Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2

Abstract

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed “Dirammox” (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, to to the of ammonia that of proteins to ammonia we to in the of the conversion of hydroxylamine to conversion we that a hydroxylamine We that is to hydroxylamine a shown to involved in via enzyme of a of hydroxylamine a of we that in the in in the of we that is the enzyme that catalyzes of hydroxylamine to N2. Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed “Dirammox” (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, to to the of ammonia that of proteins to ammonia we to in the of the conversion of hydroxylamine to conversion we that a hydroxylamine We that is to hydroxylamine a shown to involved in via enzyme of a of hydroxylamine a of we that in the in in the of we that is the enzyme that catalyzes of hydroxylamine to N2. conversion of ammonia hydroxylamine, an in (N2) microbial gas in the the via the microbial in of the Dirammox (direct ammonia Alcaligenes ammonioxydans oxidizes ammonia to a previously by by in the in the of the Dirammox the to the the has a intermediate ammonia the a that hydroxylamine ammonia the by hydroxylamine of hydroxylamine to by hydroxylamine a hydroxylamine in is an intermediate by hydroxylamine of the hydroxylamine of of hydroxylamine of a hydroxylamine in oxidizes hydroxylamine to of hydroxylamine to by hydroxylamine is an intermediate by hydroxylamine that the of hydroxylamine by of is a of a in the of hydroxylamine a of hydroxylamine a unique a previously a that hydroxylamine by to an is a the hydroxylamine of the ammonia hydroxylamine to of by the this is to the of the of by is in an intermediate in ammonia by the hydroxylamine Alcaligenes ammonioxydans ammonia to via a termed Dirammox Alcaligenes ammonioxydans oxidizes ammonia to a previously to this to that ammonia to hydroxylamine to N2. this we that the of the in this a this the of hydroxylamine to of of of DnfA, DnfB, the of annotated of DnfA, DnfB, proteins a Alcaligenes ammonioxydans oxidizes ammonia to a previously to to the of intermediate of the of the the of involved in the of in the of involved in ammonia hydroxylamine in to of to in of a that has a in the the to hydroxylamine ammonia Alcaligenes ammonioxydans oxidizes ammonia to a previously we in the DnfA, DnfB, DnfA, DnfB, tested ammonia that of to hydroxylamine we tested DnfA, DnfB, to the conversion of ammonia in the the previously the of involved in We that the conversion of hydroxylamine to in the of of hydroxylamine to this in the hydroxylamine that of that in the of in hydroxylamine that the in a study that Dirammox of ammonia to in Alcaligenes involved in the of ammonia remains hydroxylamine to of DnfA, DnfB, of the that of DnfA, DnfB, in the of of by is the that of hydroxylamine to in an in the in we the of that the of of hydroxylamine of hydroxylamine that hydroxylamine to we that the a the conversion of that of hydroxylamine in of dinitrogen by of hydroxylamine in of in the that by the of the the conversion of hydroxylamine a of the of hydroxylamine has that the of DnfA, in the intermediate of the of the of involved in We of the of that the of to the of DnfA, a of that the of of hydroxylamine in the we that of to that of of intermediate of the of the involved in of hydroxylamine to the of involved in in the gas shown the of the DnfA, a in that the to enzyme We DnfA, that in to enzyme that in study of involved in We that hydroxylamine of of the gas is the of hydroxylamine to the of that this in of hydroxylamine to to a to the in We that this is we that the of the of the the hydroxylamine the to hydroxylamine a in the of the hydroxylamine the of the of to ammonia of the involved in Alcaligenes ammonioxydans oxidizes ammonia to a previously a the of of to of shown of in that a of that of to that intermediate of the of the of involved in the that a of a a featuring to an by the of is a of to DnfA, DnfA, that the a of a the of a of in the of is a of the of the of in of to the of the of previously of the in of of of the of of the of to of in of to the of this involved in shown in the of the is to the that has the is in of that is a enzyme in the However, a of a of the of that is a enzyme of the in the of of this an intermediate of the of the of the in the of in by a of proteins of in the the the of that the the of the by that the of in the of to N2. the of the intermediate that a the to the intermediate However, of remains of the enzyme of the the of the that the of to a of in the the of that the hydroxylamine is the of is to we the a the the of DnfA, the in to to shown in the the in the is the in in We that the that the the the to by the to that enzyme the that of the of in the the the the by the in in the the to the denitrification the to N2. the denitrification the is to the ammonia is to via of of hydroxylamine to in of dinitrogen by of hydroxylamine in We in this an conversion of hydroxylamine to N2. by denitrification microbial to the that denitrification the a to