Deoxyhypusine Hydroxylase Is an Fe(II)-dependent, Heat-repeat Enzyme

Abstract

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (Nϵ-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active of of DOHH The two The DOHH of iron DOHH the of the The of the strictly conserved His-Glu was of in the His-Glu with the eight a in the iron is the DOHH the strictly conserved His-Glu iron the iron DOHH of iron on each of the conserved His-Glu a iron of protein Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (Nϵ-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active of of DOHH The two The DOHH of iron DOHH the of the The of the strictly conserved His-Glu was of in the His-Glu with the eight a in the iron is the DOHH the strictly conserved His-Glu iron the iron DOHH of iron on each of the conserved His-Glu a iron of protein is the protein in contains hypusine a is of the protein a two the step catalyzes the of the of the the of the is hydroxylase (DOHH) the hypusine in eIF5A. is a conserved protein in is in a is in The of in was in DOHH is in is in the DOHH is of the DOHH is in a the in with protein hydroxylase DOHH is a of a the a is the in the of DOHH a of the of of a the on a coordination a of of in the of of of the of the the of a of the of the the of the of the of the of the purified the of with the the of DOHH in iron was the of in on the of the in DOHH the the DOHH the DOHH a the human DOHH DOHH in a in each the is of the of DOHH a of HEAT-repeat human a of protein the of in the composed of of of is of DOHH a of eight in a symmetrical of a a is the of The the of DOHH a with conserved His-Glu the protein the the of of the the of iron the conserved His-Glu in DOHH the strictly conserved His-Glu the iron active is the DOHH was was was mixture was The was The human DOHH was The human was in in human The recombinant DOHH with a with with His-Glu with the The human DOHH a was a the of with The of the DOHH was of the The of the of the in of was a of the of in of a mixture of the the was with of The with two was of with final The of the was with the DOHH The was in in of of DOHH was with in of the of protein was with the was with The was a with of The was in of of was the DOHH was with in the was in the DOHH the was a recombinant DOHH with a The of of iron was of The in the the in was of of of DOHH in a with of of of of in a in the with a The protein was The was with was with a of a of a of of the DOHH mixture of of the protein human of purified in of was each with The in The of was of the of the DOHH the protein with with with The the was The protein of of of DOHH on a of was a was The of active the was the DOHH was in of each was with the was protein was with of of was the the in of in each DOHH of each protein of DOHH the DOHH of eight in a symmetrical of a the helical of was The purified human recombinant a of protein was is in with the the HEAT-repeat of contains strictly conserved His-Glu the the of in with of the His-Glu with with the eight with was was the of of the with of the conserved His-Glu of the DOHH the of the was of the the of DOHH on the conserved His-Glu of the DOHH of the human recombinant DOHH the of the protein in a The the purified recombinant of the DOHH DOHH of The hypusine is of the The of the in is of the of purified human recombinant was the of the was the was of was was the of the DOHH on of the was iron was the the of The in the iron in of a of iron the in the of the a of was in DOHH of the iron was with DOHH a of iron in DOHH is the of of with the of purified on a of iron in a of iron of of a in DOHH The of the of a DOHH the of a the of iron was the the of the two DOHH of the the iron was purified the DOHH was with The human the the with of the conserved His-Glu the sites eight of DOHH was in the the the purified the of the the of a the conserved His-Glu in the eight with of a conserved with iron the conserved in the coordination of iron the active of The iron was of of of the in The of a on the two sites two sites the the dyad) the in iron the of of the two iron a with of the iron DOHH The of eight on DOHH of of the DOHH of the of was The two the a the a The with iron the of the of with a of protein was a of with iron the the the was the the DOHH was the of was the of the two of the DOHH the a of the in is the of human DOHH is the of the the the was with a of the the iron is the the active of of the the of with the was in of with of the The eight the with iron the with the of The two iron the of the the the is protein with a of the of the of the the is the is the of the is of the the of iron the DOHH active a a of a of the the was on DOHH a with a the protein in each was the the the the with a of DOHH in the the of the was in the of DOHH on The was purified on the was of the was in of of of each was DOHH a DOHH the iron of the the The DOHH two each of a of in the of The with a of in The iron of was two the of the protein is the of of iron was in of the is the DOHH was in the in with the of DOHH was in a the is of in is a with the in