Arylsulfotransferase

Details Arylsulfotransferase Arylsulfotransferase

1999-01-08

2001-05-01

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S183000, C530S350000

Reexamination Certificate

active

06225100

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a novel arylsulfotransferase.
BACKGROUND OF THE INVENTION
Several enzymatic sulfation reactions occur in the human body as post-translational modification of a variety of proteins and peptides. The sulfoconjugation of endogenous and exogenous phenolic compounds is an important detoxification mechanism. Such sulfoconjugation reactions mainly occur in the liver, kidney, lung, erythrocytes, and intestinal epithelial cells. Additionally, a number of peptide hormones, such as cholecystokinin, caerulein and phyllocaerulein, require sulfation of tyrosine residues for full activity. The sulfation of both phenolic compounds and tyrosylpeptides is carried out by arylsulfotransferases (AST). A number of arylsulfotransferases have been described in mammals, fungi, and other eukaryotic organisms. Mammalian arylsulfotransferases transfer the sulfate group from 3′-phosphoadenosine-5′-phosphosulfate to a phenolic acceptor molecule. Recently, it has been shown that arylsulfotransferases from intestinal bacteria are involved in the sulfation and metabolism of some phenolic compounds. The donor substrate specificity of these bacterial arylsulfotransferases is different from that of the mammalian enzymes. The bacterial arylsulfotransferases do not utilize 3′-phosphoadenosine-5′-phosphosulfate as a sulfate donor, but instead can utilize a variety of aryl sulfate donors such as p-nitrophenylsulfate to transfer a sulfate group to both phenolic acceptors and tyrosylpeptides.
Synthetic production of some sulfated proteins is commercially valuable. For example, the tyrosylpeptide cholecystokinin (CCK) is used clinically in association with cholecystography. CCK has also been demonstrated to inhibit food intake in several animal species. CCK and a C-terminal peptide of CCK, CCK-8, are stimulators of exocrine and endocrine pancreatic secretion and gall bladder contraction in humans. To be biologically active, CCK and CCK-8 require sulfation at tyrosine residues.
Current chemical methods for sulfation of tyrosylpeptides are expensive and relatively inefficient. For example, excessive and non-specific side reactions which will occur during the chemical sulfation must be prevented by protecting the amino acid residues of the peptides. Instability of the peptides during the deprotection processes, as well as a general inefficiency of the sulfation process, result in low yields of the final tyrosylpeptide product. Therefore, there is a need for the development of a biocatalytic route for sulfation of peptides and proteins.
SUMMARY OF THE INVENTION
The present invention relates to a novel arylsulfotransferase (AST). More particularly, the present invention relates to a Clostridium arylsulfotransferase, and more particularly to the arylsulfotransferase produced by
Clostridium innocuum
554 ATCC 55803 (Strain 554WT). In preferred embodiments, an arylsulfotransferase of the present invention catalyzes the transfer of a sulfate group to cholecystokinin-8 with a yield of greater than about 35% and/or maintains greater than about 40% activity for a given substrate for about 2 weeks when stored at 4° C. without being immobilized or otherwise protected.
Another embodiment of the present invention relates to an arylsulfotransferase having a high donor substrate specificity for p-nitrophenylsulfate, estrone sulfate and indoxyl sulfate as substrate donors.
In another embodiment, an arylsulfotransferase of the present invention has all the identifying characteristics of an arylsulfotransferase produced by
Clostridium innocuum
554 ATCC 55803 (Strain 554WT).
In yet another embodiment, an arylsulfotransferase of the present invention has all the identifying characteristics of an arylsulfotransferase produced by
Eubacterium nodatum
552 ATCC 55802 (Strain 552WT).
In yet another embodiment, a arylsulfotransferase of the present invention has identifying characteristics selected from a total molecular weight of about 320 kDaltons, 4 subunits having a molecular weight of about 80 kDaltons each, an optimum pH for sulfation activity at about 8.2, a specific activity of greater than about 15.0, maintains greater than about 40% activity for about 2 weeks when stored at 4° C. without being immobilized or otherwise protected, and has an ability to use a wide variety of aryl sulfate donors.


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