Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site

Abstract

Background: Glutaminyl cyclase (QC) forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins. We previously proposed the mammalian QC has some features in common with zinc aminopeptidases. We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP) and mutated the apparent active site residues to assess their role in QC catalysis.

Results: The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide. Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering) and the two glutamates (201 and 202), while the two aspartates (159 and 248) appear to play no catalytic role. ICP-MS analysis shows less than stoichiometric zinc (0.3:1) in the purified enzyme.

Conclusions: We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues. In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity.

Figure 1

Ribbon diagram of hQC theoretical model. Ribbon diagram of secondary and tertiary structures of Vibrio aminopeptidase (PDB 1AMP) and human glutaminyl cyclase (PDB 1MOI). Structures are oriented so that active sites are on the right of the molecule. Illustration was generated with DeepView 3.7 using solid open GL rendering and colored by secondary structure succession.

Figure 2

Sequence alignment of hQC and structural templates. Alignment of amino acid sequence of Vibrio aminopeptidase (PDB code 1AMP), Streptomyces aminopeptidase (PDB code 1XJO) Pseudomonas carboxypeptidase G2 (PDB code 1CG2) and human pituitiary glutaminyl cyclase (bottom line). Numbers along the top indicate the alignment position. Numbers in parentheses on the left hand side indicate the sequence numbering for the first amino acid in on that line for each sequence. Bars indicate secondary structure (dark bars for helices, light bars for strands) for those sequences with experimentally determined structures. Bars underneath the alignment (labeled "Psipred") indicate predicted secondary structure for the human QC sequence.

Figure 3

Predicted active site of human glutaminyl cyclase. Figure 5 represents the five conserved human QC residues corresponding to the zinc ligands of the bacterial aminopeptidase plus the conserved glutamate corresponding to the general base of the aminopeptidase. QC numbering from the top (clockwise): His 330, Glu 202, Glu 201, His 140, Asp 248, Asp 159. See Table 1 for comparisons with 1AMP.

Figure 4

Circular dichroism spectrum of DES hQC. Circular dichroism of 6 μM DES hQC in 5 mM sodium phosphate buffer, pH 6.0 from 196 to 260 nm.