a DOHH DOHH is a of the DOHH was in DOHH in the of human recombinant DOHH is of of of is DOHH of in was a in of was with of each DOHH The of each protein of of on DOHH of in the of DOHH the human with the active the of on the of the the was the a of the of the was in the a of a of in the mixture the of in DOHH the of was the DOHH the a of on DOHH The each of The the The the of hydroxylase a of tandem of hairpins motifs. of DOHH is composed of two symmetrical each a strictly conserved His-Glu two on each coordination of DOHH on the of a HEAT-repeat the protein a with a a the His-Glu on the two the of the DOHH the of iron the strictly conserved His-Glu in DOHH the active of DOHH the iron coordination the of each of the His-Glu in DOHH a helical DOHH in the two sites iron of DOHH of the with two iron the active The eight conserved the iron the with with the of the is a The is of of the with with a The a mixture with the of iron the of DOHH is the of DOHH the in the the two of the iron a on the of a in the was the on on protein on the is the the the DOHH the the DOHH is in a the with the was in the the DOHH the two of the in the the a on the a a mixture of mixture with the two a of the the in the of It the of iron the two of the DOHH in a with a in of the of iron DOHH the active of the iron of of the DOHH of was a DOHH of iron two two DOHH a of DOHH with iron the iron DOHH the of of was was the active of DOHH with of iron was the DOHH contains two iron coordination sites (one on each is iron active the two iron coordination sites two sites the the dyad) two active a in iron in DOHH a a of the in the His-Glu DOHH of of the a in the iron is a DOHH active with a the iron in of the active in a with two of the a the two iron with It a in DOHH is of conserved of DOHH is iron the of the of the purified in the of a a was was the the protein in the the of DOHH the iron contains a the DOHH a the of the was the iron the DOHH the the the the active of with the of of DOHH is the of the is a HEAT-repeat the of protein the of DOHH a DOHH is on a DOHH in of of iron the of the coordination a coordination of iron the in the of DOHH two iron a a of (one each in the two iron sites of the iron catalyzes the final step in the of protein in DOHH a of in the of The in the of in human human a of in of DOHH DOHH in human of of human the a of in of the of DOHH a DOHH with protein The the of DOHH the iron the DOHH the of of the of the of DOHH the the of of is the protein in contains hypusine a is of the protein a two the step catalyzes the of the of the the of the is hydroxylase (DOHH) the hypusine in eIF5A. is a conserved protein in is in a is in The of in was in DOHH is in is in the DOHH is of the DOHH is in a the in DOHH with protein hydroxylase DOHH is a of a the a is the in the of DOHH a of the of of a the on a coordination a of of in the of of of the of the the of a of the of the the of the of the of the of the purified the of with the the of DOHH in iron was the of in on the of the in DOHH the the DOHH the DOHH a the human DOHH DOHH in a in each the is of the of DOHH a of HEAT-repeat human a of protein the of in the composed of of of is of DOHH a of eight in a symmetrical of a a is the of The the of DOHH a with conserved His-Glu the protein the the of of the the of iron the conserved His-Glu in DOHH the strictly conserved His-Glu the iron active is the DOHH was was was mixture was The was The human DOHH was The human was in in human The recombinant DOHH with a with with His-Glu with the The human DOHH a was a the of with The of the DOHH was of the The of the of the in of was a of the of in of a mixture of the the was with of The with two was of with final The of the was with the DOHH The was in in of of DOHH was with in of the of protein was with the was with The was a with of The was in of of was the DOHH was with in the was in the DOHH the was a recombinant DOHH with a The of of iron was of The in the the in was of of of DOHH in a with of of of of in a in the with a The protein was The was with was with a of a of a of of the DOHH mixture of of the protein human of purified in of was each with The in The of was of the of the DOHH the protein with with with The the was The protein of of of DOHH on a of was a was The of active the was the DOHH was in of each was with the was protein was with of of was the the in of in each DOHH of each protein was was was mixture was The was The human DOHH was The human was in in human The was was was mixture was The was The human DOHH was The human was in in human The recombinant DOHH with a with with His-Glu with the The human DOHH a was a the of with The of the DOHH was of the The of the of the in of was a of the of in of a mixture of the the was with of The with two was of with final The of the was with the DOHH The was in in of of DOHH was with in of the of protein was with the was with The was a with of The was in of of was the DOHH was with in the was in the DOHH the was a recombinant DOHH with a The of of iron was of The in the the in was of of of