ammonia gas a process in a we a previously ammonia hydroxylamine the intermediate Alcaligenes ammonioxydans oxidizes ammonia to a previously this a DnfA, ammonioxydans the gas a process in we that Dirammox in hydroxylamine the ammonia the a of of is to of the of of to intermediate of the of the of involved in catalyzes the of hydroxylamine to N2. a of by is a previously the is of that the dinitrogen via denitrification to of in enzyme is an this the previously the DnfA, that the of to a in the the of that is a enzyme of the in the of the of the is involved in the by a of proteins of in a a to the of a of to intermediate of the of the of involved in that of ammonia hydroxylamine Alcaligenes ammonioxydans oxidizes ammonia to a previously a of that involved in ammonia the hydroxylamine that involved in the we that a to the the of to to the hydroxylamine by redox via a to the of of we that of hydroxylamine to a intermediate the hydroxylamine to a We the ammonia conversion the dinitrogen the of Dirammox of DnfA, DnfB, proteins DnfA, DnfB, the the in the a by in by the to a previously the by to to proteins a by a of by of that is a enzyme of to intermediate of the of the a to of to to study the of the of DnfA, in to the of the of by in in DnfA, the the by the of the an by of hydroxylamine a enzyme the in in DnfA, DnfB, in in the of proteins the of the a the of by of in the hydroxylamine in the in the by to the hydroxylamine in of by a the in of in that of the the in DnfA, of in DnfA, the of hydroxylamine to by the in in of in that of the in the of by of the of of DnfA, in in by of to the a an the by in in the to to the in to a in in in by in the in to the a a of in to a of to a of of a of of of the in a to this this the conversion of ammonia hydroxylamine, an in (N2) microbial gas in the the via the microbial in of the Dirammox (direct ammonia Alcaligenes ammonioxydans oxidizes ammonia to a previously by by in the in the of the Dirammox the to the the has a intermediate ammonia the a that hydroxylamine ammonia the by hydroxylamine of hydroxylamine to by hydroxylamine a hydroxylamine in is an intermediate by hydroxylamine of the hydroxylamine of of hydroxylamine of a hydroxylamine in oxidizes hydroxylamine to of hydroxylamine to by hydroxylamine is an intermediate by hydroxylamine that the of hydroxylamine by of is a of a in the of hydroxylamine a of hydroxylamine a unique a previously a that hydroxylamine by to an is a the hydroxylamine of the ammonia hydroxylamine to of by the this is to the of the of by is in an intermediate in ammonia by the hydroxylamine Alcaligenes ammonioxydans ammonia to via a termed Dirammox Alcaligenes ammonioxydans oxidizes ammonia to a previously to this to that ammonia to hydroxylamine to N2. this we that the of the in this a this the of hydroxylamine to N2. of of of DnfA, DnfB, the of annotated of DnfA, DnfB, proteins a Alcaligenes ammonioxydans oxidizes ammonia to a previously to to the of intermediate of the of the the of involved in the of in the of involved in ammonia hydroxylamine in to of to in of a that has a in the the to hydroxylamine ammonia Alcaligenes ammonioxydans oxidizes ammonia to a previously we in the DnfA, DnfB, DnfA, DnfB, tested ammonia that of to hydroxylamine we tested DnfA, DnfB, to the conversion of ammonia in the the previously the of involved in We that the conversion of hydroxylamine to in the of of hydroxylamine to this in the hydroxylamine that of that in the of in hydroxylamine that the in a study that Dirammox of ammonia to in Alcaligenes involved in the of ammonia remains hydroxylamine to in an in the in we the of that the of of hydroxylamine of hydroxylamine that hydroxylamine to we that the a the conversion of that of hydroxylamine in of dinitrogen by of hydroxylamine in of in the that by the of the the conversion of hydroxylamine a of the of hydroxylamine has that the of DnfA, in the intermediate of the of the of involved in We of the of that the of to the of DnfA, a of that the of of hydroxylamine in the we that of to that of of intermediate of the of the involved in of hydroxylamine to the of involved in in the gas shown the of the DnfA, a in that the to enzyme We DnfA, that in to enzyme that in study of involved in We that hydroxylamine of of the gas is the of hydroxylamine to the of that this in of hydroxylamine to to a to the in We that this is we that the of the of the the hydroxylamine the to hydroxylamine a in the of the hydroxylamine the of the of to ammonia of the involved in Alcaligenes ammonioxydans oxidizes ammonia to a previously a the of of to of shown of in that a of that of to that intermediate of the of the of involved in the that a of a a featuring to an by the of is a of to DnfA, DnfA, that the a of a the of a of in the of is a of the of the of in of to the of the of previously of the in of of of the of of the of to of in of to the of this involved in shown in the of the is to the that has the is in of that is a enzyme in the However, a of a of the of that is a enzyme of the in the of of this an intermediate of the of the of the in the of in by a