title: Pyruvic oxime dioxygenase from heterotrophic nitrifier Alcaligenes faecalis is a nonheme Fe(II)-dependent enzyme homologous to class II aldolase.
abstract: Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn(II)-dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe(II). Affinity and catalytic constants were estimated at 470 μM and 4.69 sec-1, respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe(II), not Zn(II), is essential for POD activity and that Mn(II)could partially fulfill the function of Fe(II). A mutant POD with replacement of His183, corresponding to one of three Zn(II)-binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe(II)as a catalytic center instead of Zn(II). A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe(II)-dependent dioxygenase.
authors.affiliation: Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
authors.forename: Shuhei
authors.initials: S
authors.lastname: Tsujino
authors.affiliation: Department of Biological Sciences, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan.
authors.forename: Chisato
authors.initials: C
authors.lastname: Uematsu
authors.affiliation: Instrumental Research Support Office, Research Institute of Green Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Dohra
authors.affiliation: Department of Environment and Energy Systems, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
authors.forename: Taketomo
authors.initials: T
authors.lastname: Fujiwara
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D049308:Dioxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D005634:Fructose-Bisphosphate Aldolase; D007501:Iron / Q000378:metabolism*; D008970:Molecular Weight; D058465:Nitrification; D010802:Phylogeny; D055503:Protein Multimerization
chemical_list: D001426:Bacterial Proteins; D004492:Edetic Acid; D007501:Iron; D049308:Dioxygenases; D005634:Fructose-Bisphosphate Aldolase