Publications

• The quinohaemoprotein lupanine hydroxylase from Pseudomonas putida.

  Hopper D.J., Kaderbhai M.A.

  Lupanine hydroxylase catalyses the first reaction in the catabolism of the alkaloid lupanine by Pseudomonas putida. It dehydrogenates the substrate, which can then be hydrated. It is a monomeric protein of M(r) 72,000 and contains a covalently bound haem and a molecule of PQQ. The gene for this en ... >> More

  Lupanine hydroxylase catalyses the first reaction in the catabolism of the alkaloid lupanine by Pseudomonas putida. It dehydrogenates the substrate, which can then be hydrated. It is a monomeric protein of M(r) 72,000 and contains a covalently bound haem and a molecule of PQQ. The gene for this enzyme has been cloned and sequenced and the derived protein sequence has a 26 amino acid signal sequence at the N-terminal for translocation of the protein to the periplasm. Many of the features seen in the sequence of lupanine hydroxylase are common with other quinoproteins including the W-motifs that are characteristic of the eight-bladed propeller structure of methanol dehydrogenase. However, the unusual disulfide bridge between adjacent cysteines that is present in some PQQ-containing enzymes is absent in lupanine hydroxylase. The C-terminal domain contains characteristics of a cytochrome c and overall the sequence shows similarities with that of the quinohaemoprotein, alcohol dehydrogenase from Comamonas testosteroni. The gene coding for lupanine hydroxylase has been successfully expressed in Escherichia coli and a procedure has been developed to renature and reactivate the enzyme, which was found to be associated with the inclusion bodies. Reactivation required addition of PQQ and was dependent on calcium ions. << Less

  Biochim. Biophys. Acta 1647:110-115(2003) [PubMed] [EuropePMC]

• Lupanine hydroxylase, a quinocytochrome c from an alkaloid-degrading Pseudomonas sp.

  Hopper D.J., Rogozinski J., Toczko M.

  Lupanine 17-hydroxylase, the first enzyme in the pathway for bacterial degradation of the alkaloid, lupanine, was purified from a Pseudomonas sp. The enzyme acts by initial dehydrogenation of the substrate, and cytochrome c was used as electron acceptor in assays. It had an Mr of 66,000 by ultrace ... >> More

  Lupanine 17-hydroxylase, the first enzyme in the pathway for bacterial degradation of the alkaloid, lupanine, was purified from a Pseudomonas sp. The enzyme acts by initial dehydrogenation of the substrate, and cytochrome c was used as electron acceptor in assays. It had an Mr of 66,000 by ultracentrifuge studies and 74,000 by gel filtration. The visible absorption spectrum was that of a cytochrome c, and a stoicheiometry of one haem group per molecule of enzyme was calculated. SDS/PAGE gave a single band of Mr 72,000 containing the haem group. The enzyme also contained pyrroloquinoline quinone (PQQ), which could be removed by isoelectric focusing. The apoenzyme was reconstituted to full activity with addition of PQQ, and a stoicheiometry of one molecule of PQQ per molecule of enzyme was calculated. Steady-state kinetics gave values of 3.6 microM for the Km for lupanine, 21.3 microM for the Km for cytochrome c and 217 s-1 for the Kcat. << Less

  Biochem. J. 279:105-109(1991) [PubMed] [EuropePMC]