Publications

• Aspergillus fumigatus SidA is a highly specific ornithine hydroxylase with bound flavin cofactor.

  Chocklett S.W., Sobrado P.

  Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N(5)-hydroxylated l-ornithines essential for iron binding. A. fumigatus siderophore A (Af SidA) catalyzes the flavin- and NADPH-depe ... >> More

  Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N(5)-hydroxylated l-ornithines essential for iron binding. A. fumigatus siderophore A (Af SidA) catalyzes the flavin- and NADPH-dependent hydroxylation of l-ornithine in ferrichrome biosynthesis. Af SidA was recombinantly expressed and purified as a soluble tetramer and is the first member of this class of flavin monooxygenases to be isolated with a bound flavin cofactor. The enzyme showed typical saturation kinetics with respect to l-ornithine while substrate inhibition was observed at high concentrations of NADPH and NADH. Increasing amounts of hydrogen peroxide were measured as a function of reduced nicotinamide coenzyme concentration, indicating that inhibition was caused by increased uncoupling. Af SidA is highly specific for its amino acid substrate, only hydroxylating l-ornithine. An 8-fold preference in the catalytic efficiency was determined for NADPH compared to NADH. In the absence of substrate, Af SidA can be reduced by NADPH, and a C4a-(hydro)peroxyflavin intermediate is observed. The decay of this intermediate is accelerated by l-ornithine binding. This intermediate was only stabilized by NADPH and not by NADH, suggesting a role for NADP(+) in the stabilization of intermediates in the reaction of Af SidA. NADP(+) is a competitive inhibitor with respect to NADPH, demonstrating that Af SidA forms a ternary complex with NADP(+) and l-ornithine during catalysis. The data suggest that Af SidA likely proceeds by a sequential kinetic mechanism. << Less

  Biochemistry 49:6777-6783(2010) [PubMed] [EuropePMC]

• dffA gene from Aspergillus oryzae encodes L-ornithine N5-oxygenase and is indispensable for deferriferrichrysin biosynthesis.

  Yamada O., Na Nan S., Akao T., Tominaga M., Watanabe H., Satoh T., Enei H., Akita O.

  We identified and analyzed the dffA gene from Aspergillus oryzae which encodes L-ornithine N5-oxygenase involved in the biosynthesis of deferriferrichrysin, a type of siderophore which is a low-molecular-weight iron chelating compound. From among more than 20,000 clones in an A. oryzae EST (expres ... >> More

  We identified and analyzed the dffA gene from Aspergillus oryzae which encodes L-ornithine N5-oxygenase involved in the biosynthesis of deferriferrichrysin, a type of siderophore which is a low-molecular-weight iron chelating compound. From among more than 20,000 clones in an A. oryzae EST (expressed sequence tag) library, we found only one clone encoding a protein that exhibited homology to theUstilago maydis sid1 protein (Sid1) and Pseudomonas aeruginosa pvdA protein (PvdA), both known as the only examples of L-ornithine N5-oxygenase. The complete gene sequence shows that the dffA gene includes a 1575-bp open reading frame (ORF), one 66-bp intorn, which is a typical intorn length inA. oryzae, and encodes 502 amino acids with putative FAD-binding, NADP-binding, and 'FATGY' motifs, which are conserved inN-hydroxylating enzymes. As well as that of the U. maydis sid1 gene,dffA gene expression was induced under iron-limited conditions, and the promoter region has several GATA-type transcription regulator binding motifs. When the dffA gene was expressed under the control of the a-amylase promoter in A. oryzae, transformants revealed inducible high L-ornithine N5-oxygenase activities. In addition, a dffA gene disruptant showed no deferriferrichrysin production even under iron-limited conditions. These results clearly suggest that the dffA gene is indispensable for deferriferrichrysin biosynthesis in A. oryzae. << Less

  J. Biosci. Bioeng. 95:82-88(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• sid1, a gene initiating siderophore biosynthesis in Ustilago maydis: molecular characterization, regulation by iron, and role in phytopathogenicity.

  Mei B., Budde A.D., Leong S.A.

