The quinohaemoprotein lupanine hydroxylase from Pseudomonas putida.
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.
Hopper
David J
DJ
Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, UK. dvh@aber.ac.uk
Kaderbhai
Mustak A
MA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics*; D045542:PQQ Cofactor; D016958:Pseudomonas putida / Q000201:enzymology*; D015363:Quinolones / Q000737:chemistry; D011809:Quinones / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis
D001426:Bacterial Proteins; D015363:Quinolones; D011809:Quinones; D011994:Recombinant Proteins; D045542:PQQ Cofactor; C023929:lupanine hydroxylase; D000587:Oxidoreductases Acting on CH-NH Group Donors