Publications

• Studies on lysine:N6-hydroxylation by cell-free systems of Aerobacter aerogenes 62-1.

  Goh C.J., Szczepan E.W., Menhart N., Viswanatha T.

  Electron microscopic examination has revealed the vesicular nature of the membrane component, of the cell-free system of Aerobacter aerogenes 62-1, which catalyses lysine: N6-hydroxylation. Regardless of the orientation of the vesicles, N-hydroxylation process is still stimulated by pyruvate. Both ... >> More

  Electron microscopic examination has revealed the vesicular nature of the membrane component, of the cell-free system of Aerobacter aerogenes 62-1, which catalyses lysine: N6-hydroxylation. Regardless of the orientation of the vesicles, N-hydroxylation process is still stimulated by pyruvate. Both pyruvate oxidation and lysine: N6-hydroxylation were inhibited by protonophores and Gramicidin S. << Less

  Biochim Biophys Acta 990:240-245(1989) [PubMed] [EuropePMC]

• Isolation and some properties of lysine N6-hydroxylase from Escherichia coli strain EN222.

  Plattner H.J., Pfefferle P., Romaguera A., Waschutza S., Diekmann H.

  Lysine N6-hydroxylase was isolated as a soluble enzyme from the supernatant after ultrasonication of Escherichia coli strain EN222 which contained the structural gene on a multicopy plasmid (as described by Engelbrecht and Braun in 1986). The apoenzyme prepared by dialysis was purified by ammonium ... >> More

  Lysine N6-hydroxylase was isolated as a soluble enzyme from the supernatant after ultrasonication of Escherichia coli strain EN222 which contained the structural gene on a multicopy plasmid (as described by Engelbrecht and Braun in 1986). The apoenzyme prepared by dialysis was purified by ammonium sulfate precipitation and fast protein liquid chromatography using Superose 12 and Mono Q columns. The molecular mass as determined by gel filtration was 200 kDa and 50 kDa by SDS/polyacrylamide gel electrophoresis. The enzyme binds 0.79 molecule FAD/50 kDa. The activity of the enzyme is strictly dependent on NADPH. Its properties are similar to other flavoprotein monooxygenases of the EC group 1.14.13. << Less

  Biol Met 2:1-5(1989) [PubMed] [EuropePMC]

• FAD and substrate analogs as probes for lysine N6-hydroxylase from Escherichia coli EN 222.

  Macheroux P., Plattner H.J., Romaguera A., Diekmann H.

  Lysine N6-hydroxylase catalyzes the hydroxylation of the N-terminal amino function of L-lysine at the expense of NADPH and molecular oxygen. The enzyme also requires FAD for its catalytic activity. Unlike other flavoprotein monooxygenases, binding of FAD is rather weak with a Kd of 30 microM at 4 ... >> More

  Lysine N6-hydroxylase catalyzes the hydroxylation of the N-terminal amino function of L-lysine at the expense of NADPH and molecular oxygen. The enzyme also requires FAD for its catalytic activity. Unlike other flavoprotein monooxygenases, binding of FAD is rather weak with a Kd of 30 microM at 4 degrees C. The spectral properties of FAD bound to lysine N6-hydroxylase are very similar to free oxidized FAD. In the absence of substrate, the enzyme has an NADPH oxidase activity which results in the generation of hydrogen peroxide. With increasing concentration of L-lysine, the NADPH oxidase activity is enhanced up to 10-fold and the generation of hydrogen peroxide decreases. At the same time, the substrate is hydroxylated. Km values for L-lysine and FAD were determined as 105 microM and 0.7 microM, respectively. Utilizing FAD analogs, we could demonstrate that L-lysine exerts its effector role mostly on the reductive half reaction of the overall catalytic cycle. Prolonged incubation of the enzyme with either 8-chloro- or 8-fluoro-FAD gave rise to a covalently attached flavin which is formed as a result of the nucleophilic attack of a thiolate on the 8-position of the flavin. Several lines of evidence indicate that the reaction takes place in the FAD binding site of the protein. The substrate specificity was investigated using amino acids with various lengths of side chain. L-Lysine and derivatives with similar side chain length are hydroxylated by lysine N6-hydroxylase. Ornithine, the lower homolog of lysine, was not hydroxylated and did not affect the NADPH oxidase activity of the enzyme. On the other hand, homolysine accelerated the rate of NADPH oxidation but was not hydroxylated. Additional requirements for efficient hydroxylation were also investigated using a variety of substrate analogs. From these studies a schematic structure of the active site of the enzyme was deduced. Sequence comparison of the FAD binding site of various flavoproteins revealed possible factors for weak binding of the cofactor in the case of lysine N6-hydroxylase. << Less

  Eur J Biochem 213:995-1002(1993) [PubMed] [EuropePMC]

• Recombinant lysine:N(6)-hydroxylase: effect of cysteine-->alanine replacements on structural integrity and catalytic competence.

