Amidation of bioactive peptides: the structure of the lyase domain of the amidating enzyme

Abstract

Many neuropeptides and peptide hormones require amidation of their carboxy terminal for full biological activity. The enzyme peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5) catalyzes the second and last step of this reaction, N-dealkylation of the peptidyl-alpha-hydroxyglycine to generate the alpha-amidated peptide and glyoxylate. Here we report the X-ray crystal structure of the PAL catalytic core (PALcc) alone and in complex with the nonpeptidic substrate alpha-hydroxyhippuric acid. The structures show that PAL folds as a six-bladed beta-propeller. The active site is formed by a Zn(II) ion coordinated by three histidine residues; the substrate binds to this site with its alpha-hydroxyl group coordinated to the Zn(II) ion. The structures also reveal a tyrosine residue (Tyr(654)) at the active site as the catalytic base for hydroxyl deprotonation, an unusual role for tyrosine. A reaction mechanism is proposed based on this structural data and validated by biochemical analysis of site-directed PALcc mutants.

Figure 1. The sequential stereospecific reactions of PAM catalyzed by PHM and PAL

Figure 2. Crystal structure of His₆-PALcc

The PAL enzyme folds as a six-bladed β-propeller; the figure shows two views: oriented with the central cavity normal to the page, left, and with the central cavity in plane of the page, right. Four strands form each of the blades, numbered 1–6 around the periphery. The Zn(II) and Ca(II) ions are indicated as blue and green balls, respectively (Zn-Ca distance = 11 Å).

Figure 3. (A) Active site of PALcc

The zinc(II) (blue ball) ion is in close proximity to the essential tyrosine, Y654, and a key arginine, R706, and is coordinated in a very distorted tetrahedral polyhedron liganded by three histidines and one acetate bound in a monodentate fashion. Relevant distances include: Zn-Nε_H585 = 2.1 Å; Zn-Nε_H690 = 2.1 Å; Zn-Nε_H786 = 2.1 Å; Zn-O1_acetate = 2.0 Å; Zn-O2_acetate = 3.3 Å; Zn-OH_Y654 = 3.2 Å. Carbons are colored green, nitrogens blue, and oxygens red. The gray mesh represents the 2Fo-Fc electron density contoured at 1σ. (B) Calcium site of PALcc. The calcium(II) ion (green ball) is bound in the middle of the central cavity by three residues that belong to three different β-strands: the carboxylate of D787 (as a monodentate ligand), and the main-chain carbonyl groups of V520 and L587. Three water molecules and a glycerol complete the coordination sphere (Ca-O_D787 = 2.45 Å; Ca-O_V520 = 2.5 Å; Ca-O_L587 = 2.3 Å; Ca-O_W= 2.5 Å; Ca-O_W= 2.6 Å; Ca-O_W= 2.7 Å; Ca-O_glycerol = 2.75 Å). Carbons are colored green, nitrogens blue, and oxygens red. The gray mesh represents the 2Fo-Fc electron density contoured at 1σ.

Figure 4. PAL-substrate complex

A: PALcc-HydHipA (α-hydroxyhippuric acid) complex structure showing the substrate bound to the metal, Arg⁵³³ and Tyr⁶⁵⁴. B: A different view of the PALcc-HydHipA structure showing also the substrate interacting with Met⁷⁸⁴. C: PAL-tripeptide [Ala-Ala-Gly(OH)] model complex showing the most relevant interactions, as seen in figure B. See text for further discussion. Carbons are colored gray, nitrogens blue, and oxygens red. A purple mesh represents the calculated sigmaA weighted mFo-DFc omit map (Refmac5) of the substrate, contoured at 2σ (Figures A and B). The dashed lines represent relevant interactions between the substrate and the protein (see text).

Figure 5. Site-directed mutagenesis studies on PALcc

PEAK-Rapid cells were transiently transfected with vectors encoding wild-type PALcc or each of the mutants indicated. After 24 hours, cells were rinsed and incubated in complete serum-free medium (m) and cells (c) were harvested in TES-mannitol-Triton buffer with protease inhibitors. Samples were analyzed by Western blot (0.8% of the medium; 4% of the cell extract) using an anti-peptide polyclonal antibody and aliquots were assayed for PAL activity. The activity of each sample is shown at the top of each gel lane.

Figure 6. PAL reaction mechanism

A mechanism for PAL is proposed on the basis of structural and biochemical data. After substrate binding to the active site through coordination of the substrate a-hydoxyl group to the Zn(II) ion, the Tyr⁶⁵⁴ (as phenolate) deprotonates the substrate α-OH, triggering Cα-N bond cleavage and formation of the amidated peptide and glyoxylate.