Isolation of a Multiheme Protein with Features of a Hydrazine-Oxidizing Enzyme from an Anaerobic Ammonium-Oxidizing Enrichment Culture

Abstract

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V max and K m for hydrazine are 6.2 ؎ 0.3 mol/min · mg and 5.5 ؎ 0.6 M, respectively. Hydrazine (25 M) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 M) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of "Candidatus Kuenenia stuttgartiensis" (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.The anaerobic ammonium oxidation (anammox) process is a fascinating subject of study, not only because it is an innovative technological advance for the removal of nitrogenous contaminants from wastewater (14,17,27), but also because it includes interesting and novel biochemical reactions. In the anammox reaction, ammonium and nitrite contribute in equimolar amounts to the formation of dinitrogen gas (N 2 ) according to the following equation (26): NH 4 ϩ ϩ NO 2 Ϫ 3 N 2 ϩ 2H 2 O; G°ϭ Ϫ358 kJ/mol ammonium. This biological reaction is performed under anoxic conditions by novel autotrophic bacteria, which have not yet been isolated but which are identified as deeply branching planctomycetes, based on nucleotide sequences of 16S rRNA genes (17,22,26). The first anammox bacterium to be identified, discovered in The Netherlands, has been provisionally named "Candidatus Brocadia anammoxidans" (15). An increasing number of 16S rRNA gene sequences from other putative anaerobic ammonium-oxidizing bacteria have been reported to date from laboratories (6,21,29,31) and from natural marine habitats such as the Black Sea (4, 5, 13, 16).We designed a continuous-flow reactor shielded from light in which to enrich cultures for bacteria having the ability to catalyze the anammox reaction from a seed of denitrifying sludge originating in Kumamoto City, Japan. In the reactor, the culture turned a distinctly red color and yielded large amounts of sludge that could remove ammonium and nitrite with a relatively low production of nitrate at ratios strongly resembling that of the anammox reaction (20). To investigate the bacterial composition of the enriched culture community, 16S rRNA gene sequences were amplified by PCR and cloned. The dominant clones had an identical sequence, which has 92.2% identity with the 16S rRNA gene of "Candidatus B...

Activity, abundance and community structure of anammox bacteria along depth profiles in three different paddy soils

“…(van der Star et al, 2008), thus making this genus more abundant and active in the habitats with high substrates input such as paddy soils. Furthermore, a recent study on the biogeography of anammox bacteria based on data from both natural and engineered ecosystems indicated that Candidatus Brocadia and Kuenenia are the closest co-occurring groups among the five genera, which implies that these genera would share similar habitats (Sonthiphand et al, 2014). Comparing to other four genera, Candidatus Scalindua was observed to be favorable to high salinity environments such as oceans and marine sediments (Dale et al, 2009), which partially explained its absence in the tested soils in this study.…”

“…The preference of alkaline soils by anammox bacteria could be explained by the characteristic of their enzymes. For instance, the hydrazine oxidizing enzyme (HZO), which catalyzes the final step of anammox process, was reported to be stable between pH 6.0 and 9.0, and was more active between pH 7.5 and 8.5 (Shimamura et al, 2007). In addition to soil pH, our results also indicated that soil EC, a parameter reflecting soil salinity, had a positive relationship with anammox rates and the functional gene abundance.…”

“…Similarly, there was variation in detection of Candidatus Jettenia in two recent studies in which Candidatus Jettenia was observed in river water columns but was absent in another study in a freshwater wetland (Lee et al, 2014;Sun et al, 2014). Unlike the other four genera which were largely observed in natural habitats, the Candidatus Jettenia group was typically detected in engineered environments such as bioreactors and wastewater treatment plants (Sonthiphand et al, 2014). Such pattern implied that this genus might favor conditions with high level of substrates load.…”

Anaerobic ammonium oxidation for treatment of ammonium-rich wastewaters

“…It was formed from oxidation of ammonia with the help of hydrazine hydrolase, and then converted to N 2 through hydrazine-oxidizing enzyme or hydroxylamine oxidoreductase (van de Graaf et al, 1997;Strous et al, 2006). The two enzymes were proposed to be different in (Strous et al, 1999a) Wastewater Candidatus Brocadia fulgida (Kartal et al, 2004) Wastewater Kuenenia Candidatus Kuenenia stuttgartiensis (Penton et al, 2006) Wastewater Scalindua Candidatus Scalindua brodae Wastewater Candidatus Scalindua wagneri Wastewater Candidatus Scalindua sorokinii Seawater Others Candidatus Jettenia asiatica (Tsushima et al, 2007) Not reported Candidatus Anammoxoglobus propionicus (Kartal et al, 2007) Synthetic water their specificities (Shimamura et al, 2007). Cytochromes were important electron transporters between enzymes (Huston et al, 2007).…”

Transformações do nitrogênio e diversidade de Planctomycetes em sedimentos de manguezais

“…As enzimas hirazina-hidrolase estão envolvidas nesta via (sintetizadas pelo gene hh) (STROUS et al, 2006), como também a enzima hidroxilamina oxidorredutase (produto do gene hao), e a enzima hidrazina-oxidante (codificada pelo gene hzo) (SHIMAMURA et al, 2007). Desde a descrição do processo anammox (STROUS et al, 1999), várias bactérias foram identificadas como capazes de realizar essa transformação, todas pertencentes à ordem dos Planctomycetales, distribuídos em um clado monofilético com uma grande distância evolutiva de outros Planctomycetes.…”