Publications

• Kinetic and Structural Characterization of a Flavin-Dependent Putrescine N-Hydroxylase from Acinetobacter baumannii.

  Lyons N.S., Bogner A.N., Tanner J.J., Sobrado P.

  <i>Acinetobacter baumannii</i> is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of <i>A. baumannii</i> has limited the use of standard antibiotics, highlighting a need for new drugs ... >> More

  <i>Acinetobacter baumannii</i> is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of <i>A. baumannii</i> has limited the use of standard antibiotics, highlighting a need for new drugs that exploit novel mechanisms of pathogenicity. Disrupting iron acquisition by inhibiting the biosynthesis of iron-chelating molecules (siderophores) secreted by the pathogen is a potential strategy for developing new antibiotics. Here we investigated FbsI, an <i>N</i>-hydroxylating monooxygenase involved in the biosynthesis of fimsbactin A, the major siderophore produced by <i>A. baumannii.</i> FbsI was characterized using steady-state and transient-state kinetics, spectroscopy, X-ray crystallography, and small-angle X-ray scattering. FbsI was found to catalyze the <i>N</i>-hydroxylation of the aliphatic diamines putrescine and cadaverine. Maximum coupling of the reductive and oxidative half-reactions occurs with putrescine, suggesting it is the preferred (<i>in vivo</i>) substrate. FbsI uses both NADPH and NADH as the reducing cofactor with a slight preference for NADPH. The crystal structure of FbsI complexed with NADP⁺ was determined at 2.2 Å resolution. The structure exhibits the protein fold characteristic of Class B flavin-dependent monooxygenases. FbsI is most similar in 3D structure to the cadaverine <i>N</i>-hydroxylases DesB and DfoA. Small-angle X-ray scattering shows that FbsI forms a tetramer in solution like the <i>N</i>-hydroxylating monooxygenases of the SidA/IucD/PvdA family. A model of putrescine docked into the active site provides insight into substrate recognition. A mechanism for the catalytic cycle is proposed where dehydration of the C4a-hydroxyflavin intermediate is partially rate-limiting, and the hydroxylated putrescine product is released before NADP⁺. << Less

  Biochemistry 61:2607-2620(2022) [PubMed] [EuropePMC]

• Identification of a gene cluster that directs putrebactin biosynthesis in Shewanella species: PubC catalyzes cyclodimerization of N-hydroxy-N-succinylputrescine.

  Kadi N., Arbache S., Song L., Oves-Costales D., Challis G.L.

  Putrebactin is a dihydroxamate iron chelator produced by the metabolically versatile marine bacterium Shewanella putrefaciens. It is a macrocyclic dimer of N-hydroxy-N-succinyl-putrescine (HSP) and is structurally related to desferrioxamine E, which is a macrocyclic trimer of N-hydroxy-N-succinyl- ... >> More

  Putrebactin is a dihydroxamate iron chelator produced by the metabolically versatile marine bacterium Shewanella putrefaciens. It is a macrocyclic dimer of N-hydroxy-N-succinyl-putrescine (HSP) and is structurally related to desferrioxamine E, which is a macrocyclic trimer of N-hydroxy-N-succinyl-cadaverine (HSC). We recently showed that DesD, a member of the NIS synthetase superfamily, catalyzes the key step in desferrioxamine E biosynthesis: ATP-dependent trimerisation and macrocylization of HSC. Here we report identification of a conserved gene cluster in the sequenced genomes of several Shewanella species, including Shewanella putrefaciens, which is hypothesized to direct putrebactin biosynthesis from putrescine, succinyl-CoA and molecular oxygen. The pubC gene within this gene cluster encodes a protein with 65% similarity to DesD. We overexpressed pubC from Shewanella species MR-4 and MR-7 in E. coli. The resulting His6-PubC fusion proteins were purified by Ni-NTA affinity and gel filtration chromatography. The recombinant proteins were shown to catalyze ATP-dependent cyclodimerization of HSP to form putrebactin. The uncyclized dimer of HSP pre-putrebactin was shown to be an intermediate in the conversion of two molecules of HSP to putrebactin. The data indicate that pre-putrebactin is converted to putrebactin via PubC-catalyzed activation of the carboxyl group by adenylation, followed by PubC-catalyzed nucleophilic attack of the amino group on the carbonyl carbon of the acyl adenylate. This mechanism for macrocycle formation is very different from the mechanism involved in the biosynthesis of many other macrocyclic natural products, where already-activated acyl thioesters are converted by thioesterase domains of polyketide synthases and nonribosomal peptide synthetases to macrocycles via covalent enzyme bound intermediates. The results of this study demonstrate that two closely related enzymes, PubC and DesD, catalyze specific cyclodimerization and cyclotrimerization reactions, respectively, of structurally similar substrates, raising intriguing questions regarding the molecular mechanism of specificity. << Less

  J. Am. Chem. Soc. 130:10458-10459(2008) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Functional Identification of Putrescine C- and N-Hydroxylases.

  Li B., Lowe-Power T., Kurihara S., Gonzales S., Naidoo J., MacMillan J.B., Allen C., Michael A.J.

  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 longe ... >> More

  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. << Less

  ACS Chem. Biol. 11:2782-2789(2016) [PubMed] [EuropePMC]