Identification and characterization of gamma-glutamylamine cyclotransferase, an enzyme responsible for gamma-glutamyl-epsilon-lysine catabolism

Abstract

Gamma-glutamylamine cyclotransferase (GGACT) is an enzyme that converts gamma-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward gamma-glutamyl-epsilon-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in gamma-glutamylcyclotransferase (GGCT), an enzyme with activity toward gamma-glutamyl-alpha-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu(82)). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.

FIGURE 1.

Models of GGACT in the vicinity of the active site shown in stick form with 2mF_o − DF_c electron density, contoured at 1σ shown in chicken wire representation. A and B, GGACT and GGACT, respectively, in complex with 5-oxo-l-proline; C, GGACT E82Q mutant.

FIGURE 2.

A, schematic representation of GGACT, with bound nitrate groups represented in stick form. The N and C termini are labeled. B, topology diagram of GGACT. Interactions between β-strands are indicated by black rectangles. The broken rectangles adjacent to strands β2a and β4 indicate the wrapping of the β-sheet to form a barrel.

FIGURE 3.

A, structure of the active site of GGACT in complex with 5-oxo-l-proline; B, model of GGACT in complex with l-γ-glutamyl-l-ϵ-lysine; C, model of GGCT in complex with 5-oxo-l-proline; D, model of GGCT in complex with l-γ-glutamyl-l-α-cysteine. In all cases, carbon atoms of the protein and ligand are yellow and green, respectively. Potential hydrogen bonds are shown as thin black lines.

FIGURE 4.

Sequence alignment of Protein Data Bank structures adopting the cyclotransferase fold. Sequences are labeled by Protein Data Bank codes, except for human GGACT (hGGACT) and GGCT (hGGCT). Protein Data Bank code 2QIK contains two cyclotransferase domains that have been split into 2QIK-1 and 2QIK-2. Secondary structure elements of human GGACT are denoted above the sequences. Where significant structural homology exists, the residues are shown in uppercase letters. Residues lining the active-site cavity are indicated with green triangles. Possible catalytic glutamate residues are highlighted with a red star. Completely conserved (black) and highly conserved (gray) residues are highlighted.

FIGURE 5.

Neighbor joining tree depicting the probable phylogenetic relationships between selected proteins with the γ-glutamylcyclotransferase fold. Representative structures from each subfamily are shown.

FIGURE 6.

Proposed reaction mechanism for GGACT and GGCT. Glu represents Glu⁸² (GGACT) or Glu⁹⁸ (GGCT).

FIGURE 7.

Surface of GGACT (A) and GGCT (B) with substrate modeled in the active site. Electrostatic potential at the surface is represented as blue (positive potential) and red (negative potential).