Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypep NH3 + 02 + 2e-+ 2H+ --NH20H + H20 Hydroxylamine is then oxidized to nitrite in an energyyielding dehydrogenase reaction by the periplasmic enzyme hydroxylamine oxidoreductase (1, 9). The acceptor for the four electrons generated in the latter reaction is the tetraheme cytochrome C554, which is thought to carry the electrons in two-electron steps (2). Two of the four electrons are destined for an oxidative electron transfer chain and cytochrome aa3 terminal oxidase (5); the other two must be used in the AMO reaction.Little is known about AMO. It can be irreversibly inhibited by acetylene, a suicide substrate for many monooxygenases, and inhibition with [14C]acetylene radioactively labels a 28-kDa polypeptide that is associated with the membrane fraction (16). AMO is also inhibited by metal chelators, such as thiourea and allylthiourea (10, 20). The fact that these chemicals bind several metals (28), with a preference for copper, has been considered suggestive evidence that AMO might contain copper (3,30).AMO activity has been demonstrated with a crude membrane fraction (35, 36), but no further purification has been accomplished. The electron donor for AMO in vivo is unknown, although with the crude membrane fraction it was found that the addition of cytochrome C554 stimulated ammonia oxidation activity (35), indicating that cytochrome C554 can serve as at least an indirect electron donor for AMO.AMO is significant because of the key role that it plays in the nitrogen cycle and because it is able to degrade a wide range of hydrocarbons and halogenated hydrocarbons (14,39,40). The study of AMO is also important as a model for the related enzyme methane monooxygenase. Methane mono-* Corresponding author. oxygenase exists in a soluble form and a membrane form in the methanotrophic bacteria, with the membrane form being more widespread (3). It is thought that AMO and the membrane form of methane monooxygenase are probably homologous enzymes because, in addition to both being membrane enzymes, they have very similar substrate specificities (both enzymes can oxidize both ammonia and methane) and similar inhibitor profiles (3, 4). Also, the membrane methane monooxygenase may, like AMO, be a coppercontaining enzyme (27,31,34).In this paper we report the partial purification of the acetylene-binding polypeptide of AMO and the cloning and sequencing of the gene for this polypeptide. We also report the cloning and partial sequence of a second gene in this operon which codes for a polypeptide that copurifies with the acetylene-binding polypeptide.
We performed the first field-scale atrazine remediation study in the United States using chemically killed, recombinant organisms. This field study compared biostimulation methods for enhancing atrazine degradation with a novel bioaugmentation protocol using a killed and stabilized whole-cell suspension of recombinant Escherichia coli engineered to overproduce atrazine chlorohyrolase, AtzA. AtzA dechlorinates atrazine, producing non-toxic and non-phytotoxic hydroxyatrazine. Soil contaminated by an accidental spill of atrazine (up to 29,000 p.p.m.) supported significant populations of indigenous microorganisms capable of atrazine catabolism. Laboratory experiments indicated that supplementing soil with carbon inhibited atrazine biodegradation, but inorganic phosphate stimulated atrazine biodegradation. A subsequent field-scale study consisting of nine (0.75m3) treatment plots was designed to test four treatment protocols in triplicate. Control plots contained moistened soil; biostimulation plots received 300p.p.m. phosphate; bioaugmentation plots received 0.5% (w/w) killed, recombinant E. coli cells encapsulating AtzA; and combination plots received phosphate plus the enzyme-containing cells. After 8 weeks, atrazine levels declined 52% in plots containing killed recombinant E. coli cells, and 77% in combination plots. In contrast, atrazine levels in control and biostimulation plots did not decline significantly. These data indicate that genetically engineered bacteria overexpressing catabolic genes significantly increased degradation in this soil heavily contaminated with atrazine.
The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.
Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.
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