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A hydroxylamine oxidoreductase (HAO) was purified from anammox sludge in which an anammox bacterium, strain KSU-1, was dominant. The enzyme was a 118-kDa homodimer composed of a 53-kDa subunit. With phenazine methosulfate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide as electron acceptors, the V(max) and K(m) for hydroxylamine were determined as 9.6+/-0.2 micromol/min x mg and 33+/-2 microM, while those for hydrazine were 0.54+/-0.0 micromol/min x mg and 25+/-2 microM, respectively. The HAO had a P468 chromophore. These enzymatic properties were different from those of the hydrazine-oxidizing enzyme (HZO), a multiheme protein abundantly produced by the KSU-1 strain, but were similar to those of the HAO purified from Candidatus Brocadia anammoxidans. The hao gene exists upstream of the hzoB gene, which codes for the HZO. The sequence deduced from the hao gene indicated eight c-type heme binding motifs and showed 87% identity with a polypeptide encoded by an open reading frame (kustc1061) in the genome of an anammox bacterium Candidatus Kuenenia stuttgartiensis. These suggested that the HAO is an indispensable enzyme and well conserved in anammox bacteria, similar to the HZO. This enzyme might therefore be a specific hydroxylamine oxidoreductase for anammox bacteria.

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 micromol/min.mg and 5.5 +/- 0.6 microM, respectively. Hydrazine (25 microM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 microM) 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.