Mechanistic similarity and diversity among the guanidine-modifying members of the pentein superfamily

Abstract

The pentein superfamily is a mechanistically diverse superfamily encompassing both noncatalytic proteins and enzymes that catalyze hydrolase, dihydrolase and amidinotransfer reactions on guanidine substrates. Despite generally low sequence identity, they possess a conserved structural fold and display common mechanistic themes in catalysis. The structurally characterized catalytic penteins possess a conserved core of residues that include a Cys, His and two polar, guanidine-binding residues. All known catalytic penteins use the core Cys to attack the substrate's guanidine moiety to form a covalent thiouronium adduct and all cleave one or more of the guanidine C--N bonds. The mechanistic information compiled to date supports the hypothesis that this superfamily may have evolved divergently from a catalytically promiscuous ancestor.

Fig. 1

The pentein fold consists of five αββαβ subdomains symmetrically arranged around afivefold axis of pseudosymmetry. A) Top-down viewofribosome anti-association factor eIF6 (PDB ID: 1G61). B) Side view of eIF6. C) Side view of arginine deiminase (PDB ID: 2A9G). D) Side view of succinylarginine dihydrolase (PDB ID: 1YNI). E) Side view of arginine: glycine amidinotransferase (PDB ID: 4JDW). F) Side view of PAD4 (PDB ID: 1WDA). In C–F, the conserved pentein core is colored gray, while additional inserts and loops are colored by amino acid residue number.

Fig. 1

The pentein fold consists of five αββαβ subdomains symmetrically arranged around afivefold axis of pseudosymmetry. A) Top-down viewofribosome anti-association factor eIF6 (PDB ID: 1G61). B) Side view of eIF6. C) Side view of arginine deiminase (PDB ID: 2A9G). D) Side view of succinylarginine dihydrolase (PDB ID: 1YNI). E) Side view of arginine: glycine amidinotransferase (PDB ID: 4JDW). F) Side view of PAD4 (PDB ID: 1WDA). In C–F, the conserved pentein core is colored gray, while additional inserts and loops are colored by amino acid residue number.

Fig. 2

Reactions catalyzed by penteins. The guanidine substrate binds and reacts with the core Cys residue to form a thiouronium intermediate, which is used in the hydrolase, amidinotransferase and dihydrolase reactions. It is assumed in this figure that the active-site Cys is deprotonated prior to formation of the alkylthiouronium adduct. However, the protonation model at work is unclear (see Fig. 6).

Fig. 3

Generic active-site diagram. Residues not conserved are shown in gray. R₁=N^δ of arginine substrate in hydrolases, R₂=N^δ of arginine substrate in amidinotransferases.

Fig. 4

Active-site diagram showing core catalytic residues for A) ADI, AgD and PAD, B) AGAT and AIAT, C) DDAH and D) SADH. Note that the terms “lateral” and “anterior” refer to the position of the residue relative to the substrate arginine’s N^δ—C^ζ bond in the hydrolases and dihydrolases.

Fig. 5

Proposed reaction mechanisms for A) hydrolases, B) amidinotransferases, C) dihydrolases.

Fig. 6

Proposed protonation states of pentein core Cys.

Fig. 7

Structures of A) hydrolase (Green) and B) amidinotransferase (Orange) active sites, and C) an overlay of the two active sites. The hydrolase is ADI from P. aeruginosa (PDB ID: 2a9g) and the amidinotransferase is human AGAT (PDBID: 4jdw). Substrate is positioned at different relative angles, presumable to facilitate cleavage of a different C–N bond. In both structures, the core Cys is mutated to Ala to enable formation of a stable substrate complex.