Publications

• Purification in an active state and properties of the 3-step phytoene desaturase from Rhodobacter capsulatus overexpressed in Escherichia coli.

  Raisig A., Bartley G., Scolnik P., Sandmann G.

  The phytoene desaturase gene from Rhodobacter capsulatus was expressed in Escherichia coli and the resulting protein was purified. The purification steps involved were ammonium sulfate precipitation and ion exchange chromatography, leading to a homogenous protein of 57 kDa with high specific enzym ... >> More

  The phytoene desaturase gene from Rhodobacter capsulatus was expressed in Escherichia coli and the resulting protein was purified. The purification steps involved were ammonium sulfate precipitation and ion exchange chromatography, leading to a homogenous protein of 57 kDa with high specific enzymatic activity. The purified enzyme was characterized with respect to substrate specificity and product formation. In addition to phytoene, the intermediates, phytofluene and zeta-carotene, were both converted to neurosporene, the end product of the reaction. Furthermore, 1,2-epoxy phytoene was a suitable substrate whereas the C30 diapophytoene was not. The Km values for phytoene and zeta-carotene were determined to be 33.3 and 16.6 microM, respectively. The desaturation reaction is dependent on the cofactor FAD. Oxidized nicotine nucleotides or ATP had no positive effect. The Km value for FAD was 4.9 microM. Inhibition of the desaturation reaction was observed with diphenylamine. << Less

  J. Biochem. 119:559-564(1996) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Alteration of product specificity of Rhodobacter sphaeroides phytoene desaturase by directed evolution.

  Wang C.W., Liao J.C.

  Phytoene desaturases occurring in nature convert phytoene to either neurosporene or lycopene in most eubacteria. Approximately 10% of known phytoene desaturases, as in Rhodobacter, produce neurosporene, whereas the rest produce lycopene. These two types of enzymes, although similar in function, ha ... >> More

  Phytoene desaturases occurring in nature convert phytoene to either neurosporene or lycopene in most eubacteria. Approximately 10% of known phytoene desaturases, as in Rhodobacter, produce neurosporene, whereas the rest produce lycopene. These two types of enzymes, although similar in function, have relatively low similarity (below 60%) in terms of nucleotide or amino acid sequence. The mechanism controlling the product specificity of these enzymes is unclear. Here we used directed evolution to change the product of Rhodobacter sphaeroides phytoene desaturase (crtI gene product), a neurosporene-producing enzyme, to lycopene. Two generations of random mutagenesis were performed, from which three positive mutants were isolated and sequenced. We then used site-directed mutagenesis to determine the effect of each amino acid change. Gathering information from random mutagenesis, we further recombined the beneficial mutations by site-directed mutagenesis and increased the percent of lycopene production to 90%. << Less

  J Biol Chem 276:41161-41164(2001) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.