Analysis of Active Site Residues of the Antiviral Protein from Summer Leaves fromPhytolacca americanaby Site-Directed Mutagenesis

doi:10.1006/bbrc.1998.9815

Biochemical and Biophysical Research Communications

Volume 253, Issue 3, 30 December 1998, Pages 582-587

https://doi.org/10.1006/bbrc.1998.9815 Get rights and content

Abstract

The summer leaf isoform of the pokeweed (Phytolacca americana) antiviral protein, PAP II, was produced in high yields from inclusion bodies in recombinantE. coli.On the basis of its sequence similarity with the spring leaf isoform (PAP I) and with the A chain of ricin, a three-dimensional model of the protein was constructed as an aid in the design of active-site mutants. PAP II variants mutated in residues Asp 88 (D88N), Tyr 117 (Y117S), Glu 172 (E172Q), Arg 175 (R175H) and a combination of Asp 88 and Arg 175 (D88N/R175H) were produced inE. coliand assayed for their ability to inhibit protein synthesis in a rabbit reticulocyte lysate. All of these mutations had effects deleterious to the enzymatic activity of PAP II. The results were interpreted in the light of three reaction mechanisms proposed for ribosome-inactivating proteins (RIPs). We conclude that none of the proposed mechanisms is entirely consistent with the data presented here.

References (31)

J.D. Irvin
Arch. Biochem. Biophys.
(1975)
J.D. Irvin et al.
Arch. Biochem. Biophys.
(1980)
Y. Endo et al.
J. Biol. Chem.
(1987)
J.-L. Poyet et al.
FEBS Lett.
(1994)
W. Montfort et al.
J. Biol. Chem.
(1987)
A.F. Monzingo et al.
J. Mol. Biol.
(1993)
J. Ren et al.
Structure
(1994)
J. Husain et al.
FEBS Lett.
(1994)
M.V. Hosur et al.
J. Mol. Biol.
(1995)
T.H. Tahirov et al.
J. Mol. Biol.
(1995)

A.F. Monzingo et al.

J. Mol. Biol.

(1992)

S.N. Ho et al.

Gene

(1989)

J.-L. Poyet et al.

FEBS Lett.

(1997)

K. Watanabe et al.

Biochim. Biophys. Acta

(1987)

S. Weston et al.

J. Mol. Biol.

(1994)

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The three-dimensional structures of two type 1 RIPs, bouganin and lychnin, has been solved. Their adenine polynucleotide glycosylase activity was also determined together with other known RIPs: dianthin 30, PAP-R, momordin I, ricin A chain and saporin-S6. Saporin-S6 releases the highest number of adenine molecules from rat ribosomes, and poly(A), while its efficiency is similar to dianthin 30, bouganin and PAP-R on herring sperm DNA. Measures of the protein synthesis inhibitory activity confirmed that saporin-S6 is the most active. The overall structure of bouganin and lychnin is similar to the other considered RIPs and the typical RIP fold is conserved. The superimpositioning of their C_α atoms highlights some differences in the N-terminal and C-terminal domains. A detailed structural analysis indicates that the efficiency of saporin-S6 on various polynucleotides can be ascribed to a negative electrostatic surface potential at the active site and several exposed positively charged residues in the region around that site. These two conditions, not present at the same time in other examined RIPs, could guarantee an efficient interaction with the substrate and an efficient catalysis.
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Regular ArticleAnalysis of Active Site Residues of the Antiviral Protein from Summer Leaves fromPhytolacca americanaby Site-Directed Mutagenesis

Abstract

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Mol. Biol.

Structure

FEBS Lett.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Gene

FEBS Lett.

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J. Mol. Biol.

Regular Article
Analysis of Active Site Residues of the Antiviral Protein from Summer Leaves fromPhytolacca americanaby Site-Directed Mutagenesis