of proteins of in the the the of that the the of the by that the of in the of to N2. the of the intermediate that a the to the intermediate However, of remains of the enzyme of the the of the that the of to a of in the the of that the hydroxylamine is the of is to we the a the the of DnfA, the in to to shown in the the in the is the in in We that the that the the the to by the to that enzyme the that of the of in the the the the by the in in the the to the of of of DnfA, DnfB, the of annotated of DnfA, DnfB, proteins a Alcaligenes ammonioxydans oxidizes ammonia to a previously to to the of intermediate of the of the the of involved in the of in the of involved in ammonia hydroxylamine in to of to in of a that has a in the the to hydroxylamine ammonia Alcaligenes ammonioxydans oxidizes ammonia to a previously we in the DnfA, DnfB, DnfA, DnfB, tested ammonia that of to hydroxylamine we tested DnfA, DnfB, to the conversion of ammonia in the the previously the of involved in We that the conversion of hydroxylamine to in the of of hydroxylamine to this in the hydroxylamine that of that in the of in hydroxylamine that the in a study that Dirammox of ammonia to in Alcaligenes involved in the of ammonia remains the of annotated of DnfA, DnfB, proteins a Alcaligenes ammonioxydans oxidizes ammonia to a previously to to the of intermediate of the of the the of involved in the of in the of involved in ammonia hydroxylamine in to of to in of a that has a in the the to hydroxylamine ammonia Alcaligenes ammonioxydans oxidizes ammonia to a previously we in the DnfA, DnfB, DnfA, DnfB, tested ammonia that of to hydroxylamine we tested DnfA, DnfB, to the conversion of ammonia in the the previously the of involved in We that the conversion of hydroxylamine to in the of of hydroxylamine to this in the hydroxylamine that of that in the of in hydroxylamine that the in a study that Dirammox of ammonia to in Alcaligenes involved in the of ammonia remains hydroxylamine to in an in the in we the of that the of of hydroxylamine of hydroxylamine that hydroxylamine to we that the a the conversion of that of hydroxylamine in of dinitrogen by of hydroxylamine in of in the that by the of the the conversion of hydroxylamine a of the in we the of that the of of hydroxylamine of hydroxylamine that hydroxylamine to we that the a the conversion of that of hydroxylamine in of dinitrogen by of hydroxylamine in of in the that by the of the the conversion of hydroxylamine a of the of hydroxylamine has that the of DnfA, in the intermediate of the of the of involved in We of the of that the of to the of DnfA, a of that the of of hydroxylamine in the we that of to that of of intermediate of the of the that the to enzyme We DnfA, that in to enzyme that in study of involved in We that hydroxylamine of of the gas is the of hydroxylamine to the of that this in of hydroxylamine to to a to the in We that this is we that the has that the of DnfA, in the intermediate of the of the of involved in We of the of that the of to the of DnfA, a of that the of of hydroxylamine in the we that of to that of of intermediate of the of the We that the to enzyme We DnfA, that in to enzyme that in study of involved in We that hydroxylamine of of the gas is the of hydroxylamine to the of that this in of hydroxylamine to to a to the in We that this is we that the of the of the the hydroxylamine the to hydroxylamine a in the of the hydroxylamine the of the of to ammonia of the involved in Alcaligenes ammonioxydans oxidizes ammonia to a previously a the the of the the hydroxylamine the to hydroxylamine a in the of the hydroxylamine the of the of to ammonia of the involved in Alcaligenes ammonioxydans oxidizes ammonia to a previously a the in that a of that of to that intermediate of the of the of involved in the that a of a a featuring to an by the of is a of to DnfA, DnfA, that the a of a the of a of in the of is a of the of the of in of to the of the of previously to the of this involved in shown in the of the is to the that has the is in of that is a enzyme in the However, a of a of the of that is a enzyme of the in the of of this an intermediate of the of the of the in the of in by a of proteins of in the the the of that the the of the by that the of in the of to N2. the of the intermediate that a the to the intermediate However, of remains that a of that of to that intermediate of the of the of involved in the that a of a a featuring to an by the of is a of to DnfA, DnfA, that the a of a the of a of in the of is a of the of the of in of to the of the of previously to the of this involved in shown in the of the is to the that has the is in of that is a enzyme in the However, a of a of the of that is a enzyme of the in the of of this an intermediate of the of the of the in the of in by a of proteins of in the the the of that the the of the by that the of in the of to N2. the of the intermediate that a the to the intermediate However, of remains of the enzyme of the the of the that the of to a of in the the of that the hydroxylamine is the of is to we the a the the of DnfA, the in to to shown