stronger homology with their eukaryotic than with their eubacterial equivalents on the polypeptide sequence level. All the analyzed genes are preceded by ribosome binding sites of eubacterial type. In addition to known putative promoter sequences, conserved structural elements of the DNA were detected surrounding the transcription initiation sites of the mcr genes.

Comparative analysis of genes encoding methyl coenzyme M reductase in methanogenic bacteria.

The sequence of the gene cluster encoding the methyl coenzyme M reductase (MCR) in Methanococcus voltae was determined. It contains five open reading frames (ORF), three of which encode the known enzyme subunits. Putative ribosome binding sites were found in front of all ORFs. They differ in their degrees of complementarity to the 3' end of the 16 S rRNA, which is discussed in terms of different translation efficiencies of the respective genes. The codon usage bias is different in the subunit encoding genes compared with the two other ORFs in the cluster and two other known genes of Mc. voltae. This is interpreted in terms of increased translational accuracy of the highly expressed MCR subunit genes. The derived polypeptide sequences encoded by the five ORFs of the MCR cluster were compared to those of the respective genes in Methanobacterium thermoautotrophicum Marburg and Methanosarcina barkeri. Conserved regions were detected in the enzyme subunits, which are candidates for factor binding domains. Conserved hydrophobic sequences found in the alpha and beta subunits are discussed with respect to the membrane association of the enzyme.

Cloning and characterization of the methyl coenzyme M reductase genes from Methanobacterium thermoautotrophicum.

The genes coding for methyl coenzyme M reductase were cloned from a genomic library of Methanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, alpha, beta, and gamma. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes.

Phosphorylation and phosphate-ATP exchange catalyzed by the ATP synthase isolated from Wolinella succinogenes.

The ATP synthase, isolated from Wolinella (formerly Vibrio) succinogenes could be fully incorporated into liposomes without significant cleavage of the enzyme or loss of activity. These proteoliposomes, but not the isolated enzyme, catalyzed phosphate-ATP exchange and the phosphorylation of ADP which was driven by an artificially imposed delta mu H across the liposomal membrane. Phosphorylation driven by light was catalyzed by proteoliposomes containing also bacteriorhodopsin. The three activities were similarly sensitive to protonophores or dicyclohexylcarbodiimide. This sensitivity was similar to that of the electron-transport-driven phosphorylation catalyzed by bacterial membrane vesicles. With a delta mu H value 280 mV to drive phosphorylation the turnover number of the enzyme was in the same order of magnitude as that measured in the electron-transport-driven phosphorylation catalyzed by the bacterial membrane. When the delta mu H was below 150 mV, the phosphorylation activity of the incorporated enzyme was two orders of magnitude slower, and was about as fast as light-driven phosphorylation or as the exchange reaction.

Structural properties of the proteoliposomes catalyzing electron transport from formate to fumarate.

The electron-transport chain catalyzing fumarate reduction by formate has recently been reconstituted from the formate dehydrogenase complex and the fumarate reductase complex from Vibrio succinogenes, in a liposomal preparation containing vitamin K-1 (Unden, G. and Kröger, A. (1982) Biochim. Biophys. Acta 682, 258-263). We have now investigated the structural properties of this preparation. The preparation was found to consist of a homogeneous population of unilamellar proteoliposomes with an average diameter of about 100 nm and an internal volume of 2-4 ml/g phospholipid. The buoyant density (1.07 g/ml) was consistent with the protein/phospholipid ratio (0.2 g/g) of the preparation. Leakage of glucose from the internal spaces of the proteoliposomes was negligibly slow. Proteoliposomes prepared with either of the enzyme complexes showed peripheral projections mainly on the outer surface, when examined by electron microscopy after negative staining. The size, orientation and surface density of the projections were consistent with those of the enzymes. Most of the substrate and dye-reactive sites (70-90%) of the enzymes in the proteoliposomes were accessible to external non-permeant substrates. The proteoliposomes catalyzing electron transport were formed by freeze-thawing a mixture of liposomes and protein-phospholipid complexes which did not perform electron transport from formate to fumarate. Nearly the entire amount of the enzymes supplied (0.2 g protein/g phospholipid) was incorporated into the liposomes by this procedure. The transformation of liposomes into proteoliposomes was accompanied by exchange of the internal solutes with the external medium.