Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing a carotene nucleus
Reexamination Certificate
1999-04-27
2001-11-13
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing a carotene nucleus
C435S183000, C435S252300, C435S254300, C435S320100, C435S325000, C435S193000, C536S023200
Reexamination Certificate
active
06316216
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel mutant enzymes which synthesize linear prenyl diphosphates that are precursors of compounds important for organisms, such as steroids, ubiquinones, dolichols, carotenoids, prenylated proteins, animal hormones, plant hormones and the like, and to a gene and production of the enzymes.
BACKGROUND ART
Many substances having important functions in organisms are biosynthesized using isoprene (2-methyl-1,3-butadiene) as a building block. These compounds are also called isoprenoids, terpenoids, or terpenes, and are classified depending on the number of carbon atoms into hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), tetraterpenes (C40) and the like. The biosynthesis starts with the mevalonate pathway through which mevalonic acid-5-diphosphate is synthesized, followed by the synthesis of isopentenyl diphosphate (IPP) which is an active isoprene unit.
The true entity of the isoprene unit that was proposed as a putative precursor was found to be isopentenyl diphosphate, the so-called active-form isoprene unit. While dimethylallyl diphosphate (DMAPP), an isomer of isopentenyl diphosphate, is used as a substrate in the reaction of isopentenyl adenine which is known as a cytokinin of plant hormones, it is also known to undergo a condensation reaction with isopentenyl diphosphate to synthesize linear active isoprenoids such as geranyl diphosphate (GPP), neryl diphosphate, farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), geranylfarnesyl diphosphate (GFPP), hexaprenyl diphosphate (HexPP), heptaprenyl diphosphate (HepPP) and the like. There are the Z type and the E type in the condensation reaction. Geranyl diphosphate is a product of the E type condensation and neryl diphosphate of the Z type condensation.
Although, the all-E type is considered to be the active form in farnesyl diphosphate and geranylgeranyl diphosphate, the Z type condensation reaction leads to the synthesis of various polyprenols found in natural rubber, dolichols, bactoprenols (undecaprenols), and plants. They are believed to undergo the condensation reaction using the phosphate ester bond energy of the pyrophosphate and the carbon backbone present in the molecule and to produce pyrophosphate as the byproduct of the reaction.
Farnesyl diphosphate or geranylgeranyl diphosphate serves as a reaction substrate leading to the synthesis of prenylated proteins (from farnesyl diphosphate or geranylgeranyl diphosphate) represented by the G protein that is important in the mechanism of signal transduction in the cell; cell membrane lipids (from geranylgeranyl diphosphate) of archaea; squalene (from farnesyl diphosphate) which is a precursor of steroids; and of phytoene (from geranylgeranyl diphosphate) which is a precursor of carotenoids. Prenyl diphosphates from hexaprenyl diphosphate and heptaprenyl diphosphate having six and seven isoprene units, respectively, to prenyl diphosphates having ten isoprene units serve as the precursors of synthesis of ubiquinone and menaquinone (vitamin K2) that work in the electron transport system.
Furthermore, via the biosynthesis of these active-form isoprenoids, a large number of compounds that are essential for the life are synthesized. Just to mention a few, there are plant hormones, cytokinins and isopentenyl adenosine-modified tRNA which use hemiterpenes as precursor for synthesis thereof, monoterpene geraniols and its isomer of nerol that are the main components of the rose oil perfume, and a camphor tree extract, camphor, which is an insecticide. Sesquiterpenes include juvenile hormones of insects, diterpenes include plant hormone gibberellins, trail pheromones of insects, and retinols and retinals that function as brown pigment precursors, binding components of the purple membrane proteins of extremely halophilic microorganisms, and vitamin A.
Furthermore, using squalene which is a triterpene, a vast variety of steroid compounds are synthesized, including, for example, animal sex hormones, vitamin D, ecdysone which is an ecdysis hormone of insects, a plant hormone brassinolide, the components of plasma membrane. Various carotenoids of tetraterpenes that are precursors of various pigments of organisms and vitamin A are also important compounds derived from active isoprenoids. Compounds such as chlorophyll, pheophytin, tocopherol (vitamin E), and phylloquinone (vitamin K1) are also derived from tetraterpenes.
The active isoprenoid synthases that consecutively condense isopentenyl diphosphates with such allylic substrates as dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, geranylfarnesyl diphosphate, etc. are called the prenyl diphosphate synthases, and are also called, based on the name of the compound having the maximum chain length of the major reaction products, for example farnesyl diphosphate synthase (FPP synthase), geranylgeranyl diphosphate (GGPP synthase) and the like.
So far there have been reports on purification, activity measurement, gene cloning, and sequencing of the nucleotide sequences of the genes of enzymes such as farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, hexaprenyl diphosphate synthase, heptaprenyl diphosphate synthase, octaprenyl diphosphate synthase, nonaprenyl diphosphate synthase (solanesyl diphosphate synthase), undecaprenyl diphosphate synthase and the like from bacteria, archaea, fungi, plants, and animals.
These active isoprenoid synthases constituting the basis of synthesis of a great variety of compounds that are important both in the industry and in the academic field of life sciences have few practical uses in industrial applications due to their unstable nature and low specific activities. However, with the isolation of thermostable prenyl diphosphate synthases from thermophilic bacteria and archaea and the genes encoding these enzymes, their availability as the enzymes is increased.
With regard to farnesyl diphosphate synthase, a gene was isolated from a moderate thermophile,
Bacillus stearothermophilus,
and an enzyme having a moderate thermostability was prepared using
Escherichia coli
as the host cell [T. Koyama et al. (1993) J. Biochem., 113: 355-363; Japanese Unexamined Patent Publication No. 5(1993)-219961]. With regard to geranylgeranyl diphosphate synthase, a gene was isolated from an extreme thermophile such as
Sulfolobus acidocaldarius
and
Thermus thermophilus
[S. -i. Ohnuma et al., (1994) J. Biol. Chem., 269: 14792-14797; Japanese Unexamined Patent Publication No. 7(1995)-308193, and; Japanese Unexamined Patent Application No. 7(1995)-294956], and enzymes having an extreme thermostability were prepared.
Furthermore, with regard to the prenyl diphosphate synthase having the functions of both of the farnesyl diphosphate synthase and the geranylgeranyl diphosphate synthase, the enzyme and the gene encoding it have been isolated from extremely thermophilic
Methanobacterium thermoautotrophicum
[A. Chen and D. Poulter (1993) J. Biol. Chem., 268: 11002-11007; A. Chen and D. Poulter (1994) ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 314], and the thermostability of the enzyme has been demonstrated.
The enzymes that synthesize prenyl diphosphates having up to 20 carbons are homodimers, which relatively easily react in vitro and on which there have been many reports. However, it is believed that the enzymes that synthesize prenyl diphosphates having a longer chain length are heterodimers or that the enzymes may require other factors such as lipids, and hence for their industrial application to be effected it was necessary, and difficult, to find conditions that enable reorganization of the two subunits or other factors.
Thus, a technology has been desired that enables production of the thermostable and homodimer-type prenyl diphosphate synthases capable of synthesizing prenyl diphosphates having a longer chain length by artificially modifying amino acid sequences of the thermostable homodimer-type prenyl di
Hirooka Kazutake
Nakane Hiroyuki
Nishino Tokuzo
Ohnuma Shin-ichi
Ohto Chikara
Kenyon & Kenyon
Prouty Rebecca E.
Rao Manjunath N.
Toyota Jidosha & Kabushiki Kaisha
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