Characterization of recombinant biosynthetic precursors of the cysteine tryptophylquinone cofactors of l-lysine-epsilon-oxidase and glycine oxidase from Marinomonas mediterranea

Chacón-Verdú, María Dolores; Campillo-Brocal, Jonatan C.; Lucas-Elı́o, Patricia; Davidson, Victor L.; Sánchez-Amat, Antonio

doi:10.1016/j.bbapap.2014.12.018

Cited by 20 publications

(65 citation statements)

References 39 publications

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“…Expression of either active LodA or GoxA with the mature CTQ cofactor requires that lodA and goxA each be co-expressed with lodB and goxB , respectively 16 . Expression of either lodA or goxA in the absence of lodB or goxB , respectively, leads to the production of inactive precursor forms of the proteins that lack CTQ 17 .…”

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confidence: 99%

Interaction of GoxA with Its Modifying Enzyme and Its Subunit Assembly Are Dependent on the Extent of Cysteine Tryptophylquinone Biosynthesis

et al. 2016

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GoxA is a glycine oxidase bearing a protein-derived cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications catalyzed by a flavoprotein, GoxB. Two forms of GoxA were isolated. An active form with mature CTQ and an inactive precursor protein that lacked CTQ. The active GoxA was present as a homodimer with no detectable affinity for GoxB, whereas the precursor was isolated as a monomer in a tight complex with one GoxB. Thus, the interaction of GoxA with GoxB and subunit assembly of mature GoxA are each dependent on the extent of CTQ biosynthesis.

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confidence: 99%

Interaction of GoxA with Its Modifying Enzyme and Its Subunit Assembly Are Dependent on the Extent of Cysteine Tryptophylquinone Biosynthesis

et al. 2016

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“…A possible explanation for the loss of the remaining 95% of the total soluble D512A LodA is that it was the previously characterized unmodified precursor protein which is unstable under the conditions used for ion-exchange chromatography, and lost when trying to purify it further by this technique. In order to test this hypothesis, the same procedure was used with preLodA, the precursor which may be isolated by expression of lodA in the absence of lodB 25 . Consistent with this explanation, no preLodA was recovered after subjecting it to the same chromatographic protocol.…”

Section: Resultsmentioning

confidence: 99%

“…The generation of the D512A mutation was previously described 25 . Cell growth and induction, preparation of cell extracts and initial purification of the His-tagged proteins by affinity chromatography using a Ni-NTA resin were as previously described 26 .…”

Section: Methodsmentioning

confidence: 99%

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Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis

et al. 2017

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The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the auto-catalytic hydroxylation of a specific Trp residue at the C-7 position on the indole side-chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis, as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provide evidence that copper is required for the initial hydroxylation of the precursor protein; and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed the fluorescence appears and when it is added back to the protein the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the un-activated Trp side-chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.

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“…15) Chacón-Verdú et al reported that LodB is required for the complete synthesis of the LodA cofactor by mass spectrometry analysis. 19) We recently investigated the structural characteristics of the LodA variant and obtained unequivocal structural evidence for the presence of CTQ in the active site of LodA. 3) Amine dehydrogenases (QHNDH, EC 1.4.9.1) from Pseudomonas putida 20,21) and Paracoccus denitrificans 22) also CTQ, which are formed by QhpG, are responsible for posttranslational modification.…”

Section: Effects Of Lodb Coexpressed With Loda7mutmentioning

confidence: 99%

Heterologous production of l-lysine ε-oxidase by directed evolution using a fusion reporter method

Matsui

Asano

2015

Bioscience, Biotechnology, and Biochemistry

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For the heterologous production of l-lysine ε-oxidase (LodA), we constructed a new plasmid carrying LodA gene fused in-frame with an antibiotic (phleomycine) resistant gene. The new plasmid was randomly mutated and the mutated plasmids were transformed into Escherichia coli BL21 (DE3) harboring lodB, which encodes a protein (LodB) acting in posttranslational modification of LodA, and active mutants were selected by phleomycin resistance and oxidase activities. One soluble LodA variant isolated by this method contained six silent mutations and one missense mutation. At these mutation points, the codon adaptations at Lys92, Ala550, and Thr646, and the amino acid substitution at His286 to Arg contributed to the production of its functional form. The active form of LodA variant was induced by post-modification of LodB in the heterologous coexpression, and the activity increased with additional NaCl and heat treatment. This is the first report of heterologous production of LodA by random mutagenesis.

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Characterization of recombinant biosynthetic precursors of the cysteine tryptophylquinone cofactors of l-lysine-epsilon-oxidase and glycine oxidase from Marinomonas mediterranea

Cited by 20 publications

References 39 publications

Interaction of GoxA with Its Modifying Enzyme and Its Subunit Assembly Are Dependent on the Extent of Cysteine Tryptophylquinone Biosynthesis

Interaction of GoxA with Its Modifying Enzyme and Its Subunit Assembly Are Dependent on the Extent of Cysteine Tryptophylquinone Biosynthesis

Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis

Heterologous production of l-lysine ε-oxidase by directed evolution using a fusion reporter method

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