Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 11 to 14 amino acid residues in defined sequence
Reexamination Certificate
2001-03-14
2003-09-09
Weber, Jon P. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
11 to 14 amino acid residues in defined sequence
Reexamination Certificate
active
06617424
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a peptide which can impart photoreactivity to a protein, and a method for controlling metabolism of cells by introducing a sequence of the peptide into at least one protein which functions in a metabolic system so that the protein should acquire photoreactivity, thereby controlling metabolism of cells.
BACKGROUND OF THE INVENTION
Nitrile hydratase (NHase) is an enzyme which is produced in microorganisms and converts a nitrile compound into an amide compound by hydration. It is a soluble metalloprotein containing iron or cobalt atom in its active center. NHase derived from Rhodococcus sp. N-771 strain has a non-heme iron center of mononuclear low-spin six coordinate Fe(III). NHases have been isolated from several kinds of bacterial cells, and all of them consist of two kinds of subunits, &agr; and &bgr;. Both of the subunits have a molecular weight of about 23,000. NHases of Rhodococcus sp. N-771, N-774, and R312 are considered to be the same enzyme because their base sequences are identical to each another, and their enzymatic activity varies with light irradiation. Namely, when bacterial cells exhibiting high activity are left in the dark, the enzyme activity is reduced, and the activity is increased again by photo-irradiation.
It was recently revealed that photodissociation of nitric oxide (NO) which is bound to the non-heme iron center of inactive form Nhase activates the enzyme (Odaka et al., J. Am. Chem. Soc., 119, 3785-3791 (1997)). However, because the structure of the non-heme iron center which is the photoreactive site was not elucidated yet, the detailed mechanism of the photoreaction remained unclear. Therefore, the present inventors performed structural analysis of the non-heme iron center, and reported the results (for example, see Protein, Nucleic acid, Enzyme, Vol.42, No.2, p38-45 (1997)). That is, the present inventor isolated the &agr; and &bgr; subunits from the inactive form enzyme under denaturation condition, and found that the NO-binding type non-heme iron center is present on the &agr; subunit. Therefore, they further performed enzymatic degradation of the &agr; subunit with trypsin, and purified the resulting peptides by reversed phase chromatography under a neutral condition. As a result, a peptide consisting of 24 residues of
105
N to
128
K binding one iron atom and NO has been isolated. This region was well conserved in various kinds of NHases, and contained a cysteine cluster predicted to be a metal-binding site (
109
C-S-L-
112
C-S-
114
C).
By the way, development of processes for producing useful biomolecules such as amino acids, peptides, proteins, carbohydrates, lipids and the like by utilizing metabolic systems of cells including energy metabolic systems has been performed for a long time. However, in conventional metabolically controlled fermentation, fermentation system for the objective product is constructed through trials on screening and concentration of variants resistant to an analogue having a structure analogous to an objective product. Further, to control the constructed fermentation system, physical factors such as pH and temperature and chemical factors such as substrate concentration and addition of inducers must be changed. Therefore, various regulatory mechanisms of the living body are often affected, and many parameters must be determined for optimization of the production scheme. Moreover, regulatory methods of this type have a drawback that it takes a long period of time to obtain a reaction to stimulation. Therefore, if it is possible construct a method for controlling metabolism which enables proper turning on and turning off a desired intracellular metabolic system so as to produce a desired product in a necessary amount when it is required, it will greatly contribute to fermentation processes.
Accordingly, the present inventors studied out a method for artificially controlling metabolism of cells including energy metabolism by utilizing the peptide stably binding a non-heme iron, which is the photoreactive site of NHase mentioned above. That is, such a peptide as mentioned above is introduced into a protein which functions in a metabolic system of an objective product to impart photoreactivity to the protein so that the metabolic system can be controlled by presence or absence of light irradiation. In this method, photo-control of activity is realized by modifying a protein which functions in a specific reaction system in a metabolic pathway of an objective product. Because the protein to be modified can be arbitrarily selected depending on the purpose, the method has advantages that the screening step by trial and error like in the conventional method does not required, and that fermentation system of which metabolism is controlled can be precisely constructed. Furthermore, because activation of enzyme is achieved by photostimulation, fermentation operation can be performed more simply and quickly compared with the conventional methods.
However, when such a peptide sequence as mentioned above is introduced into various kinds of proteins working in intracellular substance metabolic systems or energy metabolic systems to impart photoreactivity to the metabolism or the energy metabolism, a relatively large peptide like the aforementioned peptide of 24 residues might have problems. The problems are that efficient reaction could not be obtained, or introduction of the peptide impairs the original function of the labeled protein in high possibility, because the relatively large protein contains a large fraction of sequence other than the minimum portion essential for the photoreaction.
SUMMARY OF THE INVENTION
Therefore, the object of the present invention is to provide a peptide sequence capable of efficient photoreaction, which is a peptide chain of a minimum unit capable of imparting photoreactivity, not likely to impair an original function of a labeled protein, and a method for enabling control of metabolic reaction by utilizing the peptide to impart photoreactivity to cells.
The present invention relates to a peptide having a sequence [SEQ ID NO:3] represented by the following general formula (1).
X
1
X
2
C
1
X
3
X
4
C
2
SC
3
X
5
X
6
X
7
(1)
wherein X
1
to X
7
represents an arbitrary amino acid, C
1
represents cysteine, C
2
represents cysteinesulfinic acid, C
3
represents cysteinesulfenic acid, and S represents serine.
An embodiment of the present invention is the aforementioned peptide wherein the sequence [SEQ ID NO:4] represented by the general formula (1) is represented as IVC
1
SLC
2
SC
3
TAW wherein I represents isoleucine, V represents valine, C
1
represents cysteine, C
2
represents cysteinesulfinic acid, C
3
represents cysteinesulfenic acid, S represents serine, L represents leucine, T represents threonine, A represents alanine, and W represents tryptophan.
Another embodiment of the present invention is the aforementioned peptide wherein X
1
to X
7
in the sequence represented by the general formula (1) are selected from amino acids which can maintain higher order structure of a peptide represented by IVC
1
SLC
2
SC
3
TAW in nitrile hydratase derived from Rhodococcus sp.N-771.
A further embodiment of the present invention is a peptide having a sequence represented by the following general formula (2):
C
1
X
3
X
4
C
2
SC
3
(2)
wherein X
3
and X
4
represent arbitrary amino acids, C
1
represents cysteine, C
2
represents cysteinesulfinic acid, C
3
represents cysteinesulfenic acid, and S represents serine. Examples of the above peptide include, for example, the aforementioned peptide wherein the sequence represented by the general formula (2) is represented as C
1
SLC
2
SC
3
wherein C
1
represents cysteine, C
2
represents cysteinesulfinic acid, C
3
represents cysteinesulfenic acid, S represents serine, and L represents leucine. Examples of the above peptide further include, for example, the aforementioned peptide wherein X
3
and X
4
in the sequence represented by the general formula (2) are select
Endo Isao
Nakasako Masayoshi
Odaka Masafumi
Takio Koji
Yohda Masafumi
Burns Doane Swecker & Mathis L.L.P.
The Institute of Physical and Chemical Research
Weber Jon P.
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