  Dick S., Siemann S., Frey H.E., Lepock J.R., Viswanatha T.

  Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a ... >> More

  Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a major change in the thermal stability, the affinity for L-lysine and FAD, as well as the k(cat) for mono-oxygenase activity of the protein. However, when the susceptibility to modification by either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 2,6-dichlorophenol indophenol (DPIP) serves as the criterion for monitoring conformational change(s) in rIucD and its muteins, Cys146-->Ala and Cys166-->Ala substitutions are found to induce an enhancement in the reactivity of one of the protein's remaining cysteine residues with concomitant diminution of mono-oxygenase function. In addition, the systematic study of cysteine-->alanine replacement has led to the identification of rIucD's Cys166 as the exposed residue which is detectable during the reaction of the protein with DTNB but not with iodoacetate. Substitution of Cys51 of rIucD with alanine results in an increase in mono-oxygenase activity (approx. 2-fold). Such replacement, unlike those of other cysteine residues, also enables the covalent DPIP conjugate of the protein to accommodate FAD in its catalytic function. A possible role of rIucD's Cys51 in the modulation of its mono-oxygenase function is discussed. << Less

  Biochim Biophys Acta 1594:219-233(2002) [PubMed] [EuropePMC]

• Aerobactin biosynthesis and transport genes of plasmid ColV-K30 in Escherichia coli K-12.

  de Lorenzo V., Bindereif A., Paw B.H., Neilands J.B.

  The iron-regulated aerobactin operon, about 8 kilobase pairs in size, of the Escherichia coli plasmid ColV-K30 was shown by deletion and subcloning analyses to consist of at least five genes for synthesis (iuc, iron uptake chelate) and transport (iut, iron uptake transport) of the siderophore. The ... >> More

  The iron-regulated aerobactin operon, about 8 kilobase pairs in size, of the Escherichia coli plasmid ColV-K30 was shown by deletion and subcloning analyses to consist of at least five genes for synthesis (iuc, iron uptake chelate) and transport (iut, iron uptake transport) of the siderophore. The gene order iucABCD iutA was established. The genes were mapped within restriction nuclease fragments of a cloned 16.3-kilobase-pair HindIII fragment. Stepwise deletion and subsequent minicell analysis of the resulting plasmids allowed assignment of four of the five genes to polypeptides of molecular masses 63,000, 33,000 53,000, and 74,000 daltons, respectively. The 74-kilodalton protein, the product of gene iutA, is the outer membrane receptor for ferric aerobactin, whereas the remaining three proteins are involved in biosynthesis of aerobactin. The 33-kilodalton protein, the product of gene iucB, was identified as N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase (acetylase) by comparison of enzyme activity in extracts from various deletion mutants. The 53-kilodalton protein, the product of gene iucD, is required for oxygenation of lysine. The 63-kilodalton protein, the product of gene iucA, is assigned to the first step of the aerobactin synthetase reaction. The product of gene iucC, so far unidentified, performs the second and final step in this reaction. This is based on the chemical characterization of two precursor hydroxamic acids (N epsilon-acetyl-N epsilon-hydroxylysine and N alpha-citryl-N epsilon-acetyl-N epsilon-hydroxylysine) isolated from a strain carrying a 0.3-kilobase-pair deletion in the iucC gene. The results support the existence of a biosynthetic pathway in which aerobactin arises by oxygenation of lysine, acetylation of the N epsilon-hydroxy function, and condensation of 2 mol of the resulting aminohydroxamic acid with citric acid. << Less

  J. Bacteriol. 165:570-578(1986) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Physico-chemical characterization of a recombinant cytoplasmic form of lysine: N6-hydroxylase.

  Thariath A.M., Fatum K.L., Valvano M.A., Viswanatha T.

  A recombinant cytoplasmic preparation of lysine: N6-hydroxylase, IucD398, with a deletion of 47 amino acids at the N-terminus, was purified to homogeneity. IucD398 is capable of N-hydroxylation of L-lysine upon supplementation with FAD and NADPH. The enzyme is stringently specific with L-lysine an ... >> More

  A recombinant cytoplasmic preparation of lysine: N6-hydroxylase, IucD398, with a deletion of 47 amino acids at the N-terminus, was purified to homogeneity. IucD398 is capable of N-hydroxylation of L-lysine upon supplementation with FAD and NADPH. The enzyme is stringently specific with L-lysine and (S)-2-aminoethyl-L-cysteine serving as substrates. Protonophores, FCCP and CCCP, as well as cinnamylidene, have been found to serve as potent inhibitors of lysine: N6-hydroxylation by virtue of their ability to interfere in the reduction of the flavin cofactor. << Less

  Biochim Biophys Acta 1203:27-35(1993) [PubMed] [EuropePMC]