  Iron uptake in Ustilago maydis is mediated by production of extracellular hydroxamate siderophores. L-Or-nithine N5-oxygenase catalyzes hydroxylation of L-ornithine, which is the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. We have characterized sid1, a gene cod ... >> More

  Iron uptake in Ustilago maydis is mediated by production of extracellular hydroxamate siderophores. L-Or-nithine N5-oxygenase catalyzes hydroxylation of L-ornithine, which is the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. We have characterized sid1, a gene coding for this enzyme, by complementation in trans, gene disruption, and DNA sequence analysis. A comparison of genomic DNA and cDNA sequences has shown that the gene is interrupted by three introns. The putative amino acid sequence revealed similarity with Escherichia coli lysine N6-hydroxylase, which catalyzes the hydroxylation of lysine, the first step in biosynthesis of aerobactin. Two transcription initiation points have been determined, both by PCR amplification of the 5' end of the mRNA and by primer extension. A 2.3-kb transcript which accumulates in cells grown under low iron conditions was detected by Northern hybridization. A less abundant 2.7-kb transcript was observed in cells grown in iron-containing medium. By contrast, constitutive accumulation of the 2.3-kb transcript was observed in a mutant carrying a disruption of urbs1, a gene involved in regulation of siderophore biosynthesis. Analysis of the pathogenicity of mutants carrying a null allele of sid1 suggests that the biosynthetic pathway of siderophores does not play an essential role in the infection of maize by U. maydis. << Less

  Proc. Natl. Acad. Sci. U.S.A. 90:903-907(1993) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural insight into the mechanism of oxygen activation and substrate selectivity of flavin-dependent N-hydroxylating monooxygenases.

  Franceschini S., Fedkenheuer M., Vogelaar N.J., Robinson H.H., Sobrado P., Mattevi A.

  SidA from the human pathogen Aspergillus fumigatus catalyzes the generation of N(5)-hydroxyornithine in the biosynthesis of siderophores, a reaction essential for virulence. The crystal structures of SidA in complex with ornithine and lysine reveal the geometry of the interactions among flavin, NA ... >> More

  SidA from the human pathogen Aspergillus fumigatus catalyzes the generation of N(5)-hydroxyornithine in the biosynthesis of siderophores, a reaction essential for virulence. The crystal structures of SidA in complex with ornithine and lysine reveal the geometry of the interactions among flavin, NADP(+), and the substrate amine group that underlie the hydroxylation reaction. The structural elucidation of the enzyme in complex with arginine provides insight into the role of electrostatics and hydrogen bonding in the mechanism of oxygen activation in this family of enzymes. << Less

  Biochemistry 51:7043-7045(2012) [PubMed] [EuropePMC]

• Comprehensive spectroscopic, steady state, and transient kinetic studies of a representative siderophore-associated flavin monooxygenase.

  Mayfield J.A., Frederick R.E., Streit B.R., Wencewicz T.A., Ballou D.P., DuBois J.L.

  Many siderophores used for the uptake and intracellular storage of essential iron contain hydroxamate chelating groups. Their biosyntheses are typically initiated by hydroxylation of the primary amine side chains of l-ornithine or l-lysine. This reaction is catalyzed by members of a widespread fam ... >> More

  Many siderophores used for the uptake and intracellular storage of essential iron contain hydroxamate chelating groups. Their biosyntheses are typically initiated by hydroxylation of the primary amine side chains of l-ornithine or l-lysine. This reaction is catalyzed by members of a widespread family of FAD-dependent monooxygenases. Here the kinetic mechanism for a representative family member has been extensively characterized by steady state and transient kinetic methods, using heterologously expressed N(5)-l-ornithine monooxygenase from the pathogenic fungus Aspergillus fumigatus. Spectroscopic data and kinetic analyses suggest a model in which a molecule of hydroxylatable substrate serves as an activator for the reaction of the reduced flavin and O(2). The rate acceleration is only ∼5-fold, a mild effect of substrate on formation of the C4a-hydroperoxide that does not influence the overall rate of turnover. The effect is also observed with the bacterial ornithine monooxygenase PvdA. The C4a-hydroperoxide is stabilized in the absence of hydroxylatable substrate by the presence of bound NADP(+) (t(½) = 33 min, 25 °C, pH 8). NADP(+) therefore is a likely regulator of O(2) and substrate reactivity in the siderophore-associated monooxygenases. Aside from the activating effect of the hydroxylatable substrate, the siderophore-associated monooxygenases share a kinetic mechanism with the hepatic microsomal flavin monooxygenases and bacterial Baeyer-Villiger monooxygenases, with which they share only moderate sequence homology and from which they are distinguished by their acute substrate specificity. The remarkable specificity of the N(5)-l-ornithine monooxygenase-catalyzed reaction suggests added means of reaction control beyond those documented in related well characterized flavoenzymes. << Less

  J. Biol. Chem. 285:30375-30388(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.