Deregulation of glutamine PRPP amidotransferase activity

Details Deregulation of glutamine PRPP amidotransferase activity Deregulation of glutamine PRPP amidotransferase activity

1998-09-09

2001-03-20

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S194000, C435S320100, C435S252300, C435S252330

Reexamination Certificate

active

06204041

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the deregulation of purine nucleotide biosynthesis. More particularly, this invention is directed to the modification of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase to decrease end-product inhibition of glutamine PRPP amidotransferase activity.
BACKGROUND
Glutamine PRPP amidotransferase catalyzes the initial reaction in de novo purine nucleotide synthesis and is the key regulatory enzyme in the pathway. Genes encoding glutamine PRPP amidotransferase have been cloned from more than 20 organisms including bacteria, eukarya, and archea. In particular, the enzymes from
Escherichia coli
and
Bacillus subtilis
have been purified to homogeneity and are well-characterized, including having the X-ray structures determined for these enzyme species. The
E. coli
and
B. subtilis
enzymes are both homotetramers and are representative of two classes of glutamine PRPP amidotransferases. Enzymes of the
B. subtilis
class are synthesized with an NH
2
terminal propeptide and an Fe—S center, whereas enzymes of the
E. coli
class have neither.
Purine nucleotide biosynthesis is regulated, at both the gene (transcription/translation) and the protein (enzymatic activity) level. Attempts at increasing the biosynthesis of purine nucleotides have focused on the deregulation of genes encoding pathway enzymes. However, the enzymatic activity of a key pathway enzyme, glutamine PRPP amidotransferase, is inhibited by the pathway end-products, adenine and guanine nucleotides. Therefore an effective strategy for enhancing host cell production of purine nucleotides includes the use of a modified glutamine PRPP amidotransferase, wherein the modification reduces the sensitivity of the enzyme to end-product inhibition by adenine and guanine nucleotides.
The two classes of glutamine PRPP amidotransferase enzymes (as represented by the
E. coli
and
B. subtilis
enzymes) exhibit different end-product inhibitory patterns by adenine and guanine nucleotides. GMP is the single strongest inhibitor of
E. coli
glutamine PRPP amidotransferase, and AMP is the strongest inhibitor of the
B. subtilis
enzyme. Notwithstanding this difference, there is one common distinctive characteristic of the inhibition by nucleotides. Certain pairs of adenine and guanine nucleotides give more than additive inhibition compared to the individual nucleotides, a phenomenon called synergistic inhibition. AMP plus GMP has been reported to be a strong synergistic nucleotide pair for the
E. coli
enzyme, although more recent experiments suggest that GDP plus AMP may be the most effective synergistic pair. The strongest synergistic pair for the
B. subtilis
enzyme has been reported to be ADP plus GMP.
The synergistic inhibition of glutamine PRPP amidotransferase implies the existence of separate binding sites for adenine and guanine nucleotides on each subunit. X-ray structures of
B subtilis
and
E. coli
enzymes have identified four nucleotide binding sites per half-tetramer, two equivalent allosteric A sites between subunits, each with an adjacent catalytic C sites. It has been reported that synergistic binding of GMP to the A site and AMP to the C site could account for the synergistic inhibition of the
E. coli
enzyme.
A more systematic study of the mechanism for synergistic inhibition of the
B. subtilis
glutamine PRPP amidotransferase, as reported herein, confirms that ADP and GMP are the most synergistic pair and demonstrates that synergistic inhibition results from synergistic binding. An X-ray structure of a ternary enzyme ADP-GMP complex establishes that ADP binds to the A site and GMP to the C site and that synergism results from a specific interaction between the &bgr;-phosphate of a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site. These results establish the mechanistic basis for synergism.
SUMMARY OF THE INVENTION
Purine nucleotide production in bacteria is controlled and limited by regulation of gene expression as well as regulation of the enzymatic activity of pathway enzymes such as glutamine PRPP amidotransferase. This regulation limits production yields during commercial biosynthesis of inosine, as well as adenine and guanine nucleosides and nucleotides. Typically, commercial production of the nucleosides and nucleotides is carried out using Bacillus species that are genetically deregulated. However, due to feedback inhibition of glutamine PRPP amidotransferase, nucleotide overproduction in these genetically deregulated strains is less than maximal. The elimination, or at least the reduction, of nucleotide end-product inhibition of glutamine PRPP amidotransferase activity will further enhance the production yield of purine nucleotides.
In accordance with one embodiment of the present invention a modified glutamine PRPP amidotransferase is prepared that is desensitized to inhibition by purine nucleotides. The modified enzyme has at least one amino acid of the allosteric A sites or the catalytic C sites of said amidotransferase substituted with a non-native amino acid, wherein the substitution reduces the sensitivity of the enzyme to end product inhibition relative to the native glutamine PRPP amidotransferase enzyme. Furthermore, the present invention encompasses gene sequences that encode for the modified glutamine PRPP amidotransferase.
In accordance with one embodiment a method is provided for producing purine nucleotides. The method comprises the steps of culturing a host cell that comprises gene sequences encoding for a modified glutamine PRPP amidotransferase, wherein the modified glutamine PRPP amidotransferase has reduced sensitivity to end-product inhibition.


REFERENCES:
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