Ammonia Oxidation Pathway

The oxidation of NH3 to NO2" is carried out in two steps (Figure 1) (Hooper, 1989). NH3 is first oxidized to hydroxylamine (NH2OH) by ammonia monooxygenase (AMO). NH2OH is subsequently oxidized to NO2" by hydroxylamine oxidoreductase (HAO). In the reaction catalyzed by AMO, one O from O2 is inserted into NH3 while the second O is reduced to H2O. This reaction requires two additional electrons. Because NH3 is the only source of reductant for these bacteria, the electrons required for the formation of H2O must come from the subsequent oxidation of NH2OH. Of the four electrons released in the oxidation of NH2OH, two must be directed towards the oxidation of NH3 and the remaining two are used for other reductant-requiring cellular processes such as biosynthesis and ATP generation (Wood 1986).



Figure 1 Ammonia oxidation pathway

3. Ammonia Monooxygenase

Although AMO has not yet been purified to homogeneity in an active form, considerable information has accumulated regarding its structure and activities. AMO is a membrane-bound enzyme which probably consists of three polypeptides. A 27 kDa polypeptide (AmoA) is labeled with 14C when AMO activity is inactivated with the mechanism-based inactivator, 14C2H2 (Hyman, Wood 1985). A 38 kDa polypeptide (AmoB) co-purifies with the 27 kDa polypeptide (McTavish et al. 1993a). A 31.4 kDa polypeptide (AmoC) is coded by a gene contiguous with AmoA (Sayavedra-Soto et al. 1998). AMO most likely contains Cu because the inhibitor profile of the enzyme includes Cu selective chelators (Bedard, Knowles 1989) and because Cu can activate AMO activities in lysates of N. europaea (Ensign et al. 1993). The substrate range of AMO is remarkably nonspecific. In addition to the oxidation of N in NH3, AMO can catalyze the oxidation of C-H bonds to alcohols (Hyman, Wood 1983), C=C bonds to epoxides (Hyman, Wood 1984b), C^C bonds to oxirenes (presumably) (Hyman et al 1988) and sulfides to sulfoxides (Juliette et al. 1993a, 1993b). The substrates for these reactions include alkyl (Hyman et al. 1988) and aryl hydrocarbons (Hyman, Wood 1985), halogenated hydrocarbons (Hyman, Wood 1984a; Rasche et al. 1991; Vannelli et al. 1990), aromatic molecules (Keener, Arp 1994) and other compounds.

The genes amoCAB, coding for the structural proteins of AMO, were isolated and sequenced (Bergmann, Hooper 1994; McTavish et al. 1993a; Sayavedra-Soto et al. 1994). The genes amoC, amoA and amoB are contiguous (Klotz et al. 1997; Sayavedra-Soto et al. 1994) and are transcribed as a single mRNA (Sayavedra-Soto et al. 1994). Two nearly identical (>99%) copies of amoCAB exist in the genome of TV! europaea (McTavish et al. 1993a, 1993b). A third, somewhat divergent copy (60% identity) of amoC is also present (Sayavedra-Soto et al. 1994). Mutants of N. europaea with either copy of amoA inactivated will grow, which indicates that both copies can be expressed (Hommes et al. 1998). However, mutants in copy

1 grew about 25% more slowly than wild type cells, while mutants in copy

2 grew at rates similar to wild type (Hommes et al. 1998). Ammonia-dependent O2 uptake rates, [14C] incorporation into AmoA, as well as the amo transcript levels in the mutants showed a pattern similar to their growth rates. Transcripts corresponding to amoC and amoAB as well as amoCAB have been identified in N. europaea (Sayavedra-Soto et al. 1994, 1996) (Figure 2). Of these the amoC mRNA is very stable, probably because it can form stable stem-loop structures which protect it from degradation. The roles of the different amo mRNAs are unknown and they may originate from amoCAB mRNA processing or from transcription from amoC and amoA (Sayavedra-Soto et al. 1994). A potential transcription start site for amo A was identified 114 bp upstream of the start codon in the intergenic region between amoC and amo A (Hommes et al. 2001). Transcript analysis for amoC showed two potential transcript start sites located 166 and 103 bp upstream of the amoC start codon (Hommes et al. 2001). All three transcript start sites had putative a70 promoter sequences associated with them. The DNA sequence of the regions upstream of amoC and amoA, including the putative promoter sequences, were identical for the two copies of amoCAB.

amoCj H

antAj |

anvBj f-



anvC? hH

atwAi I

anvB? ^

Fb P

1 kb

Fb P

1 kb

Fig. 2. Map of genesfor AMO, HAO, and Cytochromes

4. Regulation of amo Expression

Although AMO is essential to the growth of N. europaea, the specific rate of NH3 oxidation varies. For example, when cells taken from early stationary phase were suspended in fresh medium, AMO activity increased two-fold or more in two hours (Hyman, Arp 1992). Ammonia-starved cells recovered ammonia-oxidizing activity in a process which required de novo protein synthesis (Gerards et al. 1998). Following treatment with light or C2H2 (which both specifically inactivate AMO), cells of N. europaea eventually recover the ability to oxidize NH3. This recovery corresponds to the synthesis of a limited set of proteins, which includes the 27 kDa protein of AMO (Hyman, Arp 1992, 1995).