DOHH in a with of of of of in a in the with a The protein was The was with was with a of a of a of of the DOHH mixture of of the protein human of purified in of was each with The in The of was of the of the DOHH the protein with with with The the was The protein of of of DOHH on a of was a was The of active the was the DOHH was in of each was with the was protein was with of of was the the in of in each DOHH of each protein recombinant DOHH with a with with His-Glu with the The human DOHH a was a the of with The of the DOHH was of the The of the of the in of was a of the of in of a mixture of the the was with of The with two was of with final The of the was with the DOHH The was in in of of DOHH was with in of the of protein was with the was with The was a with of The was in of of was the DOHH was with in the was in the DOHH the was a recombinant DOHH with a The of of iron was of The in the the in was of of of DOHH in a with of of of of in a in the with a The protein was The was with was with a of a of a of of the DOHH DOHH mixture of of the protein human of purified in of was each with The in The of was of the of the DOHH the protein with with with The the was The protein of of of DOHH on a of was a was The of active the was the DOHH was in of each was with the was protein was with of of was the the in of in each DOHH of each protein of DOHH the DOHH of eight in a symmetrical of a the helical of was The purified human recombinant a of protein was is in with the the HEAT-repeat of contains strictly conserved His-Glu the the of in with of the His-Glu with with the eight with was was the of of the with of the conserved His-Glu of the DOHH the of the was of the the of DOHH on the conserved His-Glu of the DOHH of the human recombinant DOHH the of the protein in a The the purified recombinant the of the DOHH on of the was iron was the the of The in the iron in of a of iron the in the of the a of was in DOHH of the iron was with DOHH a of iron in DOHH is the of of with the of purified on a of iron in a of iron of of a in DOHH The of the of a DOHH the of a the of iron was the the of the two DOHH of the the iron was purified the DOHH was with The human the the with of the conserved His-Glu the sites eight of DOHH was in the the the purified the of the the of a the conserved His-Glu in the eight with of a conserved with iron the conserved in the coordination of iron the active of The iron was of of of the in The of a on the two sites two sites the the dyad) the in iron the of of the two iron a with of the iron DOHH The of eight on DOHH of of the DOHH of the of was The two the a the a The with iron the of the of with a of protein was a of with iron the the the was the the DOHH was the of was the of the two of the DOHH the a of the in is the of human DOHH is the of the the the was with a of the the iron is the the active of of the the of with the was in of with of the The eight the with iron the with the of The two iron the of the the the is protein with a of the of the of the the is the is the of the is of the the of iron the DOHH active a a of a of the the was on DOHH a with a the protein in each was the the the the with a of DOHH in the the of the was in the of DOHH on The was purified on the was of the was in of of of each was DOHH a DOHH the iron of the the The DOHH two each of a of in the of The with a of in The iron of was two the of the protein is the of of iron was in of the is the DOHH was in the in with the of DOHH was in a the is of in is a with the in a DOHH DOHH is a of the DOHH was in DOHH in the of human recombinant DOHH is of of of is DOHH of in was a in of was with of each DOHH The of each protein of of on DOHH of in the of DOHH the human with the active the of on the of the the was the a of the of the was in the a of a of in the mixture the of in DOHH the of was the DOHH the a of on DOHH The each of The the The the of The of DOHH the DOHH of eight in a symmetrical of a the helical of was The purified human recombinant a of protein was is in with the the HEAT-repeat of DOHH contains strictly conserved His-Glu the the of in with of the His-Glu with with the eight with was was the of of the with of the conserved His-Glu of the DOHH the of the was of the the of DOHH on the conserved His-Glu of the DOHH of the human recombinant DOHH the of the protein in a The the purified recombinant the of the DOHH on of the was iron was the the of The in the iron in of a of iron the in the of the a of was in DOHH of the iron was with DOHH a The of iron in DOHH is the of of with the of purified on a of iron in a of iron of of a in DOHH The of the of a DOHH the of a the of iron was the the of the two the the with of the conserved His-Glu the sites eight of DOHH was in the the the purified the of the the of a the conserved His-Glu in the eight with of a conserved with iron the conserved in the coordination of iron the active of The iron was of of of the in The of a on the two sites two sites the the dyad) the in iron the of of the two iron a with of the iron DOHH The of eight on