Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1995-11-28
1998-09-01
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435136, 435189, 435190, 435191, 43525411, 4352543, 4352523, 4353201, 435325, 536 232, 935 22, C12P 700, C12P 740, C12N 902, C07H 2104
Patent
active
058010246
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The invention relates to a new gene, a recombinant DNA molecule comprising at least a part of that gene, an RNA molecule derived therefrom and a new polypeptide (or protein), as well as a host cell transformed at least with such recombinant DNA molecule, in particular a transformed filamentous fungus. In addition, the invention relates to processes for the use of the new protein or the new host cell in enzymatic conversions using monooxygenases, more specifically enzymes from the cytochrome P450 superfamily.
In particular, the invention relates to the isolation, characterization and use of a gene coding for an NADPH cytochrome P450 oxidoreductase from filamentous fungi. Further, this application discloses the use of this gene for increasing cytochrome P450-mediated enzymatic activities.
In nature, monooxygenase reactions are responsible for a large number of conversions of both endogenous and exogenous compounds. The monooxygenases can be divided into many different classes of enzymes. One of those classes is the family of cytochrome P450 monooxygenases. These are enzymes occurring in both prokaryotes and eukaryotes.
The spatial structure of these proteins has been highly conserved during evolution. The most characteristic part of each cytochrome P450 is the presence of a haem group which is covalently bound to the protein by a sulfur-iron bond. The sulfur always originates from a cysteine molecule. The iron atom is located in the middle of a porphyrin ring with which the atom has four bonds. The sixth ligand of the iron atom is involved in the catalysis of the reaction. An oxygen is bound to it during the process. Accordingly, the manner in which the haem group is bound to the protein is characteristic of this class of proteins and provides for a specific absorption peak at approximately P450 nanometer (CO reduced form).
Cytochrome P450 proteins are only functional in an enzyme complex together with one or two other proteins which take care of the transfer of electrons from NAP(P)H to the active center of the cytochrome P450.
Roughly, the class of cytochrome P450 enzyme systems can be divided in two subclasses, viz. the eukaryotic microsomal P450s and the class of "prokaryotic-like" P450s. The general characteristics of the eukaryotic microsomal P450s are that they are bound to the membranes of eukaryotes, that they are two component enzyme systems (reaction specific cytochrome P450 and general NADPH cytochrome P450 oxidoreductase) and that the reaction is NADPH-dependent. The general characteristic of the other subclass is that the enzyme complexes consist of three components. This subclass can be further subdivided in two groups, viz. the bacterial P450s (soluble, mostly NADH dependent, complex consisting of a reaction specific cytochrome P450 and a general Fe-S protein and NADH reductase) and the eukaryotic P450s occurring in organelles such as mitochondria (membrane-bound, mostly NADPH dependent, complex consisting of reaction specific cytochrome P450 and a general Fe-S protein and NAD(P)H reductase).
The cytochrome P450 proteins of the first subclass accordingly complex to the membrane with the protein NADPH cytochrome P450 oxidoreductase. This protein, like cytochrome P450s, is found in the microsomal (endoplasmatic reticulum) membrane of lower eukaryotes, plants and animals.
Less is known about the structure of this protein than about that of cytochrome P450s, but here, too, a high degree of structural conservation must have occurred during the evolution, in view of the fact that functional exchange of NADPH cytochrome P450 oxidoreductase has been demonstrated between yeast and mammal systems.
In microsomal enzyme systems of various eukaryotes, it has been demonstrated in different cases that particularly after induction of a specific cytochrome P450, the oxidoreductase could be present in a minor proportion. Thus, the amount of reductase would be determinative or the rate of conversion of compounds to be modified by cytochrome P450. Increasing the amount of NADPH cytoc
REFERENCES:
van Gorcom et al. (1990) Isolation and molecular characterisation of the benzoate-para-hydroxylase gene (bphA) of Aspergillus niger: A member of a new gene family of the cytochrome P450 superfamily, Mol. Gen. Genet. 223: 192-197, Dec. 1990.
Ngo et al. (1994) Computational Complexity, Protein Structure Preticition, and the Levinthal Paradox, In The Protein Folding Problem and Tertiary Structure Prediction, Eds. Merz et al., Birkhauser, Boston, MA, pp. 491-495, Jan. 1994.
van den Brink et al. (1995) Cloning and Characterization of the NADPH Cytochrome P450 Oxidoreductase Gene from the Filamentous Fungus Aspergillus niger, DNA and Cell Biology 8(14):719-729, Aug. 1995.
van den Brink Johannes Maarten
van Gorcom Robertus Franciscus
Nederlandse Organisatie voor toegepast-natuurwetenschappelijk on
Stole Einar
Wax Robert A.
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