The sulfonated osmolyte N-methyltaurine is dissimilated by Alcaligenes faecalis and by Paracoccus versutus with release of methylamine.
(9/29)
Selective enrichments yielded bacterial cultures able to utilize the osmolyte N-methyltaurine as sole source of carbon and energy or as sole source of fixed nitrogen for aerobic growth. Strain MT1, which degraded N-methyltaurine as a sole source of carbon concomitantly with growth, was identified as a strain of Alcaligenes faecalis. Stoichiometric amounts of methylamine, whose identity was confirmed by matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry, and of sulfate were released during growth. Inducible N-methyltaurine dehydrogenase, sulfoacetaldehyde acetyltransferase (Xsc) and a sulfite dehydrogenase could be detected. Taurine dehydrogenase was also present and it was hypothesized that taurine dehydrogenase has a substrate range that includes N-methyltaurine. Partial sequences of a tauY-like gene (encoding the putative large component of taurine dehydrogenase) and an xsc gene were obtained by PCR with degenerate primers. Strain N-MT utilized N-methyltaurine as a sole source of fixed nitrogen for growth and could also utilize the compound as sole source of carbon. This bacterium was identified as a strain of Paracoccus versutus. This organism also expressed inducible (N-methyl)taurine dehydrogenase, Xsc and a sulfite dehydrogenase. The presence of a gene cluster with high identity to a larger cluster from Paracoccus pantotrophus NKNCYSA, which is now known to dissimilate N-methyltaurine via Xsc, allowed most of the overall pathway, including transport and excretion, to be defined. N-Methyltaurine is thus another compound whose catabolism is channelled directly through sulfoacetaldehyde. (+info)
A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase.
(10/29)
The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO(-)(2)] reduction of the type-2 site precedes nitrite binding, at high [NO(-)(2)] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism. (+info)
Atomic description of an enzyme reaction dominated by proton tunneling.
(11/29)
We present an atomic-level description of the reaction chemistry of an enzyme-catalyzed reaction dominated by proton tunneling. By solving structures of reaction intermediates at near-atomic resolution, we have identified the reaction pathway for tryptamine oxidation by aromatic amine dehydrogenase. Combining experiment and computer simulation, we show proton transfer occurs predominantly to oxygen O2 of Asp(128)beta in a reaction dominated by tunneling over approximately 0.6 angstroms. The role of long-range coupled motions in promoting tunneling is controversial. We show that, in this enzyme system, tunneling is promoted by a short-range motion modulating proton-acceptor distance and no long-range coupled motion is required. (+info)
Fluorescence in situ hybridization for rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis recovered from cystic fibrosis patients.
(12/29)
Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients. (+info)
Atomic level insight into the oxidative half-reaction of aromatic amine dehydrogenase.
(13/29)
The quinoprotein aromatic amine dehydrogenase (AADH) uses a covalently bound tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. Recent crystal structures have provided insight into the reductive half-reaction. In contrast, no atomic details are available for the oxidative half-reaction. The TTQ O7 hydroxyl group is protonated during reduction, but it is unclear how this proton can be removed during the oxidative half-reaction. Furthermore, compared with the electron transfer from the N-quinol form, electron transfer from the non-physiological O-quinol form to azurin is significantly slower. Here we report crystal structures of the O-quinol, N-quinol, and N-semiquinone forms of AADH. A comparison of oxidized and substrate reduced AADH species reveals changes in the TTQ-containing subunit, extending from residues in the immediate vicinity of the N-quinol to the putative azurin docking site, suggesting a mechanism whereby TTQ redox state influences interprotein electron transfer. In contrast, chemical reduction of the TTQ center has no significant effect on protein conformation. Furthermore, structural reorganization upon substrate reduction places a water molecule near TTQ O7 where it can act as proton acceptor. The structure of the N-semiquinone, however, is essentially similar to oxidized AADH. Surprisingly, in the presence of substrate a covalent N-semiquinone substrate adduct is observed. To our knowledge this is the first detailed insight into a complex, branching mechanism of quinone oxidation where significant structural reorganization upon reduction of the quinone center directly influences formation of the electron transfer complex and nature of the electron transfer process. (+info)
Improvement in ammonium removal efficiency in wastewater treatment by mixed culture of Alcaligenes faecalis no. 4 and L1.
(14/29)
To improve ammonium removal efficiency in wastewater treatment, a mixed culture of Alcaligenes faecalis no. 4 and its mutant L1, both of which have heterotrophic nitrification and aerobic denitrification abilities, was performed. In a batch culture, no. 4 has a higher denitrification ability than L1, but its ammonium removal rate was lower. In a mixed continuous culture in the ammonium loading range of 750 to 3500 mg-N/l/d, the average ammonium removal rate and the average denitrification ratio were 61 mg-N/l/h and 31%, respectively. In the mixed culture, the ammonium removal rate was twofold higher than that in a single culture of no. 4, the rate was similar to that in a single culture of L1, and the denitrification ratio was very high compared with that in the single culture of L1. (+info)
New insights into the reductive half-reaction mechanism of aromatic amine dehydrogenase revealed by reaction with carbinolamine substrates.
(15/29)
Aromatic amine dehydrogenase uses a tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. In the reductive half-reaction, a proton is transferred from the substrate C1 to betaAsp-128 O-2, in a reaction that proceeds by H-tunneling. Using solution studies, kinetic crystallography, and computational simulation we show that the mechanism of oxidation of aromatic carbinolamines is similar to amine oxidation, but that carbinolamine oxidation occurs at a substantially reduced rate. This has enabled us to determine for the first time the structure of the intermediate prior to the H-transfer/reduction step. The proton-betaAsp-128 O-2 distance is approximately 3.7A, in contrast to the distance of approximately 2.7A predicted for the intermediate formed with the corresponding primary amine substrate. This difference of approximately 1.0 A is due to an unexpected conformation of the substrate moiety, which is supported by molecular dynamic simulations and reflected in the approximately 10(7)-fold slower TTQ reduction rate with phenylaminoethanol compared with that with primary amines. A water molecule is observed near TTQ C-6 and is likely derived from the collapse of the preceding carbinolamine TTQ-adduct. We suggest this water molecule is involved in consecutive proton transfers following TTQ reduction, and is ultimately repositioned near the TTQ O-7 concomitant with protein rearrangement. For all carbinolamines tested, highly stable amide-TTQ adducts are formed following proton abstraction and TTQ reduction. Slow hydrolysis of the amide occurs after, rather than prior to, TTQ oxidation and leads ultimately to a carboxylic acid product. (+info)
The enzyme mechanism of nitrite reductase studied at single-molecule level.
(16/29)