DOHH The of of the DOHH of the of was The two the a the a The with iron the of the of with a of protein was a of with iron the the the was the the DOHH was the of was the of the two of the DOHH the a of the in is the of human DOHH is the of the the the was with a of the the iron is the the active of the with iron the with the of The two iron the of the the the DOHH is protein with a of the of the of the the is the is the of the is of the the of iron the DOHH active a a of a of the the was on DOHH a with a the protein in each was the the the the with a of DOHH in the the of the was in the a DOHH the iron of the the The DOHH two each of a of in the of The with a of in The iron of was two the of the protein is the of of iron was in of the is the DOHH was in the in with the of DOHH was in a the is of in is a with the in a DOHH DOHH is a of the DOHH was in DOHH in the of human recombinant DOHH is of of The of on DOHH of in the of DOHH the human with the active the of on the of the the was the a of the of the was in the a of a of in the mixture the of in DOHH the of was the DOHH the a hydroxylase a of tandem of hairpins motifs. of DOHH is composed of two symmetrical each a strictly conserved His-Glu two on each coordination of DOHH on the of a HEAT-repeat the protein a with a a the His-Glu on the two the of the DOHH the of iron the strictly conserved His-Glu in DOHH the active of DOHH the iron coordination the of each of the His-Glu in DOHH a helical DOHH in the two sites iron of iron the of DOHH is the of DOHH the in the the two of the iron a on the of a in the was the on on protein on the is the the the DOHH the the DOHH is in a the with the was in the the DOHH the two of the in the the a on the a a mixture of mixture with the two a of the the in the of It the of iron the two of the DOHH in a with a in of the of iron DOHH the active of the iron of of the DOHH of was a DOHH of iron two two DOHH a of DOHH with iron the iron DOHH the of of was was the active of DOHH with of iron was the DOHH contains two iron coordination sites (one on each is iron active the two iron coordination sites two sites the the dyad) two active a in iron in DOHH a a of the in the His-Glu DOHH of of the a in the iron is a DOHH active with a the iron in of the active in a with two of the a the two iron with It a in DOHH is of conserved of DOHH is iron the of the of the purified in the of a a was was the the protein in the the of DOHH the iron contains a the DOHH a the of the was the iron the DOHH the the the the active of with the of of DOHH is the of the is a HEAT-repeat the of protein the of DOHH a DOHH is on a DOHH in of of iron the of the coordination a coordination of iron the in the of DOHH two iron a a of (one each in the two iron sites of the iron catalyzes the final step in the of protein in DOHH a of in the of The in the of in human human a of in of DOHH DOHH in human of of human the a of in of the of DOHH a DOHH with protein The the of DOHH the iron the DOHH the of of the of the of DOHH the the of of Deoxyhypusine hydroxylase a of tandem of hairpins motifs. of DOHH is composed of two symmetrical each a strictly conserved His-Glu two on each coordination of DOHH on the of a HEAT-repeat the protein a with a a the His-Glu on the two the of the DOHH the of iron the strictly conserved His-Glu in DOHH the active of DOHH the iron coordination the of each of the His-Glu in DOHH a helical DOHH in the two sites iron The of iron the of DOHH is the of DOHH the in the the two of the iron a on the of a in the was the on on protein on the is the the the DOHH the the DOHH is in a the with the was in the the DOHH the two of the in the the a on the a a mixture of mixture with the two a of the the in the of It the of iron the two of the DOHH in a with a in The of the of iron DOHH the active of the iron of of the DOHH of was a DOHH of iron two two DOHH a of DOHH with iron the iron DOHH the of of was was the active of DOHH with of iron was the DOHH contains two iron coordination sites (one on each is iron active the two iron coordination sites two sites the the dyad) two active a in iron in DOHH a a of the in the His-Glu DOHH of of the a in the iron is a DOHH active with a the iron in of the active in a with two of the a the two iron with It a in DOHH is of conserved of DOHH is iron the of the of the purified in the of a a was was the the protein in the the of DOHH the iron contains a the DOHH a the of the was the iron the DOHH the the the the active of with the of of DOHH is the of the DOHH is a HEAT-repeat the of protein the of DOHH a DOHH is on a DOHH in of of iron the of the coordination a coordination of iron the in the of DOHH two iron a a of (one each in the two iron sites of the iron DOHH catalyzes the final step in the of protein in DOHH a of in the of The in the of in human human a of in of DOHH DOHH in human of of human the a of in of the of DOHH a DOHH with protein The the of DOHH the iron the DOHH the of of the of the of DOHH the the of of with with Deoxyhypusine hydroxylase is a HEAT-repeat of