<jats:title>ABSTRACT</jats:title><jats:p>The recently described ammonia-oxidizing archaea of the phylum<jats:named-content content-type="genus-species">Thaumarchaeota</jats:named-content>are highly abundant in marine, geothermal, and terrestrial environments. All characterized representatives of this phylum are aerobic chemolithoautotrophic ammonia oxidizers assimilating inorganic carbon via a recently described thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle. Although some genes coding for the enzymes of this cycle have been identified in the genomes of<jats:named-content content-type="genus-species">Thaumarchaeota</jats:named-content>, many other genes of the cycle are not homologous to the characterized enzymes from other species and can therefore not be identified bioinformatically. Here we report the identification and characterization of malonic semialdehyde reductase Nmar_1110 in the cultured marine thaumarchaeon<jats:named-content content-type="genus-species">Nitrosopumilus maritimus</jats:named-content>. This enzyme, which catalyzes the reduction of malonic semialdehyde with NAD(P)H to 3-hydroxypropionate, belongs to the family of iron-containing alcohol dehydrogenases and is not homologous to malonic semialdehyde reductases from<jats:named-content content-type="genus-species">Chloroflexus aurantiacus</jats:named-content>and<jats:named-content content-type="genus-species">Metallosphaera sedula</jats:named-content>. It is highly specific to malonic semialdehyde (<jats:italic>K<jats:sub>m</jats:sub></jats:italic>, 0.11 mM;<jats:italic>V</jats:italic><jats:sub>max</jats:sub>, 86.9 μmol min<jats:sup>−1</jats:sup>mg<jats:sup>−1</jats:sup>of protein) and exhibits only low activity with succinic semialdehyde (<jats:italic>K<jats:sub>m</jats:sub></jats:italic>, 4.26 mM;<jats:italic>V</jats:italic><jats:sub>max</jats:sub>, 18.5 μmol min<jats:sup>−1</jats:sup>mg<jats:sup>−1</jats:sup>of protein). Homologues of<jats:named-content content-type="genus-species">N. maritimus</jats:named-content>malonic semialdehyde reductase can be found in the genomes of all<jats:named-content content-type="genus-species">Thaumarchaeota</jats:named-content>sequenced so far and form a well-defined cluster in the phylogenetic tree of iron-containing alcohol dehydrogenases. We conclude that malonic semialdehyde reductase can be regarded as a characteristic enzyme for the thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle.</jats:p>