Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1.

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35 degrees C and 8, respectively. The apparent Km for IDA was 4.0 mM with a kcat of 5.3 s(-1). When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.

Evidence for a chemiosmotic model of dehalorespiration in Desulfomonile tiedjei DCB-1.

Desulfomonile tiedjei DCB-1, a sulfate-reducing bacterium, conserves energy for growth from reductive dehalogenation of 3-chlorobenzoate by an uncharacterized chemiosmotic process. Respiratory electron transport components were examined in D. tiedjei cells grown under conditions for reductive dehalogenation, pyruvate fermentation, and sulfate reduction. Reductive dehalogenation was inhibited by the respiratory quinone inhibitor 2-heptyl-4-hydroxyquinoline N-oxide, suggesting that a respiratory quinoid is a component of the electron transport chain coupled to reductive dehalogenation. Moreover, reductive dehalogenation activity was dependent on 1, 4-naphthoquinone, a possible precursor for a respiratory quinoid. However, no ubiquinone or menaquinone could be extracted from D. tiedjei. Rather, a UV-absorbing quinoid which is different from common respiratory quinones in chemical structure according to mass spectrometric and UV absorption spectroscopic analyses was extracted. ATP sulfurylase, adenosine phosphosulfate reductase, and desulfoviridin sulfite reductase, enzymes involved in sulfate reduction, were constitutively expressed in the cytoplasm of D. tiedjei cells grown under all three metabolic conditions. A periplasmic hydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. A membrane-bound, periplasm-oriented formate dehydrogenase was detected only in cells grown with formate as electron donor, while a cytoplasmic formate dehydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. Results from dehalogenation assays with D. tiedjei whole-cell suspensions and cell extracts suggest that the membrane-bound reductive dehalogenase is cytoplasm oriented. The data clearly demonstrate an enzyme topology in D. tiedjei which produces protons directly in the periplasm, generating a proton motive force by a scalar mechanism.

Purification, characterization and gene sequence analysis of a novel cytochrome c co-induced with reductive dechlorination activity in Desulfomonile tiedjei DCB-1.

The sulfate-reducing bacterium, Desulfomonile tiedjei DCB-1, conserves energy for growth from reductive dechlorination of 3-chlorobenzoate via halorespiration. To understand this respiratory process better, we examined electron carriers from different cellular compartments of D. tiedjei. A 50-kDa cytochrome from the membrane fraction was found to be co-induced with dechlorination activity. This inducible cytochrome was extracted from the membrane fractions by Tris-HCl buffer containing ammonium sulfate at 35% saturation and was purified to electrophoretic homogeneity by phenyl superose, Mono Q, and hydroxyapatite chromatography. The purified cytochrome had a high-spin absorption spectrum. In a pH titration experiment, the absorption spectrum of the inducible cytochrome shifted to low spin at pH 13.2. The midpoint potential of the inducible cytochrome at pH 7.0 was -342 mV. The NH2-terminal amino acid sequence of the inducible cytochrome was determined and was used to obtain inverse PCR products containing the sequence of the gene encoding the inducible cytochrome. The ORF was 1398 bp and coded for a protein of 52.6 kDa. Two c-type heme-binding domains were identified in the COOH-terminal half of the protein. A putative signal peptide of 26 residues was found at the NH2-terminal end. The protein sequence was not found to have substantial sequence similarity to any other sequence in GenBank. We conclude that this is a c-type cytochrome substantially different from previously characterized c-type cytochromes.