title: Functional Identification of Putrescine C- and N-Hydroxylases.
abstract: The small polyamine putrescine (1,4-diaminobutane) is ubiquitously and abundantly found in all three domains of life. It is a precursor, through N-aminopropylation or N-aminobutylation, for biosynthesis of the longer polyamines spermidine, sym-homospermidine, spermine, and thermospermine and longer and branched chain polyamines. Putrescine is also biochemically modified for purposes of metabolic regulation and catabolism, e.g. N-acetylation and N-glutamylation, and for incorporation into specialized metabolites, e.g. N-methylation, N-citrylation, N-palmitoylation, N-hydroxylation, and N-hydroxycinnamoylation. Only one example is known where putrescine is modified on a methylene carbon: the formation of 2-hydroxyputrescine by an unknown C-hydroxylase. Here, we report the functional identification of a previously undescribed putrescine 2-hydroxylase, a Rieske-type nonheme iron sulfur protein from the β-proteobacteria Bordetella bronchiseptica and Ralstonia solanacearum. Identification of the putrescine 2-hydroxylase will facilitate investigation of the physiological functions of 2-hydroxyputrescine. One known role of 2-hydroxyputrescine has direct biomedical relevance: its role in the biosynthesis of the cyclic hydroxamate siderophore alcaligin, a potential virulence factor of the causative agent of whooping cough, Bordetella pertussis. We also report the functional identification of a putrescine N-hydroxylase from the γ-proteobacterium Shewanella oneidensis, which is homologous to FAD- and NADPH-dependent ornithine and lysine N-monooxygenases involved in siderophore biosynthesis. Heterologous expression of the putrescine N-hydroxylase in E. coli produced free N-hydroxyputrescine, never detected previously in a biological system. Furthermore, the putrescine C- and N-hydroxylases identified here could contribute new functionality to polyamine structural scaffolds, including C-H bond functionalization in synthetic biology strategies.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: Deptartment of Plant Pathology, University of Wisconsin , Madison, Wisconsin, United States.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Lowe-Power
authors.forename: Shin
authors.initials: S
authors.lastname: Kurihara
authors.forename: Stephen
authors.initials: S
authors.lastname: Gonzales
authors.forename: Jacinth
authors.initials: J
authors.lastname: Naidoo
authors.forename: John B
authors.initials: JB
authors.lastname: MacMillan
authors.affiliation: Deptartment of Plant Pathology, University of Wisconsin , Madison, Wisconsin, United States.
authors.forename: Caitilyn
authors.initials: C
authors.lastname: Allen
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Michael
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D007536:Isomerism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D015394:Molecular Structure; D011700:Putrescine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006899:Mixed Function Oxygenases; D011700:Putrescine