Abstract

Transglutaminases (TGases) are ubiquitous enzymes that catalyze selective cross-linking between protein-bound glutamine and lysine residues; the resulting isopeptide bond confers high resistance to proteolysis. Phytophthora sojae, a pathogen of soybean, secretes a Ca(2+)-dependent TGase (GP42) that is activating defense responses in both host and non-host plants. A GP42 fragment of 13 amino acids, termed Pep-13, was shown to be absolutely indispensable for both TGase and elicitor activity. GP42 does not share significant primary sequence similarity with known TGases from mammals or bacteria. This suggests that GP42 has evolved novel structural and catalytic features to support enzymatic activity. We have solved the crystal structure of the catalytically inactive point mutant GP42 (C290S) at 2.95 Å resolution and identified residues involved in catalysis by mutational analysis. The protein comprises three domains that assemble into an elongated structure. Although GP42 has no structural homolog, its core region displays significant similarity to the catalytic core of the Mac-1 cysteine protease from Group A Streptococcus, a member of the papain-like superfamily of cysteine proteases. Proteins that are taxonomically related to GP42 are only present in plant pathogenic oomycetes belonging to the order of the Peronosporales (e.g. Phytophthora, Hyaloperonospora, and Pythium spp.) and in marine Vibrio bacteria. This suggests that a lateral gene transfer event may have occurred between bacteria and oomycetes. Our results offer a basis to design and use highly specific inhibitors of the GP42-like TGase family that may impair the growth of important oomycete and bacterial pathogens.

Highlights

  • The Phytophthora sojae GP42 transglutaminase induces defense responses in parsley and potato

  • Our results offer a basis to design and use highly specific inhibitors of the GP42like TGase family that may impair the growth of important oomycete and bacterial pathogens

  • The reaction mechanism of TGases and cysteine proteases is based on a catalytic triad formed by Cys, His, and Asp residues and proceeds via an acyl-enzyme intermediate in which the active site cysteine is covalently bound to the ligand [1]

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Summary

Background

The Phytophthora sojae GP42 transglutaminase induces defense responses in parsley and potato. GP42 does not share significant primary sequence similarity with known TGases from mammals or bacteria This suggests that GP42 has evolved novel structural and catalytic features to support enzymatic activity. The reaction mechanism of TGases and cysteine proteases is based on a catalytic triad formed by Cys, His, and Asp residues and proceeds via an acyl-enzyme intermediate in which the active site cysteine is covalently bound to the ligand [1]. This is a striking example of the conservation in evolutionary distant taxa of a tertiary structure in proteins mechanistically related but with highly diverging primary sequences. It was suggested that animal TGases have evolved from

Structure of a Phytophthora Transglutaminase
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
No of atoms
Relative TGase activity
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