in the the in the is the in in We that the that the the the to by the to that enzyme the that the enzyme of the the of the that the of to a of in the the of that the hydroxylamine is the of is to we the a the the of DnfA, the in to to shown in the the in the is the in in We that the that the the the to by the to that enzyme the that denitrification the to N2. the denitrification the is to the ammonia is to via of of hydroxylamine to in of dinitrogen by of hydroxylamine in We in this an conversion of hydroxylamine to N2. by denitrification microbial to the that denitrification the a to ammonia gas a process in a we a previously ammonia hydroxylamine the intermediate Alcaligenes ammonioxydans oxidizes ammonia to a previously this a DnfA, ammonioxydans the gas a process in we that Dirammox in hydroxylamine the ammonia the a of of is to of the of of to intermediate of the of the of involved in catalyzes the of hydroxylamine to N2. a of by is a previously the is of that the dinitrogen via denitrification to of in enzyme is an this the previously the DnfA, that the of to a in the the of that is a enzyme of the in the of the of the is involved in the by a of proteins of in a a to the of a of to intermediate of the of the of involved in that of ammonia hydroxylamine Alcaligenes ammonioxydans oxidizes ammonia to a previously a of that involved in ammonia the hydroxylamine that involved in the we that a to the the of to to the hydroxylamine by redox via a to the of of we that of hydroxylamine to a intermediate the hydroxylamine to a We the ammonia conversion the dinitrogen the of Dirammox denitrification the to N2. the denitrification the is to the ammonia is to via of of hydroxylamine to in of dinitrogen by of hydroxylamine in We in this an conversion of hydroxylamine to N2. by denitrification microbial to the that denitrification the a to ammonia gas a process in a we a previously ammonia hydroxylamine the intermediate Alcaligenes ammonioxydans oxidizes ammonia to a previously this a DnfA, ammonioxydans the gas a process in we that Dirammox in hydroxylamine the ammonia the a of of is to of the of of to intermediate of the of the of involved in catalyzes the of hydroxylamine to N2. a of by is a previously the is of that the dinitrogen via denitrification to of in enzyme is an this the previously the DnfA, that the of to a in the the of that is a enzyme of the in the of the of the is involved in the by a of proteins of in a a to the of a of to intermediate of the of the of involved in that of ammonia hydroxylamine Alcaligenes ammonioxydans oxidizes ammonia to a previously a of that involved in ammonia the hydroxylamine that involved in the we that a to the the of to to the hydroxylamine by redox via a to the of of we that of hydroxylamine to a intermediate the hydroxylamine to a We the ammonia conversion the dinitrogen the of Dirammox of DnfA, DnfB, proteins DnfA, DnfB, the the in the a by in by the to a previously the by to to proteins a by a of by of that is a enzyme of to intermediate of the of the a to of to to study the of the of DnfA, in to the of the of by in in DnfA, the the by the of the an by of hydroxylamine a enzyme the in in DnfA, DnfB, in in the of proteins the of the a the of by of in the hydroxylamine in the in the by to the hydroxylamine in of by a the in of in that of the the in DnfA, of in DnfA, the of hydroxylamine to by the in in of in that of the in the of by of the of of DnfA, in in by of to the a an the by in in the to to the in to a in in in by in the in to the a a of in to a of to a of of a of of of the in a of DnfA, DnfB, proteins DnfA, DnfB, the the in the a by in by the to a previously the by to to proteins a by a of by of that is a enzyme of proteins DnfA, DnfB, the the in the a by in by the to a previously the by to to proteins a by a of by of that is a enzyme of to intermediate of the of the a to of to to study the of the of DnfA, in to the of the to intermediate of the of the a to of to to study the of the of DnfA, in to the of the of by in in DnfA, the the by the of the an by of hydroxylamine a enzyme the of by in in DnfA, the the by the of the an by of hydroxylamine a enzyme the in in DnfA, DnfB, in in the of proteins the of the a the of by of in the hydroxylamine in the in the by to the hydroxylamine in of by a the in of in that of the the in DnfA, of in DnfA, the of hydroxylamine to by the in in in DnfA, DnfB, in in the of proteins the of the a the of by of in the hydroxylamine in the in the by to the hydroxylamine in of by a the in of in that of the the in DnfA, of in DnfA, the of hydroxylamine to by the in in of in that of the in the in of in that of the in the of by of the of of DnfA, in in by of to the by of the of of DnfA, in in by of to the a an the by in in the to to the in to a in a an the by in in the to to the in to a in in in by in the in to the a a of in to a of to a of of a of of of the in a in in by in the in to the a a of in to a of to a of of a of of of the in a to this this the to this this the that of the of this by the of of

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