A question arises as to what signal the cells respond to when synthesizing new AMO proteins. NH3 is an obvious candidate to provide this signal but NH3 also provides both a source of energy and N for these cells. Thus, it is difficult to assign a specific role to NH3 as a signaling molecule. We investigated the effects of NH3 on both protein synthesis and gene transcription in N. europaea. AmoA (27 kDa) was synthesized when cells were incubated simultaneously with NH4+ (as a potential inducer of AMO synthesis and nitrogen source), C2H2 (so NH3 cannot serve as an energy source), NH2OH (as an energy source) and 14C02 (as a radiotracer to detect de novo protein synthesis in this autotrophic bacterium). The synthesis of AmoA required NH3 and was strongly influenced by changes in the free NH3 concentration (Hyman, Arp 1995). Other potential N sources (amino acids, nitrite) did not induce synthesis. The regulation of expression of AmoA is influenced by the concentration of NH3 available to the cells.

We also investigated the regulation of transcription of amoA and hao by NH/ (Sayavedra-Soto et al. 1994). In 14C02 labeling experiments the response to NH3 appeared to be global (detected as a smear of RNA) at the transcription level (Sayavedra-Soto et al. 1996) in contrast to the few synthesized proteins (defined bands) observed at the translational level (Hyman, Arp 1995). When C2H2-treated cells (AMO inactivated) were subsequently exposed to NH/, transcripts for amoA and hao were produced even if the incubations were carried out in the continued presence of C2H2 (Sayavedra-Soto et al. 1996). However, neither transcripts for amo A nor hao were produced in the absence of NH/. Unlike amoA, a very stable mRNA encoding amoC can be found for at least 72 hours after NH/ is removed (Sayavedra-Soto et al. 1998). Analysis of the two identified transcription start sites for amoC showed that they responded differently to the addition of NH/. Whereas in the absence of NH/ transcripts starting at both potential promoters were found (i.e. derived from the stable amoC mRNA), in the presence of Ni l/ transcripts from the distal promoter greatly predominated (Hommes et al. 2001).

5. Hydroxylamine Oxidoreductase

This remarkable periplasmic enzyme is a homotrimer (subunit size 60 kDa), and each subunit harbors eight c-type hemes (Hooper et al. 1997; Igarashi et al. 1997). Seven of these hemes are each covalently bound to the protein by two thioether linkages typical of c-type hemes. The eighth heme has a third point of covalent attachment to the protein through a tyrosine residue. This unusual heme, designated P460, is at the site of hydroxylamine oxidation. The crystal structure of HAO has been determined and has revealed the orientation of the hemes in each subunit (Igarashi et al. 1997). The eight hemes group into four clusters. The structure also revealed that the subunits of HAO are covalently cross-linked through one of the hemes. All eight hemes have different mid-point potentials (Hooper et al. 1997), but it is not yet known which potential is associated with which heme.

The gene that codes for HAO Qiao) is expressed as a monocistronic transcript (Sayavedra-Soto et al. 1994). The genome of N. europaea encodes three copies of hao (McTavish et al. 1993a) (Figure 2). The coding regions for the three copies are nearly identical (Bergmann et al. 1994). Mutants with any one copy disrupted grew with no discernible difference from the wild type

(Hommes et al. 1996). We investigated the regulation of transcription of hao by NH4+ (Sayavedra-Soto et al. 1996). HAO mRNA was induced under the same conditions as AMO mRNA but to a lower extent. HAO activity increased 5% to 20% under these conditions. DNA sequencing of the flanking regions upstream of the three copies of hao was done (Hommes et al. 2001). The sequences of hao/ and hao2 were found to be nearly identical for 160 bp upstream. The sequence of hao3 copy diverged from the other two copies 15 bp upstream of the start codon. Transcript analysis identified putative transcript start sites for haoj and hao2 71 bp upstream of the start codon, and 54 bp upstream of the start codon for hao3 (Hommes et al. 2001). All of these transcript start sites had

a promoter sequences associated with them.

The gene for HAO has also been examined in the closely related strain Nitrosomonas sp. ENI-11 (Yamagata et al. 2000). Gene mapping revealed that haoi was located about 23 kb upstream of amoCAB ยก, hao2 was located about 15 kb downstream from amoCAB2 and hao3 was located about 75 kb upstream of amoCAB2. The two copies of amoCAB were more than 360 kb apart. Unlike N. europaea, three single hao mutants were created in Nitrosomonas sp. ENI-11 which had 68 to 75% of wild type growth rates and 58 to 89% wild type HAO activity (NH2OH dependent N02" formation) (Yamagata et al. 2000).

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